This article was downloaded by: [Biblioteca Universitaria], [Giulia Amagliani] On: 19 January 2012, At: 07:17 Publisher: Taylor & Francis Informa Ltd Registered in England and Wales Registered Number: 1072954 Registered office: Mortimer House, 37-41 Mortimer Street, London W1T 3JH, UK International Journal of Environmental Health Research Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/cije20 Molecular detection of Pseudomonas aeruginosa in recreational water Giulia Amagliani a b , Maria Lorella Parlani c , Giorgio Brandi a b , Giuseppe Sebastianelli c , Vilberto Stocchi a b & Giuditta Fiorella Schiavano a a Dipartimento di Scienze Biomolecolari, Università degli Studi di Urbino “Carlo Bo”, Urbino, Italy b Facoltà di Scienze Motorie, Università degli Studi di Urbino “Carlo Bo”, Urbania, Italy c Laboratorio di Igiene e Sanità Pubblica, Dipartimento di Prevenzione, ASUR ZT2, Urbania, Italy Available online: 13 Jun 2011 To cite this article: Giulia Amagliani, Maria Lorella Parlani, Giorgio Brandi, Giuseppe Sebastianelli, Vilberto Stocchi & Giuditta Fiorella Schiavano (2012): Molecular detection of Pseudomonas aeruginosa in recreational water, International Journal of Environmental Health Research, 22:1, 60-70 To link to this article: http://dx.doi.org/10.1080/09603123.2011.588325 PLEASE SCROLL DOWN FOR ARTICLE Full terms and conditions of use: http://www.tandfonline.com/page/terms-and- conditions This article may be used for research, teaching, and private study purposes. Any substantial or systematic reproduction, redistribution, reselling, loan, sub-licensing, systematic supply, or distribution in any form to anyone is expressly forbidden. The publisher does not give any warranty express or implied or make any representation that the contents will be complete or accurate or up to date. The accuracy of any instructions, formulae, and drug doses should be independently verified with primary sources. The publisher shall not be liable for any loss, actions, claims, proceedings,
Transcript
This article was downloaded by [Biblioteca Universitaria] [Giulia Amagliani]On 19 January 2012 At 0717Publisher Taylor amp FrancisInforma Ltd Registered in England and Wales Registered Number 1072954 Registeredoffice Mortimer House 37-41 Mortimer Street London W1T 3JH UK
International Journal of EnvironmentalHealth ResearchPublication details including instructions for authors andsubscription informationhttpwwwtandfonlinecomloicije20
Molecular detection of Pseudomonasaeruginosa in recreational waterGiulia Amagliani a b Maria Lorella Parlani c Giorgio Brandi a b Giuseppe Sebastianelli c Vilberto Stocchi a b amp Giuditta FiorellaSchiavano aa Dipartimento di Scienze Biomolecolari Universitagrave degli Studi diUrbino ldquoCarlo Bordquo Urbino Italyb Facoltagrave di Scienze Motorie Universitagrave degli Studi di UrbinoldquoCarlo Bordquo Urbania Italyc Laboratorio di Igiene e Sanitagrave Pubblica Dipartimento diPrevenzione ASUR ZT2 Urbania Italy
Available online 13 Jun 2011
To cite this article Giulia Amagliani Maria Lorella Parlani Giorgio Brandi Giuseppe SebastianelliVilberto Stocchi amp Giuditta Fiorella Schiavano (2012) Molecular detection of Pseudomonasaeruginosa in recreational water International Journal of Environmental Health Research 22160-70
To link to this article httpdxdoiorg101080096031232011588325
PLEASE SCROLL DOWN FOR ARTICLE
Full terms and conditions of use httpwwwtandfonlinecompageterms-and-conditions
This article may be used for research teaching and private study purposes Anysubstantial or systematic reproduction redistribution reselling loan sub-licensingsystematic supply or distribution in any form to anyone is expressly forbidden
The publisher does not give any warranty express or implied or make any representationthat the contents will be complete or accurate or up to date The accuracy of anyinstructions formulae and drug doses should be independently verified with primarysources The publisher shall not be liable for any loss actions claims proceedings
demand or costs or damages whatsoever or howsoever caused arising directly orindirectly in connection with or arising out of the use of this material
Dow
nloa
ded
by [
Bib
liote
ca U
nive
rsita
ria]
[G
iulia
Am
aglia
ni]
at 0
717
19
Janu
ary
2012
Molecular detection of Pseudomonas aeruginosa in recreational water
Giulia Amaglianiab Maria Lorella Parlanic Giorgio BrandiabGiuseppe Sebastianellic Vilberto Stocchiab and Giuditta Fiorella Schiavanoa
aDipartimento di Scienze Biomolecolari Universita degli Studi di Urbino lsquolsquoCarlo Borsquorsquo Urbino ItalybFacolta di Scienze Motorie Universita degli Studi di Urbino lsquolsquoCarlo Borsquorsquo Urbania ItalycLaboratorio di Igiene e Sanita Pubblica Dipartimento di Prevenzione ASUR ZT2 Urbania Italy
(Received 21 March 2011 final version received 3 May 2011)
The aim of this study was the development of a new molecular assay forPseudomonas aeruginosa identification in recreational water The method includesbacterial cell concentration through membrane filtration a short (6 h) culture-enrichment step DNA extraction and its amplification through a Real-Time PCRassay The performance of the molecular approach was evaluated on 44 samples ofswimming pool water and compared with the reference method UNI EN ISO162662008 Positivity rates of 6 and 74 in pool and inlet water respectivelywith the standard culture method and of 23 and 74 with the molecular methodwere found Statistical analysis indicated lsquolsquosubstantial agreementrsquorsquo (Cohenrsquos Kappaindex 06831) between the two approaches RAPD typing of P aeruginosa isolatesshowed identical fingerprint profiles indicating their epidemiological correlationThe developed protocol showed very high specificity and a detection limit of 10genomic units This technique has the potential to screen large numbers ofenvironmental samples and could be proposed as part of a self-monitoring plan forrecreational facilities improving surveillance and early warning systems
Keywords Pseudomonas aeruginosa ecfX Real-Time PCR recreational waterrandom amplification of polymorphic DNA (RAPD)
Introduction
The use of swimming pools and other recreational waters is associated with benefitsto health and well-being However a variety of microorganisms which mayconstitute a microbiological hazard can be found in such waters and their relatedfacilities (World Health Organisation [WHO] 2006) Associated adverse healthoutcomes including skin and ear infections can arise from opportunistic pathogensshed by bathers or naturally present in the aquatic environment and transmitted viasurfaces and contaminated water These pathogens can also accumulate in biofilmsthus becoming resistant to biocides and other disinfection agents (Goeres et al2004) Recreational water is a documented environmental source of Pseudomonasaeruginosa (Moore et al 2002 Papadopoulou et al 2008 Guida et al 2009)therefore it may represent a health risk for some user categories Swimming pools areused by a large variety of people varying in age health and hygienic standards
International Journal of Environmental Health Research
Vol 22 No 1 February 2012 60ndash70
ISSN 0960-3123 printISSN 1369-1619 online
2012 Taylor amp Francis
httpdxdoiorg101080096031232011588325
httpwwwtandfonlinecom
Dow
nloa
ded
by [
Bib
liote
ca U
nive
rsita
ria]
[G
iulia
Am
aglia
ni]
at 0
717
19
Janu
ary
2012
Pregnant women babies the elderly and disabled people who may attend specialcourses are potentially predisposed to contracting infections from pathogens andopportunistic bacteria (Leoni et al 1999) Moreover immunocompromised andcystic fibrosis patients are at particular risk (Barben et al 2005 Mena and Gerba2009) as well as athletes under competitive stress who may have weakened immunesystems (Pond 2005 WHO 2006)
In swimming pools the primary health effect associated with P aeruginosa isotitis externa although folliculitis dermatitis conjunctivitis and pneumonia havealso been reported (WHO 2006) Indeed waterborne pseudomonas-infection can berecognised in patients presenting a history of participation in water sportsactivities(Wang et al 2005)
Recently published guidelines from WHO (2006) are intended to be used as abasis for the development of international and national approaches for the controlof hazards that may be encountered in such environments In Italy in order toimplement an appropriate monitoring program and ensure adequate surveillanceLocal Sanitary Authorities (ASUR Azienda Sanitaria Unica Regionale) have beenmade responsible for official controls of recreational water Moreover programmesof self-monitoring according to the HACCP approach are to be applied byswimming pool managers as an lsquolsquointernal controlrsquorsquo (Italian Republic 2003)
Currently monitoring of P aeruginosa in swimming pool water is conducted inaccordance with the EU Drinking Water Directive (European Union [EU] 1998) andits Italian counterparts (Italian Republic 2001 2002) For P aeruginosa a maximumpermissible standard of zero per 100 ml of inlet water and acceptability limits of 1cfu (colony-forming units) per 100 ml of pool water have been fixed by ItalianGuidelines (Italian Republic 2003)
The reference analytical method for P aeruginosa detection (UNI EN ISO162662008) although sensitive and specific requires at least 48 h for the isolation ofsuspected positive colonies and additional time is needed for confirmation testsMoreover it is unable to detect the presence of viable but non-culturable (VBNC) formsof P aeruginosa (Oliver 2005) Consequently rapid accurate and culture-independentalternatives are being investigated to enable monitoring of this opportunistic pathogenin water A molecular method could reduce analysis time while maintaining sensitivityand specificity at very high levels and offering the additional capability of VBNCdetection Hence the aim of this study was the development of a new molecular methodfor P aeruginosa identification in recreational water
The entire testing process yielding a definitive result in one working dayincluded bacterial cell concentration through membrane filtration a short (6 h)culture-enrichment step DNA extraction and its amplification in Real-Time PCRIn order to validate the developed molecular assay and assess its applicability in aself monitoring plan of a swimming pool its performance was evaluated andcompared to the reference culture method carried out by ASUR
Methods
Collection and processing of water samples
The swimming pool investigated for this study was an indoor pool used by peopleof all ages for recreational purposes and by athletes for training A total of 44 watersamples (17 pool water and 27 inlet water) were collected in sterile bottlescontaining 10 sodium thiosulfate Water temperature pH free and total chlorine
International Journal of Environmental Health Research 61
Dow
nloa
ded
by [
Bib
liote
ca U
nive
rsita
ria]
[G
iulia
Am
aglia
ni]
at 0
717
19
Janu
ary
2012
were recorded at the moment of sampling (PoolTester Lovibond DortmundGermany)
Water analysis with standard method
Pseudomonas aeruginosa was isolated and characterised from water specimensaccording to the official reference method (UNI EN ISO 162662008) 100 ml waterwas filtered through a 045 mm gridded cellulose ester membrane of 47 mm diameter(Millipore Billerica MA USA) the membrane was placed on Pseudomonas AgarBase (Oxoid Basingstoke UK) with Pseudomonas CN Selective Supplement(Oxoid) and 10 ml glycerol l71 and incubated at 36+ 28C for 44+ 4 h Coloniesthat clearly showed pyocyanin production (blue-green fluorescent colonies) wereconsidered positive for P aeruginosa counted and expressed as cfu per 100 ml Allcolonies were confirmed in accordance with the reference method
Molecular characterisation of P aeruginosa isolates
Each bacterial isolate obtained on Pseudomonas CN agar was also tested for speciesconfirmation by amplification of a 528 bp specific fragment of the ecfX gene(GenBank Accession No AE004091 nt 1409949-1410476) as described by Laveniret al (2007) with the HotStarTaq Plus DNA Polymerase kit (Qiagen HildenGermany) and primers ECF1 (50-ATGGATGAGCGCTTCCGTG-30) -ECF2(50-TCATCCTTCGCCTCCCTG-30)
DNA was extracted from each isolate using DNeasy Blood and Tissue Kit(Qiagen) and its concentration was estimated through Quant-iT dsDNA HS AssayKit on Qubit fluorometer (both from Invitrogen Paisley UK) RAPD (randomamplification of polymorphic DNA) typing was conducted according to Lanotteet al (2004) with 272 primer (50-AGCGGGCCAA-30) PCR products were run on15 agarose gel with 100 bp Ladder Plus (MBI Fermentas Burlington Canada)and analysed on a Gel Doc 2000 apparatus using Quantity One QuantitationSoftware (Bio-Rad) In order to test the discriminatory power of RAPD typing with272 primer four additional P aeruginosa strains (Table 1) were used along withisolates obtained from water samples
Molecular detection of P aeruginosa in water
Specificity and sensitivity of Real-Time PCR
The Real-Time PCR assay was performed according to Anuj et al (2009) using Hot-Rescue Real-Time PCR Kit FP (Diatheva Fano Italy) with 5 mmol l71 MgCl204 mmol l71 each primer ecfXF (50-CGCATGCCTATCAGGCGTT-30) ecfXR(50-GAACTGCCCAGGTGCTTGC-30) 016 mmol l71 probe ecf-X-TM (50-FAM-ATGGCGAGTTGCTGCGCTTCCT-BHQ1-30) and 08 units DNA polymerase ina final reaction volume of 25 ml The amplification was carried out in a Rotor-Gene3000 (Corbett Research Sydney Australia) with the following thermal protocoldenaturation at 958C 10 min 50 cycles at 958C 15 s and 608C 1 min The expectedproduct size was 63 bp
Assay specificity was tested on a panel of 22 strains of non-target species and fourstrains of P aeruginosa (Table 1) The sensitivity was determined with serial dilutionsof P aeruginosa ATCC 10145 DNA extracted and quantified as above calculating
62 G Amagliani et al
Dow
nloa
ded
by [
Bib
liote
ca U
nive
rsita
ria]
[G
iulia
Am
aglia
ni]
at 0
717
19
Janu
ary
2012
genomic units based on bacterial genome size at httpwwwncbinlmgovgenomeslprokscgi DNA was diluted in dH2O and amplified in the range 106-1 genomicequivalents (ge) per PCR in triplicate Efficiency was calculated by plotting Ct(threshold cycle) values against log of genome copy number and using the slope ofthe resulting curve in the following equation
Efficiency frac14 101=slope 1
Artificially contaminated pool water samples
Preliminary tests to assess method sensitivity were carried out on artificiallycontaminated pool water samples Neutralised pool water was contaminated withvarious concentrations of P aeruginosa as follows P aeruginosa ATCC 10145 wasgrown in Brain Heart Infusion (BHI Oxoid) at 378C to an A600 of 018corresponding to 108 cfu ml71 The bacterial suspension was serially diluted and1-ml aliquots used for water contamination in the range 10ndash103 cfu100 ml Bacterialtitre was confirmed by plating 100 ml of each dilution on Pseudomonas AgarBase CN Water samples were filtered as described above and each membrane wasincubated for culture-enrichment in 20 ml of Pseudomonas Selective Brothsupplemented with 1 glycerol (Biolife Milan Italy) at 378C in agitation After
Table 1 Bacterial strains used for Real-Time PCR specificity test and corresponding results
International Journal of Environmental Health Research 63
Dow
nloa
ded
by [
Bib
liote
ca U
nive
rsita
ria]
[G
iulia
Am
aglia
ni]
at 0
717
19
Janu
ary
2012
6 h membranes were removed and the cultures were centrifuged at 6000 g for 20min Total DNA was extracted from the bacterial pellets either through a column-based kit (DNeasy Blood and Tissue Kit) or by magnetic extraction (MagPrep HSMerck Darmstadt Germany) with 1 mg nanoparticles per sample according tomanufacturer protocol under mild acidic conditions and 5 ml of each sample wasused for the Real-Time PCR assay
Analysis of field samples
The same 44 water samples analysed with the standard method were also examinedwith the molecular protocol starting from 1-l aliquots filtration culture enrichmentcolumn-based DNA extraction and Real-Time PCR were carried out as describedfor artificially contaminated water samples
Statistical analysis
Accordance of results obtained by both reference and molecular methods wascalculated with DAG_Stat software (Mackinnon 2000) providing sensitivityspecificity Positive and Negative Agreement (PA and NA) indices The k statisticwas used to estimate concordance (Landis and Koch 1977)
Results
Development of a molecular method for the detection of P aeruginosa
Specificity and sensitivity of Real-Time PCR
The specificity of the Real-Time PCR assay was confirmed over a panel of 22bacterial strains of species likely to be present in water environments and four ofP aeruginosa (Table 1) Assay sensitivity showed a detection limit of 10 ge with anamplification efficiency of 105 (R2 099453) (Figure 1)
Artificially contaminated pool water samples
The application of the entire molecular procedure on pool water samples artificiallycontaminated with 10ndash103 cfu100 ml made it possible to assess the sensitivity of the
Figure 1 Real-Time PCR sensitivity and efficiency Bacterial DNA dilutions (106-1 genomeequivalents ge) of the target species were amplified as described in the Methods section Ctvalues were plotted against DNA concentrations and the slope of the linear curve was used foramplification efficiency calculation Efficiency data with respective correlation coefficients aresummarised in the inset
64 G Amagliani et al
Dow
nloa
ded
by [
Bib
liote
ca U
nive
rsita
ria]
[G
iulia
Am
aglia
ni]
at 0
717
19
Janu
ary
2012
developed protocol Column-based extraction proved to be more efficient thanmagnetic nanoparticles for the isolation of bacterial DNA from water samples witha sensitivity of 10 cfu and 103 cfu respectively Ct values were as follows Qiagen kit0 cfu100 ml tested negative 10 cfu100 ml Ct 3565 102 cfu100 ml Ct 3278103 cfu100 ml Ct 2872 Merck magnetic particles 0 cfu100 ml 10 cfu100 ml and102 cfu100 ml tested negative 103 cfu100 ml Ct 3741
Analysis of recreational water samples
One out of 17 pool water samples and 2027 inlet water samples tested positive forP aeruginosa when analysed using the reference method Contamination titersranged between 1 and 45 cfu per 100 ml A high bacterial concentration above 100cfu was detected on two occasions When the same samples were analysed usingthe molecular method positivity rates of 417 and 2027 in pool and inlet waterrespectively were found (Table 2)
Statistical analysis
Test reliability was estimated by statistical analysis of result accordance With regardto the standard method the molecular assay showed a sensitivity of 9048 and aspecificity of 7826 PA was 844 (95 CI 0730ndash0958) NA 837 (95 CI0718ndash0956) Cohenrsquos Kappa index was 06831 which indicates lsquolsquosubstantialagreementrsquorsquo according to Landis and Koch (1977) classification
RAPD fingerprinting
The efficacy of the RAPD typing scheme was shown with bacterial strains of thesame species but different origins These included two additional isolates that hadoriginally been recovered from other swimming pools (environmental isolates 1 and2) one collection strain ATCC 10145 and one strain used as quality control strainby the ASUR (CQXVIII) (Table 1) As shown in Figure 2 panel B RAPDfingerprinting allowed us to differentiate all these distinct strains of P aeruginosaWhen all the P aeruginosa isolated from water samples were tested using the sameprotocol they were found to possess identical RAPD fingerprint profiles (Figure 2panel A)
Discussion
P aeruginosa is the second most common cause of recreational disease outbreaks(Craun et al 2005) Ear infection and otitis externa caused by this microorganism inswimming pools are probably more common than reported (Guida et al 2009)P aeruginosa presence in recreational water has been investigated by few authors(Barben et al 2005 Papadopoulou et al 2008 Guida et al 2009 Nikaeen et al2009) and additional data have been provided by Mena and Gerba (2009) Howevernone of these studies made use of a PCR-based method for the detection of thisspecies
More recently Villarreal et al (2010) described culture-independent techniquesapplied to drinking water surveillance Perkins et al (2009) to shower water andLavenir et al (2007) to environmental water while the use of PCR for pathogen
International Journal of Environmental Health Research 65
Dow
nloa
ded
by [
Bib
liote
ca U
nive
rsita
ria]
[G
iulia
Am
aglia
ni]
at 0
717
19
Janu
ary
2012
Table 2 Comparison of results obtained through reference and molecular methods forP aeruginosa detection in pool and inlet water
aColony count after membrane growth on Pseudomonas Agar Base CN (see Materials and methods)
66 G Amagliani et al
Dow
nloa
ded
by [
Bib
liote
ca U
nive
rsita
ria]
[G
iulia
Am
aglia
ni]
at 0
717
19
Janu
ary
2012
detection in swimming pools has been limited to the identification of adenoviruses(Heerden et al 2005) enteric protozoa (Fournier et al 2002) and free-living amoebae(Gianinazzi et al 2009) To the best of our knowledge the application of a molecularassay for the detection of P aeruginosa in recreational water has yet to be reported
The aim of this study was the development of a new molecular method forP aeruginosa identification in recreational water and the evaluation of itsperformance in the monitoring of a swimming pool in comparison with thereference culture method (UNI EN ISO 162662008) In order to make such acomparison inlet and pool water samples were collected from a local swimming pooland examined by both systems
Results of microbiological analysis indicated higher positivity rates in inlet water(74) compared to pool water (6) with higher levels of P aeruginosa (147 vs1 cfu100 ml mean values) in accordance with values reported by Guida et al(2009) These results may be explained by the presence of biofilm in the inlet waterpipeline and are supported by RAPD typing of isolates which indicate theirepidemiological correlation A longer period of contact with chlorine and thedilution process may have led to lower levels of P aeruginosa in pool water (Guidaet al 2009) Based on criteria provided by Italian Guidelines (Italian Republic 2003)fixing acceptability limits of 1 cfu100 ml in pool water and 0 cfu100 ml of inletwater in this study only the samples of pool water proved to be in compliance
lsquolsquoSubstantial agreementrsquorsquo (Landis and Koch 1977) was found between thedeveloped molecular method and the reference protocol The higher sensitivity of themolecular approach compared to the reference method in testing pool water samplescould be ascribed to the greater starting volume used for the analysis Moreoverbacteria in the pool water may have been in a subvital state due to chlorine watertreatment and able to recover culturability after a short enrichment in liquid
Figure 2 Discrimination of P aeruginosa by RAPD typing The ability of RAPDfingerprinting with primer 272 to cluster identical isolates (Panel A) and differentiatedistinct isolates within the P aeruginosa species (Panel B) is shown M 100 bp Ladder PlusFermentas Lanes 1ndash25 P aeruginosa isolated from recreational water 26 P aeruginosaenvironmental isolate 1 27 P aeruginosa environmental isolate 2 28 CQXVIII 29P aeruginosa ATCC 10145
International Journal of Environmental Health Research 67
Dow
nloa
ded
by [
Bib
liote
ca U
nive
rsita
ria]
[G
iulia
Am
aglia
ni]
at 0
717
19
Janu
ary
2012
medium thus providing resuscitation of injured or stressed cells However it iscommonly accepted that culture-dependent methods can fail in the identification ofbacteria in VBNC state (Oliver 2005)
It has been reported that the examination of colony morphology after membranegrowth on Cetrimide agar is not conclusive for P aeruginosa identification andconfirmation tests are needed for the correct identification of presumptive positivecolonies (Casanovas-Massana et al 2010) The need for such tests was illustrated bythe interlaboratory proficiency test carried out by the Italian National Health Service(Bonadonna and Ottaviani 2007) owing to the high rate (64) of false positiveresults obtained Conversely the Real-Time PCR assay specificity previously testedby Anuj et al (2009) on 101 target and non-target isolates and confirmed with 26further target and non-target strains in the present study proved to be very highConsidering that the assay has been successfully applied for the screening ofsuspected positive colonies on Pseudomonas Agar Base CN it may constitute aviable alternative to conventional time-consuming confirmation tests with a briefReal-Time PCR assay
A limitation for the broad application of molecular assays for testingenvironmental samples stems from the presence of substances of environmentalorigin such as humic acids and other organic molecules well known as PCRinhibitors (Wilson 1997) Hence the development of a DNA isolation system able toeffectively remove such contaminating molecules is a key step In a first attemptDNA direct isolation from filter membrane was carried out although the sensitivityobtained was not sufficient to be in compliance with National standards provided forrecreational water (not shown) After introducing a short (6 h) enrichment step twodifferent DNA extraction strategies were compared silica-based spin columns andmagnetic nanoparticles However the magnetic procedure for DNA isolation wasnot able to eliminate water-derived PCR inhibitors or did not provide sufficientrecovery especially with low bacterial concentrations On the other hand thecolumn devices proved effective and appropriate in this application
The employment of Real-Time PCR for the detection of P aeruginosa in wateroffers high specificity and sensitivity levels with a reduction of analysis time Inaddition this technique has the potential to screen large numbers of environmentalsamples limiting the application of the conventional culture techniques only toPCR-positive samples Hence the method could be proposed as part of a self-monitoring plan for swimming pool facilities to evaluate the quality of water usedfor recreational purposes improving surveillance and early warning systemsAlthough the use of PCR-based methods has not been recognised by AuthoritiesEU Directive 9883EC (EU 1998) allows the application of molecular diagnosis if itsequivalence with the reference protocol is demonstrated However a validationprogramme is needed to assess the level of accordance of the described system withthe reference method in order to verify the equivalence of results required byEuropean legislation
Acknowledgements
The authors would like to thank Dr G Cappuccini Director of the Department of Preventionand Head of the Hygiene and Public Health Service ASUR ZT 2 Urbino the personnel at theswimming pool facility of the Faculty of Health and Physical Exercise University of UrbinolsquolsquoCarlo Borsquorsquo for their collaboration and Professor Timothy Bloom of the University ofUrbino for a critical reading of the manuscript
68 G Amagliani et al
Dow
nloa
ded
by [
Bib
liote
ca U
nive
rsita
ria]
[G
iulia
Am
aglia
ni]
at 0
717
19
Janu
ary
2012
References
Anuj SN Whiley DM Kidd TJ Bell SC Wainwright CE Nissen MD Sloots TP 2009Identification of Pseudomonas aeruginosa by a duplex real-time polymerase chain reactionassay targeting the ecfX and the gyrB genes Diagn Microbiol Infect Dis 63 127ndash131
Barben J Hafen G Schmid J 2005 Pseudomonas aeruginosa in public swimming pools andbathroom water of patients with cystic fibrosis J Cystic Fibros 4227ndash231
Bonadonna L Ottaviani M 2007 Reference analytical methods for water intended forhuman consumption according to the Italian Legislative Decree 312001 Microbiologicalmethods Rapporti ISTISAN 075 Rome Istituto Superiore di Sanita (National HealthService)
Casanovas-Massana A Lucena F Blanch AR 2010 Identification of Pseudomonas aeruginosain water-bottling plants on the basis of procedures included in ISO 162662006J Microbiol Meth 811ndash5
Craun GF Calderon RL Craun MF 2005 Outbreaks associated with recreational water inthe United States Int J Environ Health Res 15243ndash262
European Union (EU) 1998 Council Directive 9883EC of 3 November 1998 on the qualityof water intended for human consumption
Fournier S Dubrou S Liguory O Gaussin F Santillana-Hayat M Sarfati C Molina JMDerouin F 2002 Detection of microsporidia cryptosporidia and giardia in swimmingpools a one-year prospective study FEMS Immunol Med Microbiol 33209ndash213
Gianinazzi C Schild M Wuthrich F Muller N Schurch N Gottstein B 2009 Potentiallyhuman pathogenic Acanthamoeba isolated from a heated indoor swimming pool inSwitzerland Exp Parasitol 121180ndash186
Goeres DM Palys T Sandel BB Geiger J 2004 Evaluation of disinfectant efficacy againstbiofilm and suspended bacteria in a laboratory swimming pool model Water Res383103ndash3109
Guida M Galle F Mattei ML Anastasi D Liguori G 2009 Microbiological quality of thewater of recreational and rehabilitation pools a 2-year survey in Naples Italy PublicHealth 123448ndash451
Heerden J Ehlers MM Grabow WOK 2005 Detection and risk assessment of adenovirusesin swimming pool water J Appl Microbiol 991256ndash1264
Italian Republic 2001 Legislative Decree No 31 2 February implementing Directive 9883EC on the quality of water intended for human consumption Official Gazette No 52 of3rd March 2001 Rome
Italian Republic 2002 Legislative Decree No 27 2 February amending and supplementingLegislative Decree No 31 of 2 February 2001 implementing Directive 9883EC on thequality of water intended for human consumption Official Gazette No 58 of 9th of March2002 Rome
Italian Republic 2003 Permanent Council State-Regions and Autonomous ProvincesAccordo Stato ndash Regioni e Province Autonome 16-1-2003 Official Gazette No 51 of 3rdof March 2003 Rome
Landis JR Koch GG 1977 The measurement of observer agreement for categorical dataBiometrics 33159ndash174
Lanotte P Watt S Mereghetti L Dartiguelongue N Rastegar-Lari A Goudeau A QuentinR 2004 Genetic features of Pseudomonas aeruginosa isolates from cystic fibrosis patientscompared with those of isolates from other origins J Med Microbiol 5373ndash81
Lavenir R Jocktane D Laurent F Nazaret S Cournoyer B 2007 Improved reliability ofPseudomonas aeruginosa PCR detection by the use of the species-specific ecfX gene targetJ Microbiol Meth 7020ndash29
Leoni E Legnani P Guberti E Masotti A 1999 Risk of infection associated withmicrobiological quality of public swimming pools in Bologna Italy Public Health113227ndash232
Mackinnon A 2000 A spreadsheet for the calculation of comprehensive statistics for theassessment of diagnostic tests and inter-rater agreement Comput Biol Med 30127ndash134
Mena KD Gerba CP 2009 Risk assessment of Pseudomonas aeruginosa in water RevEnviron Contam Toxicol 20171ndash115
Moore JE Heaney N Millar BC Crowe M Elborn JS 2002 Incidence of Pseudomonasaeruginosa in recreational and hydrotherapy pools Commun Dis Public Health 523ndash26
International Journal of Environmental Health Research 69
Dow
nloa
ded
by [
Bib
liote
ca U
nive
rsita
ria]
[G
iulia
Am
aglia
ni]
at 0
717
19
Janu
ary
2012
Nikaeen M Hatamzadeh M Vahid Dastjerdi M Hassanzadeh A 2009 Predictive indicatorsof the safety of swimming pool waters Water Sci Technol 603101ndash3107
Oliver JD 2005 The viable but nonculturable state in bacteria J Microbiol 4393ndash100Papadopoulou C Economou V Sakkas H Gousia P Giannakopoulos X Dontorou C
Filioussis G Gessouli H Karanis P Leveidiotou S 2008 Microbiological quality ofindoor and outdoor swimming pools in Greece investigation of the antibiotic resistanceof the bacterial isolates Int J Hyg Environ Health 211385ndash397
Perkins SD Mayfield J Fraser V Angenent LT 2009 Potentially pathogenic bacteria inshower water and air of a stem cell transplant unit Appl Environ Microbiol 755363ndash5372
Pond K 2005 Water recreation and disease Plausibility of associated infections acute effectssequelae and mortality London WHO IWA Publishing
UNI EN ISO 162662008 ndash 18092008 ndashWater quality ndash Detection and enumeration ofPseudomonas aeruginosa ndash Method by membrane filtration
Villarreal JV Schwartz T Obst U 2010 Culture-independent techniques applied to foodindustry water surveillance ndash a case study Int J Food Microbiol 141(Suppl 1)S147ndash55
Wang MC Liu CY Shiao AS Wang T 2005 Ear problems in swimmers J Chin Med Assoc68347ndash352
World Health Organisation (WHO) 2006 Guidelines for safe recreational water environ-ments Vol 2 Swimming pools and similar environments Geneva WHO Press
Wilson IG 1997 Inhibition and facilitation of nucleic acid amplification Appl EnvironMicrobiol 633741ndash3751
70 G Amagliani et al
Dow
nloa
ded
by [
Bib
liote
ca U
nive
rsita
ria]
[G
iulia
Am
aglia
ni]
at 0
717
19
Janu
ary
2012
demand or costs or damages whatsoever or howsoever caused arising directly orindirectly in connection with or arising out of the use of this material
Dow
nloa
ded
by [
Bib
liote
ca U
nive
rsita
ria]
[G
iulia
Am
aglia
ni]
at 0
717
19
Janu
ary
2012
Molecular detection of Pseudomonas aeruginosa in recreational water
Giulia Amaglianiab Maria Lorella Parlanic Giorgio BrandiabGiuseppe Sebastianellic Vilberto Stocchiab and Giuditta Fiorella Schiavanoa
aDipartimento di Scienze Biomolecolari Universita degli Studi di Urbino lsquolsquoCarlo Borsquorsquo Urbino ItalybFacolta di Scienze Motorie Universita degli Studi di Urbino lsquolsquoCarlo Borsquorsquo Urbania ItalycLaboratorio di Igiene e Sanita Pubblica Dipartimento di Prevenzione ASUR ZT2 Urbania Italy
(Received 21 March 2011 final version received 3 May 2011)
The aim of this study was the development of a new molecular assay forPseudomonas aeruginosa identification in recreational water The method includesbacterial cell concentration through membrane filtration a short (6 h) culture-enrichment step DNA extraction and its amplification through a Real-Time PCRassay The performance of the molecular approach was evaluated on 44 samples ofswimming pool water and compared with the reference method UNI EN ISO162662008 Positivity rates of 6 and 74 in pool and inlet water respectivelywith the standard culture method and of 23 and 74 with the molecular methodwere found Statistical analysis indicated lsquolsquosubstantial agreementrsquorsquo (Cohenrsquos Kappaindex 06831) between the two approaches RAPD typing of P aeruginosa isolatesshowed identical fingerprint profiles indicating their epidemiological correlationThe developed protocol showed very high specificity and a detection limit of 10genomic units This technique has the potential to screen large numbers ofenvironmental samples and could be proposed as part of a self-monitoring plan forrecreational facilities improving surveillance and early warning systems
Keywords Pseudomonas aeruginosa ecfX Real-Time PCR recreational waterrandom amplification of polymorphic DNA (RAPD)
Introduction
The use of swimming pools and other recreational waters is associated with benefitsto health and well-being However a variety of microorganisms which mayconstitute a microbiological hazard can be found in such waters and their relatedfacilities (World Health Organisation [WHO] 2006) Associated adverse healthoutcomes including skin and ear infections can arise from opportunistic pathogensshed by bathers or naturally present in the aquatic environment and transmitted viasurfaces and contaminated water These pathogens can also accumulate in biofilmsthus becoming resistant to biocides and other disinfection agents (Goeres et al2004) Recreational water is a documented environmental source of Pseudomonasaeruginosa (Moore et al 2002 Papadopoulou et al 2008 Guida et al 2009)therefore it may represent a health risk for some user categories Swimming pools areused by a large variety of people varying in age health and hygienic standards
International Journal of Environmental Health Research
Vol 22 No 1 February 2012 60ndash70
ISSN 0960-3123 printISSN 1369-1619 online
2012 Taylor amp Francis
httpdxdoiorg101080096031232011588325
httpwwwtandfonlinecom
Dow
nloa
ded
by [
Bib
liote
ca U
nive
rsita
ria]
[G
iulia
Am
aglia
ni]
at 0
717
19
Janu
ary
2012
Pregnant women babies the elderly and disabled people who may attend specialcourses are potentially predisposed to contracting infections from pathogens andopportunistic bacteria (Leoni et al 1999) Moreover immunocompromised andcystic fibrosis patients are at particular risk (Barben et al 2005 Mena and Gerba2009) as well as athletes under competitive stress who may have weakened immunesystems (Pond 2005 WHO 2006)
In swimming pools the primary health effect associated with P aeruginosa isotitis externa although folliculitis dermatitis conjunctivitis and pneumonia havealso been reported (WHO 2006) Indeed waterborne pseudomonas-infection can berecognised in patients presenting a history of participation in water sportsactivities(Wang et al 2005)
Recently published guidelines from WHO (2006) are intended to be used as abasis for the development of international and national approaches for the controlof hazards that may be encountered in such environments In Italy in order toimplement an appropriate monitoring program and ensure adequate surveillanceLocal Sanitary Authorities (ASUR Azienda Sanitaria Unica Regionale) have beenmade responsible for official controls of recreational water Moreover programmesof self-monitoring according to the HACCP approach are to be applied byswimming pool managers as an lsquolsquointernal controlrsquorsquo (Italian Republic 2003)
Currently monitoring of P aeruginosa in swimming pool water is conducted inaccordance with the EU Drinking Water Directive (European Union [EU] 1998) andits Italian counterparts (Italian Republic 2001 2002) For P aeruginosa a maximumpermissible standard of zero per 100 ml of inlet water and acceptability limits of 1cfu (colony-forming units) per 100 ml of pool water have been fixed by ItalianGuidelines (Italian Republic 2003)
The reference analytical method for P aeruginosa detection (UNI EN ISO162662008) although sensitive and specific requires at least 48 h for the isolation ofsuspected positive colonies and additional time is needed for confirmation testsMoreover it is unable to detect the presence of viable but non-culturable (VBNC) formsof P aeruginosa (Oliver 2005) Consequently rapid accurate and culture-independentalternatives are being investigated to enable monitoring of this opportunistic pathogenin water A molecular method could reduce analysis time while maintaining sensitivityand specificity at very high levels and offering the additional capability of VBNCdetection Hence the aim of this study was the development of a new molecular methodfor P aeruginosa identification in recreational water
The entire testing process yielding a definitive result in one working dayincluded bacterial cell concentration through membrane filtration a short (6 h)culture-enrichment step DNA extraction and its amplification in Real-Time PCRIn order to validate the developed molecular assay and assess its applicability in aself monitoring plan of a swimming pool its performance was evaluated andcompared to the reference culture method carried out by ASUR
Methods
Collection and processing of water samples
The swimming pool investigated for this study was an indoor pool used by peopleof all ages for recreational purposes and by athletes for training A total of 44 watersamples (17 pool water and 27 inlet water) were collected in sterile bottlescontaining 10 sodium thiosulfate Water temperature pH free and total chlorine
International Journal of Environmental Health Research 61
Dow
nloa
ded
by [
Bib
liote
ca U
nive
rsita
ria]
[G
iulia
Am
aglia
ni]
at 0
717
19
Janu
ary
2012
were recorded at the moment of sampling (PoolTester Lovibond DortmundGermany)
Water analysis with standard method
Pseudomonas aeruginosa was isolated and characterised from water specimensaccording to the official reference method (UNI EN ISO 162662008) 100 ml waterwas filtered through a 045 mm gridded cellulose ester membrane of 47 mm diameter(Millipore Billerica MA USA) the membrane was placed on Pseudomonas AgarBase (Oxoid Basingstoke UK) with Pseudomonas CN Selective Supplement(Oxoid) and 10 ml glycerol l71 and incubated at 36+ 28C for 44+ 4 h Coloniesthat clearly showed pyocyanin production (blue-green fluorescent colonies) wereconsidered positive for P aeruginosa counted and expressed as cfu per 100 ml Allcolonies were confirmed in accordance with the reference method
Molecular characterisation of P aeruginosa isolates
Each bacterial isolate obtained on Pseudomonas CN agar was also tested for speciesconfirmation by amplification of a 528 bp specific fragment of the ecfX gene(GenBank Accession No AE004091 nt 1409949-1410476) as described by Laveniret al (2007) with the HotStarTaq Plus DNA Polymerase kit (Qiagen HildenGermany) and primers ECF1 (50-ATGGATGAGCGCTTCCGTG-30) -ECF2(50-TCATCCTTCGCCTCCCTG-30)
DNA was extracted from each isolate using DNeasy Blood and Tissue Kit(Qiagen) and its concentration was estimated through Quant-iT dsDNA HS AssayKit on Qubit fluorometer (both from Invitrogen Paisley UK) RAPD (randomamplification of polymorphic DNA) typing was conducted according to Lanotteet al (2004) with 272 primer (50-AGCGGGCCAA-30) PCR products were run on15 agarose gel with 100 bp Ladder Plus (MBI Fermentas Burlington Canada)and analysed on a Gel Doc 2000 apparatus using Quantity One QuantitationSoftware (Bio-Rad) In order to test the discriminatory power of RAPD typing with272 primer four additional P aeruginosa strains (Table 1) were used along withisolates obtained from water samples
Molecular detection of P aeruginosa in water
Specificity and sensitivity of Real-Time PCR
The Real-Time PCR assay was performed according to Anuj et al (2009) using Hot-Rescue Real-Time PCR Kit FP (Diatheva Fano Italy) with 5 mmol l71 MgCl204 mmol l71 each primer ecfXF (50-CGCATGCCTATCAGGCGTT-30) ecfXR(50-GAACTGCCCAGGTGCTTGC-30) 016 mmol l71 probe ecf-X-TM (50-FAM-ATGGCGAGTTGCTGCGCTTCCT-BHQ1-30) and 08 units DNA polymerase ina final reaction volume of 25 ml The amplification was carried out in a Rotor-Gene3000 (Corbett Research Sydney Australia) with the following thermal protocoldenaturation at 958C 10 min 50 cycles at 958C 15 s and 608C 1 min The expectedproduct size was 63 bp
Assay specificity was tested on a panel of 22 strains of non-target species and fourstrains of P aeruginosa (Table 1) The sensitivity was determined with serial dilutionsof P aeruginosa ATCC 10145 DNA extracted and quantified as above calculating
62 G Amagliani et al
Dow
nloa
ded
by [
Bib
liote
ca U
nive
rsita
ria]
[G
iulia
Am
aglia
ni]
at 0
717
19
Janu
ary
2012
genomic units based on bacterial genome size at httpwwwncbinlmgovgenomeslprokscgi DNA was diluted in dH2O and amplified in the range 106-1 genomicequivalents (ge) per PCR in triplicate Efficiency was calculated by plotting Ct(threshold cycle) values against log of genome copy number and using the slope ofthe resulting curve in the following equation
Efficiency frac14 101=slope 1
Artificially contaminated pool water samples
Preliminary tests to assess method sensitivity were carried out on artificiallycontaminated pool water samples Neutralised pool water was contaminated withvarious concentrations of P aeruginosa as follows P aeruginosa ATCC 10145 wasgrown in Brain Heart Infusion (BHI Oxoid) at 378C to an A600 of 018corresponding to 108 cfu ml71 The bacterial suspension was serially diluted and1-ml aliquots used for water contamination in the range 10ndash103 cfu100 ml Bacterialtitre was confirmed by plating 100 ml of each dilution on Pseudomonas AgarBase CN Water samples were filtered as described above and each membrane wasincubated for culture-enrichment in 20 ml of Pseudomonas Selective Brothsupplemented with 1 glycerol (Biolife Milan Italy) at 378C in agitation After
Table 1 Bacterial strains used for Real-Time PCR specificity test and corresponding results
International Journal of Environmental Health Research 63
Dow
nloa
ded
by [
Bib
liote
ca U
nive
rsita
ria]
[G
iulia
Am
aglia
ni]
at 0
717
19
Janu
ary
2012
6 h membranes were removed and the cultures were centrifuged at 6000 g for 20min Total DNA was extracted from the bacterial pellets either through a column-based kit (DNeasy Blood and Tissue Kit) or by magnetic extraction (MagPrep HSMerck Darmstadt Germany) with 1 mg nanoparticles per sample according tomanufacturer protocol under mild acidic conditions and 5 ml of each sample wasused for the Real-Time PCR assay
Analysis of field samples
The same 44 water samples analysed with the standard method were also examinedwith the molecular protocol starting from 1-l aliquots filtration culture enrichmentcolumn-based DNA extraction and Real-Time PCR were carried out as describedfor artificially contaminated water samples
Statistical analysis
Accordance of results obtained by both reference and molecular methods wascalculated with DAG_Stat software (Mackinnon 2000) providing sensitivityspecificity Positive and Negative Agreement (PA and NA) indices The k statisticwas used to estimate concordance (Landis and Koch 1977)
Results
Development of a molecular method for the detection of P aeruginosa
Specificity and sensitivity of Real-Time PCR
The specificity of the Real-Time PCR assay was confirmed over a panel of 22bacterial strains of species likely to be present in water environments and four ofP aeruginosa (Table 1) Assay sensitivity showed a detection limit of 10 ge with anamplification efficiency of 105 (R2 099453) (Figure 1)
Artificially contaminated pool water samples
The application of the entire molecular procedure on pool water samples artificiallycontaminated with 10ndash103 cfu100 ml made it possible to assess the sensitivity of the
Figure 1 Real-Time PCR sensitivity and efficiency Bacterial DNA dilutions (106-1 genomeequivalents ge) of the target species were amplified as described in the Methods section Ctvalues were plotted against DNA concentrations and the slope of the linear curve was used foramplification efficiency calculation Efficiency data with respective correlation coefficients aresummarised in the inset
64 G Amagliani et al
Dow
nloa
ded
by [
Bib
liote
ca U
nive
rsita
ria]
[G
iulia
Am
aglia
ni]
at 0
717
19
Janu
ary
2012
developed protocol Column-based extraction proved to be more efficient thanmagnetic nanoparticles for the isolation of bacterial DNA from water samples witha sensitivity of 10 cfu and 103 cfu respectively Ct values were as follows Qiagen kit0 cfu100 ml tested negative 10 cfu100 ml Ct 3565 102 cfu100 ml Ct 3278103 cfu100 ml Ct 2872 Merck magnetic particles 0 cfu100 ml 10 cfu100 ml and102 cfu100 ml tested negative 103 cfu100 ml Ct 3741
Analysis of recreational water samples
One out of 17 pool water samples and 2027 inlet water samples tested positive forP aeruginosa when analysed using the reference method Contamination titersranged between 1 and 45 cfu per 100 ml A high bacterial concentration above 100cfu was detected on two occasions When the same samples were analysed usingthe molecular method positivity rates of 417 and 2027 in pool and inlet waterrespectively were found (Table 2)
Statistical analysis
Test reliability was estimated by statistical analysis of result accordance With regardto the standard method the molecular assay showed a sensitivity of 9048 and aspecificity of 7826 PA was 844 (95 CI 0730ndash0958) NA 837 (95 CI0718ndash0956) Cohenrsquos Kappa index was 06831 which indicates lsquolsquosubstantialagreementrsquorsquo according to Landis and Koch (1977) classification
RAPD fingerprinting
The efficacy of the RAPD typing scheme was shown with bacterial strains of thesame species but different origins These included two additional isolates that hadoriginally been recovered from other swimming pools (environmental isolates 1 and2) one collection strain ATCC 10145 and one strain used as quality control strainby the ASUR (CQXVIII) (Table 1) As shown in Figure 2 panel B RAPDfingerprinting allowed us to differentiate all these distinct strains of P aeruginosaWhen all the P aeruginosa isolated from water samples were tested using the sameprotocol they were found to possess identical RAPD fingerprint profiles (Figure 2panel A)
Discussion
P aeruginosa is the second most common cause of recreational disease outbreaks(Craun et al 2005) Ear infection and otitis externa caused by this microorganism inswimming pools are probably more common than reported (Guida et al 2009)P aeruginosa presence in recreational water has been investigated by few authors(Barben et al 2005 Papadopoulou et al 2008 Guida et al 2009 Nikaeen et al2009) and additional data have been provided by Mena and Gerba (2009) Howevernone of these studies made use of a PCR-based method for the detection of thisspecies
More recently Villarreal et al (2010) described culture-independent techniquesapplied to drinking water surveillance Perkins et al (2009) to shower water andLavenir et al (2007) to environmental water while the use of PCR for pathogen
International Journal of Environmental Health Research 65
Dow
nloa
ded
by [
Bib
liote
ca U
nive
rsita
ria]
[G
iulia
Am
aglia
ni]
at 0
717
19
Janu
ary
2012
Table 2 Comparison of results obtained through reference and molecular methods forP aeruginosa detection in pool and inlet water
aColony count after membrane growth on Pseudomonas Agar Base CN (see Materials and methods)
66 G Amagliani et al
Dow
nloa
ded
by [
Bib
liote
ca U
nive
rsita
ria]
[G
iulia
Am
aglia
ni]
at 0
717
19
Janu
ary
2012
detection in swimming pools has been limited to the identification of adenoviruses(Heerden et al 2005) enteric protozoa (Fournier et al 2002) and free-living amoebae(Gianinazzi et al 2009) To the best of our knowledge the application of a molecularassay for the detection of P aeruginosa in recreational water has yet to be reported
The aim of this study was the development of a new molecular method forP aeruginosa identification in recreational water and the evaluation of itsperformance in the monitoring of a swimming pool in comparison with thereference culture method (UNI EN ISO 162662008) In order to make such acomparison inlet and pool water samples were collected from a local swimming pooland examined by both systems
Results of microbiological analysis indicated higher positivity rates in inlet water(74) compared to pool water (6) with higher levels of P aeruginosa (147 vs1 cfu100 ml mean values) in accordance with values reported by Guida et al(2009) These results may be explained by the presence of biofilm in the inlet waterpipeline and are supported by RAPD typing of isolates which indicate theirepidemiological correlation A longer period of contact with chlorine and thedilution process may have led to lower levels of P aeruginosa in pool water (Guidaet al 2009) Based on criteria provided by Italian Guidelines (Italian Republic 2003)fixing acceptability limits of 1 cfu100 ml in pool water and 0 cfu100 ml of inletwater in this study only the samples of pool water proved to be in compliance
lsquolsquoSubstantial agreementrsquorsquo (Landis and Koch 1977) was found between thedeveloped molecular method and the reference protocol The higher sensitivity of themolecular approach compared to the reference method in testing pool water samplescould be ascribed to the greater starting volume used for the analysis Moreoverbacteria in the pool water may have been in a subvital state due to chlorine watertreatment and able to recover culturability after a short enrichment in liquid
Figure 2 Discrimination of P aeruginosa by RAPD typing The ability of RAPDfingerprinting with primer 272 to cluster identical isolates (Panel A) and differentiatedistinct isolates within the P aeruginosa species (Panel B) is shown M 100 bp Ladder PlusFermentas Lanes 1ndash25 P aeruginosa isolated from recreational water 26 P aeruginosaenvironmental isolate 1 27 P aeruginosa environmental isolate 2 28 CQXVIII 29P aeruginosa ATCC 10145
International Journal of Environmental Health Research 67
Dow
nloa
ded
by [
Bib
liote
ca U
nive
rsita
ria]
[G
iulia
Am
aglia
ni]
at 0
717
19
Janu
ary
2012
medium thus providing resuscitation of injured or stressed cells However it iscommonly accepted that culture-dependent methods can fail in the identification ofbacteria in VBNC state (Oliver 2005)
It has been reported that the examination of colony morphology after membranegrowth on Cetrimide agar is not conclusive for P aeruginosa identification andconfirmation tests are needed for the correct identification of presumptive positivecolonies (Casanovas-Massana et al 2010) The need for such tests was illustrated bythe interlaboratory proficiency test carried out by the Italian National Health Service(Bonadonna and Ottaviani 2007) owing to the high rate (64) of false positiveresults obtained Conversely the Real-Time PCR assay specificity previously testedby Anuj et al (2009) on 101 target and non-target isolates and confirmed with 26further target and non-target strains in the present study proved to be very highConsidering that the assay has been successfully applied for the screening ofsuspected positive colonies on Pseudomonas Agar Base CN it may constitute aviable alternative to conventional time-consuming confirmation tests with a briefReal-Time PCR assay
A limitation for the broad application of molecular assays for testingenvironmental samples stems from the presence of substances of environmentalorigin such as humic acids and other organic molecules well known as PCRinhibitors (Wilson 1997) Hence the development of a DNA isolation system able toeffectively remove such contaminating molecules is a key step In a first attemptDNA direct isolation from filter membrane was carried out although the sensitivityobtained was not sufficient to be in compliance with National standards provided forrecreational water (not shown) After introducing a short (6 h) enrichment step twodifferent DNA extraction strategies were compared silica-based spin columns andmagnetic nanoparticles However the magnetic procedure for DNA isolation wasnot able to eliminate water-derived PCR inhibitors or did not provide sufficientrecovery especially with low bacterial concentrations On the other hand thecolumn devices proved effective and appropriate in this application
The employment of Real-Time PCR for the detection of P aeruginosa in wateroffers high specificity and sensitivity levels with a reduction of analysis time Inaddition this technique has the potential to screen large numbers of environmentalsamples limiting the application of the conventional culture techniques only toPCR-positive samples Hence the method could be proposed as part of a self-monitoring plan for swimming pool facilities to evaluate the quality of water usedfor recreational purposes improving surveillance and early warning systemsAlthough the use of PCR-based methods has not been recognised by AuthoritiesEU Directive 9883EC (EU 1998) allows the application of molecular diagnosis if itsequivalence with the reference protocol is demonstrated However a validationprogramme is needed to assess the level of accordance of the described system withthe reference method in order to verify the equivalence of results required byEuropean legislation
Acknowledgements
The authors would like to thank Dr G Cappuccini Director of the Department of Preventionand Head of the Hygiene and Public Health Service ASUR ZT 2 Urbino the personnel at theswimming pool facility of the Faculty of Health and Physical Exercise University of UrbinolsquolsquoCarlo Borsquorsquo for their collaboration and Professor Timothy Bloom of the University ofUrbino for a critical reading of the manuscript
68 G Amagliani et al
Dow
nloa
ded
by [
Bib
liote
ca U
nive
rsita
ria]
[G
iulia
Am
aglia
ni]
at 0
717
19
Janu
ary
2012
References
Anuj SN Whiley DM Kidd TJ Bell SC Wainwright CE Nissen MD Sloots TP 2009Identification of Pseudomonas aeruginosa by a duplex real-time polymerase chain reactionassay targeting the ecfX and the gyrB genes Diagn Microbiol Infect Dis 63 127ndash131
Barben J Hafen G Schmid J 2005 Pseudomonas aeruginosa in public swimming pools andbathroom water of patients with cystic fibrosis J Cystic Fibros 4227ndash231
Bonadonna L Ottaviani M 2007 Reference analytical methods for water intended forhuman consumption according to the Italian Legislative Decree 312001 Microbiologicalmethods Rapporti ISTISAN 075 Rome Istituto Superiore di Sanita (National HealthService)
Casanovas-Massana A Lucena F Blanch AR 2010 Identification of Pseudomonas aeruginosain water-bottling plants on the basis of procedures included in ISO 162662006J Microbiol Meth 811ndash5
Craun GF Calderon RL Craun MF 2005 Outbreaks associated with recreational water inthe United States Int J Environ Health Res 15243ndash262
European Union (EU) 1998 Council Directive 9883EC of 3 November 1998 on the qualityof water intended for human consumption
Fournier S Dubrou S Liguory O Gaussin F Santillana-Hayat M Sarfati C Molina JMDerouin F 2002 Detection of microsporidia cryptosporidia and giardia in swimmingpools a one-year prospective study FEMS Immunol Med Microbiol 33209ndash213
Gianinazzi C Schild M Wuthrich F Muller N Schurch N Gottstein B 2009 Potentiallyhuman pathogenic Acanthamoeba isolated from a heated indoor swimming pool inSwitzerland Exp Parasitol 121180ndash186
Goeres DM Palys T Sandel BB Geiger J 2004 Evaluation of disinfectant efficacy againstbiofilm and suspended bacteria in a laboratory swimming pool model Water Res383103ndash3109
Guida M Galle F Mattei ML Anastasi D Liguori G 2009 Microbiological quality of thewater of recreational and rehabilitation pools a 2-year survey in Naples Italy PublicHealth 123448ndash451
Heerden J Ehlers MM Grabow WOK 2005 Detection and risk assessment of adenovirusesin swimming pool water J Appl Microbiol 991256ndash1264
Italian Republic 2001 Legislative Decree No 31 2 February implementing Directive 9883EC on the quality of water intended for human consumption Official Gazette No 52 of3rd March 2001 Rome
Italian Republic 2002 Legislative Decree No 27 2 February amending and supplementingLegislative Decree No 31 of 2 February 2001 implementing Directive 9883EC on thequality of water intended for human consumption Official Gazette No 58 of 9th of March2002 Rome
Italian Republic 2003 Permanent Council State-Regions and Autonomous ProvincesAccordo Stato ndash Regioni e Province Autonome 16-1-2003 Official Gazette No 51 of 3rdof March 2003 Rome
Landis JR Koch GG 1977 The measurement of observer agreement for categorical dataBiometrics 33159ndash174
Lanotte P Watt S Mereghetti L Dartiguelongue N Rastegar-Lari A Goudeau A QuentinR 2004 Genetic features of Pseudomonas aeruginosa isolates from cystic fibrosis patientscompared with those of isolates from other origins J Med Microbiol 5373ndash81
Lavenir R Jocktane D Laurent F Nazaret S Cournoyer B 2007 Improved reliability ofPseudomonas aeruginosa PCR detection by the use of the species-specific ecfX gene targetJ Microbiol Meth 7020ndash29
Leoni E Legnani P Guberti E Masotti A 1999 Risk of infection associated withmicrobiological quality of public swimming pools in Bologna Italy Public Health113227ndash232
Mackinnon A 2000 A spreadsheet for the calculation of comprehensive statistics for theassessment of diagnostic tests and inter-rater agreement Comput Biol Med 30127ndash134
Mena KD Gerba CP 2009 Risk assessment of Pseudomonas aeruginosa in water RevEnviron Contam Toxicol 20171ndash115
Moore JE Heaney N Millar BC Crowe M Elborn JS 2002 Incidence of Pseudomonasaeruginosa in recreational and hydrotherapy pools Commun Dis Public Health 523ndash26
International Journal of Environmental Health Research 69
Dow
nloa
ded
by [
Bib
liote
ca U
nive
rsita
ria]
[G
iulia
Am
aglia
ni]
at 0
717
19
Janu
ary
2012
Nikaeen M Hatamzadeh M Vahid Dastjerdi M Hassanzadeh A 2009 Predictive indicatorsof the safety of swimming pool waters Water Sci Technol 603101ndash3107
Oliver JD 2005 The viable but nonculturable state in bacteria J Microbiol 4393ndash100Papadopoulou C Economou V Sakkas H Gousia P Giannakopoulos X Dontorou C
Filioussis G Gessouli H Karanis P Leveidiotou S 2008 Microbiological quality ofindoor and outdoor swimming pools in Greece investigation of the antibiotic resistanceof the bacterial isolates Int J Hyg Environ Health 211385ndash397
Perkins SD Mayfield J Fraser V Angenent LT 2009 Potentially pathogenic bacteria inshower water and air of a stem cell transplant unit Appl Environ Microbiol 755363ndash5372
Pond K 2005 Water recreation and disease Plausibility of associated infections acute effectssequelae and mortality London WHO IWA Publishing
UNI EN ISO 162662008 ndash 18092008 ndashWater quality ndash Detection and enumeration ofPseudomonas aeruginosa ndash Method by membrane filtration
Villarreal JV Schwartz T Obst U 2010 Culture-independent techniques applied to foodindustry water surveillance ndash a case study Int J Food Microbiol 141(Suppl 1)S147ndash55
Wang MC Liu CY Shiao AS Wang T 2005 Ear problems in swimmers J Chin Med Assoc68347ndash352
World Health Organisation (WHO) 2006 Guidelines for safe recreational water environ-ments Vol 2 Swimming pools and similar environments Geneva WHO Press
Wilson IG 1997 Inhibition and facilitation of nucleic acid amplification Appl EnvironMicrobiol 633741ndash3751
70 G Amagliani et al
Dow
nloa
ded
by [
Bib
liote
ca U
nive
rsita
ria]
[G
iulia
Am
aglia
ni]
at 0
717
19
Janu
ary
2012
Molecular detection of Pseudomonas aeruginosa in recreational water
Giulia Amaglianiab Maria Lorella Parlanic Giorgio BrandiabGiuseppe Sebastianellic Vilberto Stocchiab and Giuditta Fiorella Schiavanoa
aDipartimento di Scienze Biomolecolari Universita degli Studi di Urbino lsquolsquoCarlo Borsquorsquo Urbino ItalybFacolta di Scienze Motorie Universita degli Studi di Urbino lsquolsquoCarlo Borsquorsquo Urbania ItalycLaboratorio di Igiene e Sanita Pubblica Dipartimento di Prevenzione ASUR ZT2 Urbania Italy
(Received 21 March 2011 final version received 3 May 2011)
The aim of this study was the development of a new molecular assay forPseudomonas aeruginosa identification in recreational water The method includesbacterial cell concentration through membrane filtration a short (6 h) culture-enrichment step DNA extraction and its amplification through a Real-Time PCRassay The performance of the molecular approach was evaluated on 44 samples ofswimming pool water and compared with the reference method UNI EN ISO162662008 Positivity rates of 6 and 74 in pool and inlet water respectivelywith the standard culture method and of 23 and 74 with the molecular methodwere found Statistical analysis indicated lsquolsquosubstantial agreementrsquorsquo (Cohenrsquos Kappaindex 06831) between the two approaches RAPD typing of P aeruginosa isolatesshowed identical fingerprint profiles indicating their epidemiological correlationThe developed protocol showed very high specificity and a detection limit of 10genomic units This technique has the potential to screen large numbers ofenvironmental samples and could be proposed as part of a self-monitoring plan forrecreational facilities improving surveillance and early warning systems
Keywords Pseudomonas aeruginosa ecfX Real-Time PCR recreational waterrandom amplification of polymorphic DNA (RAPD)
Introduction
The use of swimming pools and other recreational waters is associated with benefitsto health and well-being However a variety of microorganisms which mayconstitute a microbiological hazard can be found in such waters and their relatedfacilities (World Health Organisation [WHO] 2006) Associated adverse healthoutcomes including skin and ear infections can arise from opportunistic pathogensshed by bathers or naturally present in the aquatic environment and transmitted viasurfaces and contaminated water These pathogens can also accumulate in biofilmsthus becoming resistant to biocides and other disinfection agents (Goeres et al2004) Recreational water is a documented environmental source of Pseudomonasaeruginosa (Moore et al 2002 Papadopoulou et al 2008 Guida et al 2009)therefore it may represent a health risk for some user categories Swimming pools areused by a large variety of people varying in age health and hygienic standards
International Journal of Environmental Health Research
Vol 22 No 1 February 2012 60ndash70
ISSN 0960-3123 printISSN 1369-1619 online
2012 Taylor amp Francis
httpdxdoiorg101080096031232011588325
httpwwwtandfonlinecom
Dow
nloa
ded
by [
Bib
liote
ca U
nive
rsita
ria]
[G
iulia
Am
aglia
ni]
at 0
717
19
Janu
ary
2012
Pregnant women babies the elderly and disabled people who may attend specialcourses are potentially predisposed to contracting infections from pathogens andopportunistic bacteria (Leoni et al 1999) Moreover immunocompromised andcystic fibrosis patients are at particular risk (Barben et al 2005 Mena and Gerba2009) as well as athletes under competitive stress who may have weakened immunesystems (Pond 2005 WHO 2006)
In swimming pools the primary health effect associated with P aeruginosa isotitis externa although folliculitis dermatitis conjunctivitis and pneumonia havealso been reported (WHO 2006) Indeed waterborne pseudomonas-infection can berecognised in patients presenting a history of participation in water sportsactivities(Wang et al 2005)
Recently published guidelines from WHO (2006) are intended to be used as abasis for the development of international and national approaches for the controlof hazards that may be encountered in such environments In Italy in order toimplement an appropriate monitoring program and ensure adequate surveillanceLocal Sanitary Authorities (ASUR Azienda Sanitaria Unica Regionale) have beenmade responsible for official controls of recreational water Moreover programmesof self-monitoring according to the HACCP approach are to be applied byswimming pool managers as an lsquolsquointernal controlrsquorsquo (Italian Republic 2003)
Currently monitoring of P aeruginosa in swimming pool water is conducted inaccordance with the EU Drinking Water Directive (European Union [EU] 1998) andits Italian counterparts (Italian Republic 2001 2002) For P aeruginosa a maximumpermissible standard of zero per 100 ml of inlet water and acceptability limits of 1cfu (colony-forming units) per 100 ml of pool water have been fixed by ItalianGuidelines (Italian Republic 2003)
The reference analytical method for P aeruginosa detection (UNI EN ISO162662008) although sensitive and specific requires at least 48 h for the isolation ofsuspected positive colonies and additional time is needed for confirmation testsMoreover it is unable to detect the presence of viable but non-culturable (VBNC) formsof P aeruginosa (Oliver 2005) Consequently rapid accurate and culture-independentalternatives are being investigated to enable monitoring of this opportunistic pathogenin water A molecular method could reduce analysis time while maintaining sensitivityand specificity at very high levels and offering the additional capability of VBNCdetection Hence the aim of this study was the development of a new molecular methodfor P aeruginosa identification in recreational water
The entire testing process yielding a definitive result in one working dayincluded bacterial cell concentration through membrane filtration a short (6 h)culture-enrichment step DNA extraction and its amplification in Real-Time PCRIn order to validate the developed molecular assay and assess its applicability in aself monitoring plan of a swimming pool its performance was evaluated andcompared to the reference culture method carried out by ASUR
Methods
Collection and processing of water samples
The swimming pool investigated for this study was an indoor pool used by peopleof all ages for recreational purposes and by athletes for training A total of 44 watersamples (17 pool water and 27 inlet water) were collected in sterile bottlescontaining 10 sodium thiosulfate Water temperature pH free and total chlorine
International Journal of Environmental Health Research 61
Dow
nloa
ded
by [
Bib
liote
ca U
nive
rsita
ria]
[G
iulia
Am
aglia
ni]
at 0
717
19
Janu
ary
2012
were recorded at the moment of sampling (PoolTester Lovibond DortmundGermany)
Water analysis with standard method
Pseudomonas aeruginosa was isolated and characterised from water specimensaccording to the official reference method (UNI EN ISO 162662008) 100 ml waterwas filtered through a 045 mm gridded cellulose ester membrane of 47 mm diameter(Millipore Billerica MA USA) the membrane was placed on Pseudomonas AgarBase (Oxoid Basingstoke UK) with Pseudomonas CN Selective Supplement(Oxoid) and 10 ml glycerol l71 and incubated at 36+ 28C for 44+ 4 h Coloniesthat clearly showed pyocyanin production (blue-green fluorescent colonies) wereconsidered positive for P aeruginosa counted and expressed as cfu per 100 ml Allcolonies were confirmed in accordance with the reference method
Molecular characterisation of P aeruginosa isolates
Each bacterial isolate obtained on Pseudomonas CN agar was also tested for speciesconfirmation by amplification of a 528 bp specific fragment of the ecfX gene(GenBank Accession No AE004091 nt 1409949-1410476) as described by Laveniret al (2007) with the HotStarTaq Plus DNA Polymerase kit (Qiagen HildenGermany) and primers ECF1 (50-ATGGATGAGCGCTTCCGTG-30) -ECF2(50-TCATCCTTCGCCTCCCTG-30)
DNA was extracted from each isolate using DNeasy Blood and Tissue Kit(Qiagen) and its concentration was estimated through Quant-iT dsDNA HS AssayKit on Qubit fluorometer (both from Invitrogen Paisley UK) RAPD (randomamplification of polymorphic DNA) typing was conducted according to Lanotteet al (2004) with 272 primer (50-AGCGGGCCAA-30) PCR products were run on15 agarose gel with 100 bp Ladder Plus (MBI Fermentas Burlington Canada)and analysed on a Gel Doc 2000 apparatus using Quantity One QuantitationSoftware (Bio-Rad) In order to test the discriminatory power of RAPD typing with272 primer four additional P aeruginosa strains (Table 1) were used along withisolates obtained from water samples
Molecular detection of P aeruginosa in water
Specificity and sensitivity of Real-Time PCR
The Real-Time PCR assay was performed according to Anuj et al (2009) using Hot-Rescue Real-Time PCR Kit FP (Diatheva Fano Italy) with 5 mmol l71 MgCl204 mmol l71 each primer ecfXF (50-CGCATGCCTATCAGGCGTT-30) ecfXR(50-GAACTGCCCAGGTGCTTGC-30) 016 mmol l71 probe ecf-X-TM (50-FAM-ATGGCGAGTTGCTGCGCTTCCT-BHQ1-30) and 08 units DNA polymerase ina final reaction volume of 25 ml The amplification was carried out in a Rotor-Gene3000 (Corbett Research Sydney Australia) with the following thermal protocoldenaturation at 958C 10 min 50 cycles at 958C 15 s and 608C 1 min The expectedproduct size was 63 bp
Assay specificity was tested on a panel of 22 strains of non-target species and fourstrains of P aeruginosa (Table 1) The sensitivity was determined with serial dilutionsof P aeruginosa ATCC 10145 DNA extracted and quantified as above calculating
62 G Amagliani et al
Dow
nloa
ded
by [
Bib
liote
ca U
nive
rsita
ria]
[G
iulia
Am
aglia
ni]
at 0
717
19
Janu
ary
2012
genomic units based on bacterial genome size at httpwwwncbinlmgovgenomeslprokscgi DNA was diluted in dH2O and amplified in the range 106-1 genomicequivalents (ge) per PCR in triplicate Efficiency was calculated by plotting Ct(threshold cycle) values against log of genome copy number and using the slope ofthe resulting curve in the following equation
Efficiency frac14 101=slope 1
Artificially contaminated pool water samples
Preliminary tests to assess method sensitivity were carried out on artificiallycontaminated pool water samples Neutralised pool water was contaminated withvarious concentrations of P aeruginosa as follows P aeruginosa ATCC 10145 wasgrown in Brain Heart Infusion (BHI Oxoid) at 378C to an A600 of 018corresponding to 108 cfu ml71 The bacterial suspension was serially diluted and1-ml aliquots used for water contamination in the range 10ndash103 cfu100 ml Bacterialtitre was confirmed by plating 100 ml of each dilution on Pseudomonas AgarBase CN Water samples were filtered as described above and each membrane wasincubated for culture-enrichment in 20 ml of Pseudomonas Selective Brothsupplemented with 1 glycerol (Biolife Milan Italy) at 378C in agitation After
Table 1 Bacterial strains used for Real-Time PCR specificity test and corresponding results
International Journal of Environmental Health Research 63
Dow
nloa
ded
by [
Bib
liote
ca U
nive
rsita
ria]
[G
iulia
Am
aglia
ni]
at 0
717
19
Janu
ary
2012
6 h membranes were removed and the cultures were centrifuged at 6000 g for 20min Total DNA was extracted from the bacterial pellets either through a column-based kit (DNeasy Blood and Tissue Kit) or by magnetic extraction (MagPrep HSMerck Darmstadt Germany) with 1 mg nanoparticles per sample according tomanufacturer protocol under mild acidic conditions and 5 ml of each sample wasused for the Real-Time PCR assay
Analysis of field samples
The same 44 water samples analysed with the standard method were also examinedwith the molecular protocol starting from 1-l aliquots filtration culture enrichmentcolumn-based DNA extraction and Real-Time PCR were carried out as describedfor artificially contaminated water samples
Statistical analysis
Accordance of results obtained by both reference and molecular methods wascalculated with DAG_Stat software (Mackinnon 2000) providing sensitivityspecificity Positive and Negative Agreement (PA and NA) indices The k statisticwas used to estimate concordance (Landis and Koch 1977)
Results
Development of a molecular method for the detection of P aeruginosa
Specificity and sensitivity of Real-Time PCR
The specificity of the Real-Time PCR assay was confirmed over a panel of 22bacterial strains of species likely to be present in water environments and four ofP aeruginosa (Table 1) Assay sensitivity showed a detection limit of 10 ge with anamplification efficiency of 105 (R2 099453) (Figure 1)
Artificially contaminated pool water samples
The application of the entire molecular procedure on pool water samples artificiallycontaminated with 10ndash103 cfu100 ml made it possible to assess the sensitivity of the
Figure 1 Real-Time PCR sensitivity and efficiency Bacterial DNA dilutions (106-1 genomeequivalents ge) of the target species were amplified as described in the Methods section Ctvalues were plotted against DNA concentrations and the slope of the linear curve was used foramplification efficiency calculation Efficiency data with respective correlation coefficients aresummarised in the inset
64 G Amagliani et al
Dow
nloa
ded
by [
Bib
liote
ca U
nive
rsita
ria]
[G
iulia
Am
aglia
ni]
at 0
717
19
Janu
ary
2012
developed protocol Column-based extraction proved to be more efficient thanmagnetic nanoparticles for the isolation of bacterial DNA from water samples witha sensitivity of 10 cfu and 103 cfu respectively Ct values were as follows Qiagen kit0 cfu100 ml tested negative 10 cfu100 ml Ct 3565 102 cfu100 ml Ct 3278103 cfu100 ml Ct 2872 Merck magnetic particles 0 cfu100 ml 10 cfu100 ml and102 cfu100 ml tested negative 103 cfu100 ml Ct 3741
Analysis of recreational water samples
One out of 17 pool water samples and 2027 inlet water samples tested positive forP aeruginosa when analysed using the reference method Contamination titersranged between 1 and 45 cfu per 100 ml A high bacterial concentration above 100cfu was detected on two occasions When the same samples were analysed usingthe molecular method positivity rates of 417 and 2027 in pool and inlet waterrespectively were found (Table 2)
Statistical analysis
Test reliability was estimated by statistical analysis of result accordance With regardto the standard method the molecular assay showed a sensitivity of 9048 and aspecificity of 7826 PA was 844 (95 CI 0730ndash0958) NA 837 (95 CI0718ndash0956) Cohenrsquos Kappa index was 06831 which indicates lsquolsquosubstantialagreementrsquorsquo according to Landis and Koch (1977) classification
RAPD fingerprinting
The efficacy of the RAPD typing scheme was shown with bacterial strains of thesame species but different origins These included two additional isolates that hadoriginally been recovered from other swimming pools (environmental isolates 1 and2) one collection strain ATCC 10145 and one strain used as quality control strainby the ASUR (CQXVIII) (Table 1) As shown in Figure 2 panel B RAPDfingerprinting allowed us to differentiate all these distinct strains of P aeruginosaWhen all the P aeruginosa isolated from water samples were tested using the sameprotocol they were found to possess identical RAPD fingerprint profiles (Figure 2panel A)
Discussion
P aeruginosa is the second most common cause of recreational disease outbreaks(Craun et al 2005) Ear infection and otitis externa caused by this microorganism inswimming pools are probably more common than reported (Guida et al 2009)P aeruginosa presence in recreational water has been investigated by few authors(Barben et al 2005 Papadopoulou et al 2008 Guida et al 2009 Nikaeen et al2009) and additional data have been provided by Mena and Gerba (2009) Howevernone of these studies made use of a PCR-based method for the detection of thisspecies
More recently Villarreal et al (2010) described culture-independent techniquesapplied to drinking water surveillance Perkins et al (2009) to shower water andLavenir et al (2007) to environmental water while the use of PCR for pathogen
International Journal of Environmental Health Research 65
Dow
nloa
ded
by [
Bib
liote
ca U
nive
rsita
ria]
[G
iulia
Am
aglia
ni]
at 0
717
19
Janu
ary
2012
Table 2 Comparison of results obtained through reference and molecular methods forP aeruginosa detection in pool and inlet water
aColony count after membrane growth on Pseudomonas Agar Base CN (see Materials and methods)
66 G Amagliani et al
Dow
nloa
ded
by [
Bib
liote
ca U
nive
rsita
ria]
[G
iulia
Am
aglia
ni]
at 0
717
19
Janu
ary
2012
detection in swimming pools has been limited to the identification of adenoviruses(Heerden et al 2005) enteric protozoa (Fournier et al 2002) and free-living amoebae(Gianinazzi et al 2009) To the best of our knowledge the application of a molecularassay for the detection of P aeruginosa in recreational water has yet to be reported
The aim of this study was the development of a new molecular method forP aeruginosa identification in recreational water and the evaluation of itsperformance in the monitoring of a swimming pool in comparison with thereference culture method (UNI EN ISO 162662008) In order to make such acomparison inlet and pool water samples were collected from a local swimming pooland examined by both systems
Results of microbiological analysis indicated higher positivity rates in inlet water(74) compared to pool water (6) with higher levels of P aeruginosa (147 vs1 cfu100 ml mean values) in accordance with values reported by Guida et al(2009) These results may be explained by the presence of biofilm in the inlet waterpipeline and are supported by RAPD typing of isolates which indicate theirepidemiological correlation A longer period of contact with chlorine and thedilution process may have led to lower levels of P aeruginosa in pool water (Guidaet al 2009) Based on criteria provided by Italian Guidelines (Italian Republic 2003)fixing acceptability limits of 1 cfu100 ml in pool water and 0 cfu100 ml of inletwater in this study only the samples of pool water proved to be in compliance
lsquolsquoSubstantial agreementrsquorsquo (Landis and Koch 1977) was found between thedeveloped molecular method and the reference protocol The higher sensitivity of themolecular approach compared to the reference method in testing pool water samplescould be ascribed to the greater starting volume used for the analysis Moreoverbacteria in the pool water may have been in a subvital state due to chlorine watertreatment and able to recover culturability after a short enrichment in liquid
Figure 2 Discrimination of P aeruginosa by RAPD typing The ability of RAPDfingerprinting with primer 272 to cluster identical isolates (Panel A) and differentiatedistinct isolates within the P aeruginosa species (Panel B) is shown M 100 bp Ladder PlusFermentas Lanes 1ndash25 P aeruginosa isolated from recreational water 26 P aeruginosaenvironmental isolate 1 27 P aeruginosa environmental isolate 2 28 CQXVIII 29P aeruginosa ATCC 10145
International Journal of Environmental Health Research 67
Dow
nloa
ded
by [
Bib
liote
ca U
nive
rsita
ria]
[G
iulia
Am
aglia
ni]
at 0
717
19
Janu
ary
2012
medium thus providing resuscitation of injured or stressed cells However it iscommonly accepted that culture-dependent methods can fail in the identification ofbacteria in VBNC state (Oliver 2005)
It has been reported that the examination of colony morphology after membranegrowth on Cetrimide agar is not conclusive for P aeruginosa identification andconfirmation tests are needed for the correct identification of presumptive positivecolonies (Casanovas-Massana et al 2010) The need for such tests was illustrated bythe interlaboratory proficiency test carried out by the Italian National Health Service(Bonadonna and Ottaviani 2007) owing to the high rate (64) of false positiveresults obtained Conversely the Real-Time PCR assay specificity previously testedby Anuj et al (2009) on 101 target and non-target isolates and confirmed with 26further target and non-target strains in the present study proved to be very highConsidering that the assay has been successfully applied for the screening ofsuspected positive colonies on Pseudomonas Agar Base CN it may constitute aviable alternative to conventional time-consuming confirmation tests with a briefReal-Time PCR assay
A limitation for the broad application of molecular assays for testingenvironmental samples stems from the presence of substances of environmentalorigin such as humic acids and other organic molecules well known as PCRinhibitors (Wilson 1997) Hence the development of a DNA isolation system able toeffectively remove such contaminating molecules is a key step In a first attemptDNA direct isolation from filter membrane was carried out although the sensitivityobtained was not sufficient to be in compliance with National standards provided forrecreational water (not shown) After introducing a short (6 h) enrichment step twodifferent DNA extraction strategies were compared silica-based spin columns andmagnetic nanoparticles However the magnetic procedure for DNA isolation wasnot able to eliminate water-derived PCR inhibitors or did not provide sufficientrecovery especially with low bacterial concentrations On the other hand thecolumn devices proved effective and appropriate in this application
The employment of Real-Time PCR for the detection of P aeruginosa in wateroffers high specificity and sensitivity levels with a reduction of analysis time Inaddition this technique has the potential to screen large numbers of environmentalsamples limiting the application of the conventional culture techniques only toPCR-positive samples Hence the method could be proposed as part of a self-monitoring plan for swimming pool facilities to evaluate the quality of water usedfor recreational purposes improving surveillance and early warning systemsAlthough the use of PCR-based methods has not been recognised by AuthoritiesEU Directive 9883EC (EU 1998) allows the application of molecular diagnosis if itsequivalence with the reference protocol is demonstrated However a validationprogramme is needed to assess the level of accordance of the described system withthe reference method in order to verify the equivalence of results required byEuropean legislation
Acknowledgements
The authors would like to thank Dr G Cappuccini Director of the Department of Preventionand Head of the Hygiene and Public Health Service ASUR ZT 2 Urbino the personnel at theswimming pool facility of the Faculty of Health and Physical Exercise University of UrbinolsquolsquoCarlo Borsquorsquo for their collaboration and Professor Timothy Bloom of the University ofUrbino for a critical reading of the manuscript
68 G Amagliani et al
Dow
nloa
ded
by [
Bib
liote
ca U
nive
rsita
ria]
[G
iulia
Am
aglia
ni]
at 0
717
19
Janu
ary
2012
References
Anuj SN Whiley DM Kidd TJ Bell SC Wainwright CE Nissen MD Sloots TP 2009Identification of Pseudomonas aeruginosa by a duplex real-time polymerase chain reactionassay targeting the ecfX and the gyrB genes Diagn Microbiol Infect Dis 63 127ndash131
Barben J Hafen G Schmid J 2005 Pseudomonas aeruginosa in public swimming pools andbathroom water of patients with cystic fibrosis J Cystic Fibros 4227ndash231
Bonadonna L Ottaviani M 2007 Reference analytical methods for water intended forhuman consumption according to the Italian Legislative Decree 312001 Microbiologicalmethods Rapporti ISTISAN 075 Rome Istituto Superiore di Sanita (National HealthService)
Casanovas-Massana A Lucena F Blanch AR 2010 Identification of Pseudomonas aeruginosain water-bottling plants on the basis of procedures included in ISO 162662006J Microbiol Meth 811ndash5
Craun GF Calderon RL Craun MF 2005 Outbreaks associated with recreational water inthe United States Int J Environ Health Res 15243ndash262
European Union (EU) 1998 Council Directive 9883EC of 3 November 1998 on the qualityof water intended for human consumption
Fournier S Dubrou S Liguory O Gaussin F Santillana-Hayat M Sarfati C Molina JMDerouin F 2002 Detection of microsporidia cryptosporidia and giardia in swimmingpools a one-year prospective study FEMS Immunol Med Microbiol 33209ndash213
Gianinazzi C Schild M Wuthrich F Muller N Schurch N Gottstein B 2009 Potentiallyhuman pathogenic Acanthamoeba isolated from a heated indoor swimming pool inSwitzerland Exp Parasitol 121180ndash186
Goeres DM Palys T Sandel BB Geiger J 2004 Evaluation of disinfectant efficacy againstbiofilm and suspended bacteria in a laboratory swimming pool model Water Res383103ndash3109
Guida M Galle F Mattei ML Anastasi D Liguori G 2009 Microbiological quality of thewater of recreational and rehabilitation pools a 2-year survey in Naples Italy PublicHealth 123448ndash451
Heerden J Ehlers MM Grabow WOK 2005 Detection and risk assessment of adenovirusesin swimming pool water J Appl Microbiol 991256ndash1264
Italian Republic 2001 Legislative Decree No 31 2 February implementing Directive 9883EC on the quality of water intended for human consumption Official Gazette No 52 of3rd March 2001 Rome
Italian Republic 2002 Legislative Decree No 27 2 February amending and supplementingLegislative Decree No 31 of 2 February 2001 implementing Directive 9883EC on thequality of water intended for human consumption Official Gazette No 58 of 9th of March2002 Rome
Italian Republic 2003 Permanent Council State-Regions and Autonomous ProvincesAccordo Stato ndash Regioni e Province Autonome 16-1-2003 Official Gazette No 51 of 3rdof March 2003 Rome
Landis JR Koch GG 1977 The measurement of observer agreement for categorical dataBiometrics 33159ndash174
Lanotte P Watt S Mereghetti L Dartiguelongue N Rastegar-Lari A Goudeau A QuentinR 2004 Genetic features of Pseudomonas aeruginosa isolates from cystic fibrosis patientscompared with those of isolates from other origins J Med Microbiol 5373ndash81
Lavenir R Jocktane D Laurent F Nazaret S Cournoyer B 2007 Improved reliability ofPseudomonas aeruginosa PCR detection by the use of the species-specific ecfX gene targetJ Microbiol Meth 7020ndash29
Leoni E Legnani P Guberti E Masotti A 1999 Risk of infection associated withmicrobiological quality of public swimming pools in Bologna Italy Public Health113227ndash232
Mackinnon A 2000 A spreadsheet for the calculation of comprehensive statistics for theassessment of diagnostic tests and inter-rater agreement Comput Biol Med 30127ndash134
Mena KD Gerba CP 2009 Risk assessment of Pseudomonas aeruginosa in water RevEnviron Contam Toxicol 20171ndash115
Moore JE Heaney N Millar BC Crowe M Elborn JS 2002 Incidence of Pseudomonasaeruginosa in recreational and hydrotherapy pools Commun Dis Public Health 523ndash26
International Journal of Environmental Health Research 69
Dow
nloa
ded
by [
Bib
liote
ca U
nive
rsita
ria]
[G
iulia
Am
aglia
ni]
at 0
717
19
Janu
ary
2012
Nikaeen M Hatamzadeh M Vahid Dastjerdi M Hassanzadeh A 2009 Predictive indicatorsof the safety of swimming pool waters Water Sci Technol 603101ndash3107
Oliver JD 2005 The viable but nonculturable state in bacteria J Microbiol 4393ndash100Papadopoulou C Economou V Sakkas H Gousia P Giannakopoulos X Dontorou C
Filioussis G Gessouli H Karanis P Leveidiotou S 2008 Microbiological quality ofindoor and outdoor swimming pools in Greece investigation of the antibiotic resistanceof the bacterial isolates Int J Hyg Environ Health 211385ndash397
Perkins SD Mayfield J Fraser V Angenent LT 2009 Potentially pathogenic bacteria inshower water and air of a stem cell transplant unit Appl Environ Microbiol 755363ndash5372
Pond K 2005 Water recreation and disease Plausibility of associated infections acute effectssequelae and mortality London WHO IWA Publishing
UNI EN ISO 162662008 ndash 18092008 ndashWater quality ndash Detection and enumeration ofPseudomonas aeruginosa ndash Method by membrane filtration
Villarreal JV Schwartz T Obst U 2010 Culture-independent techniques applied to foodindustry water surveillance ndash a case study Int J Food Microbiol 141(Suppl 1)S147ndash55
Wang MC Liu CY Shiao AS Wang T 2005 Ear problems in swimmers J Chin Med Assoc68347ndash352
World Health Organisation (WHO) 2006 Guidelines for safe recreational water environ-ments Vol 2 Swimming pools and similar environments Geneva WHO Press
Wilson IG 1997 Inhibition and facilitation of nucleic acid amplification Appl EnvironMicrobiol 633741ndash3751
70 G Amagliani et al
Dow
nloa
ded
by [
Bib
liote
ca U
nive
rsita
ria]
[G
iulia
Am
aglia
ni]
at 0
717
19
Janu
ary
2012
Pregnant women babies the elderly and disabled people who may attend specialcourses are potentially predisposed to contracting infections from pathogens andopportunistic bacteria (Leoni et al 1999) Moreover immunocompromised andcystic fibrosis patients are at particular risk (Barben et al 2005 Mena and Gerba2009) as well as athletes under competitive stress who may have weakened immunesystems (Pond 2005 WHO 2006)
In swimming pools the primary health effect associated with P aeruginosa isotitis externa although folliculitis dermatitis conjunctivitis and pneumonia havealso been reported (WHO 2006) Indeed waterborne pseudomonas-infection can berecognised in patients presenting a history of participation in water sportsactivities(Wang et al 2005)
Recently published guidelines from WHO (2006) are intended to be used as abasis for the development of international and national approaches for the controlof hazards that may be encountered in such environments In Italy in order toimplement an appropriate monitoring program and ensure adequate surveillanceLocal Sanitary Authorities (ASUR Azienda Sanitaria Unica Regionale) have beenmade responsible for official controls of recreational water Moreover programmesof self-monitoring according to the HACCP approach are to be applied byswimming pool managers as an lsquolsquointernal controlrsquorsquo (Italian Republic 2003)
Currently monitoring of P aeruginosa in swimming pool water is conducted inaccordance with the EU Drinking Water Directive (European Union [EU] 1998) andits Italian counterparts (Italian Republic 2001 2002) For P aeruginosa a maximumpermissible standard of zero per 100 ml of inlet water and acceptability limits of 1cfu (colony-forming units) per 100 ml of pool water have been fixed by ItalianGuidelines (Italian Republic 2003)
The reference analytical method for P aeruginosa detection (UNI EN ISO162662008) although sensitive and specific requires at least 48 h for the isolation ofsuspected positive colonies and additional time is needed for confirmation testsMoreover it is unable to detect the presence of viable but non-culturable (VBNC) formsof P aeruginosa (Oliver 2005) Consequently rapid accurate and culture-independentalternatives are being investigated to enable monitoring of this opportunistic pathogenin water A molecular method could reduce analysis time while maintaining sensitivityand specificity at very high levels and offering the additional capability of VBNCdetection Hence the aim of this study was the development of a new molecular methodfor P aeruginosa identification in recreational water
The entire testing process yielding a definitive result in one working dayincluded bacterial cell concentration through membrane filtration a short (6 h)culture-enrichment step DNA extraction and its amplification in Real-Time PCRIn order to validate the developed molecular assay and assess its applicability in aself monitoring plan of a swimming pool its performance was evaluated andcompared to the reference culture method carried out by ASUR
Methods
Collection and processing of water samples
The swimming pool investigated for this study was an indoor pool used by peopleof all ages for recreational purposes and by athletes for training A total of 44 watersamples (17 pool water and 27 inlet water) were collected in sterile bottlescontaining 10 sodium thiosulfate Water temperature pH free and total chlorine
International Journal of Environmental Health Research 61
Dow
nloa
ded
by [
Bib
liote
ca U
nive
rsita
ria]
[G
iulia
Am
aglia
ni]
at 0
717
19
Janu
ary
2012
were recorded at the moment of sampling (PoolTester Lovibond DortmundGermany)
Water analysis with standard method
Pseudomonas aeruginosa was isolated and characterised from water specimensaccording to the official reference method (UNI EN ISO 162662008) 100 ml waterwas filtered through a 045 mm gridded cellulose ester membrane of 47 mm diameter(Millipore Billerica MA USA) the membrane was placed on Pseudomonas AgarBase (Oxoid Basingstoke UK) with Pseudomonas CN Selective Supplement(Oxoid) and 10 ml glycerol l71 and incubated at 36+ 28C for 44+ 4 h Coloniesthat clearly showed pyocyanin production (blue-green fluorescent colonies) wereconsidered positive for P aeruginosa counted and expressed as cfu per 100 ml Allcolonies were confirmed in accordance with the reference method
Molecular characterisation of P aeruginosa isolates
Each bacterial isolate obtained on Pseudomonas CN agar was also tested for speciesconfirmation by amplification of a 528 bp specific fragment of the ecfX gene(GenBank Accession No AE004091 nt 1409949-1410476) as described by Laveniret al (2007) with the HotStarTaq Plus DNA Polymerase kit (Qiagen HildenGermany) and primers ECF1 (50-ATGGATGAGCGCTTCCGTG-30) -ECF2(50-TCATCCTTCGCCTCCCTG-30)
DNA was extracted from each isolate using DNeasy Blood and Tissue Kit(Qiagen) and its concentration was estimated through Quant-iT dsDNA HS AssayKit on Qubit fluorometer (both from Invitrogen Paisley UK) RAPD (randomamplification of polymorphic DNA) typing was conducted according to Lanotteet al (2004) with 272 primer (50-AGCGGGCCAA-30) PCR products were run on15 agarose gel with 100 bp Ladder Plus (MBI Fermentas Burlington Canada)and analysed on a Gel Doc 2000 apparatus using Quantity One QuantitationSoftware (Bio-Rad) In order to test the discriminatory power of RAPD typing with272 primer four additional P aeruginosa strains (Table 1) were used along withisolates obtained from water samples
Molecular detection of P aeruginosa in water
Specificity and sensitivity of Real-Time PCR
The Real-Time PCR assay was performed according to Anuj et al (2009) using Hot-Rescue Real-Time PCR Kit FP (Diatheva Fano Italy) with 5 mmol l71 MgCl204 mmol l71 each primer ecfXF (50-CGCATGCCTATCAGGCGTT-30) ecfXR(50-GAACTGCCCAGGTGCTTGC-30) 016 mmol l71 probe ecf-X-TM (50-FAM-ATGGCGAGTTGCTGCGCTTCCT-BHQ1-30) and 08 units DNA polymerase ina final reaction volume of 25 ml The amplification was carried out in a Rotor-Gene3000 (Corbett Research Sydney Australia) with the following thermal protocoldenaturation at 958C 10 min 50 cycles at 958C 15 s and 608C 1 min The expectedproduct size was 63 bp
Assay specificity was tested on a panel of 22 strains of non-target species and fourstrains of P aeruginosa (Table 1) The sensitivity was determined with serial dilutionsof P aeruginosa ATCC 10145 DNA extracted and quantified as above calculating
62 G Amagliani et al
Dow
nloa
ded
by [
Bib
liote
ca U
nive
rsita
ria]
[G
iulia
Am
aglia
ni]
at 0
717
19
Janu
ary
2012
genomic units based on bacterial genome size at httpwwwncbinlmgovgenomeslprokscgi DNA was diluted in dH2O and amplified in the range 106-1 genomicequivalents (ge) per PCR in triplicate Efficiency was calculated by plotting Ct(threshold cycle) values against log of genome copy number and using the slope ofthe resulting curve in the following equation
Efficiency frac14 101=slope 1
Artificially contaminated pool water samples
Preliminary tests to assess method sensitivity were carried out on artificiallycontaminated pool water samples Neutralised pool water was contaminated withvarious concentrations of P aeruginosa as follows P aeruginosa ATCC 10145 wasgrown in Brain Heart Infusion (BHI Oxoid) at 378C to an A600 of 018corresponding to 108 cfu ml71 The bacterial suspension was serially diluted and1-ml aliquots used for water contamination in the range 10ndash103 cfu100 ml Bacterialtitre was confirmed by plating 100 ml of each dilution on Pseudomonas AgarBase CN Water samples were filtered as described above and each membrane wasincubated for culture-enrichment in 20 ml of Pseudomonas Selective Brothsupplemented with 1 glycerol (Biolife Milan Italy) at 378C in agitation After
Table 1 Bacterial strains used for Real-Time PCR specificity test and corresponding results
International Journal of Environmental Health Research 63
Dow
nloa
ded
by [
Bib
liote
ca U
nive
rsita
ria]
[G
iulia
Am
aglia
ni]
at 0
717
19
Janu
ary
2012
6 h membranes were removed and the cultures were centrifuged at 6000 g for 20min Total DNA was extracted from the bacterial pellets either through a column-based kit (DNeasy Blood and Tissue Kit) or by magnetic extraction (MagPrep HSMerck Darmstadt Germany) with 1 mg nanoparticles per sample according tomanufacturer protocol under mild acidic conditions and 5 ml of each sample wasused for the Real-Time PCR assay
Analysis of field samples
The same 44 water samples analysed with the standard method were also examinedwith the molecular protocol starting from 1-l aliquots filtration culture enrichmentcolumn-based DNA extraction and Real-Time PCR were carried out as describedfor artificially contaminated water samples
Statistical analysis
Accordance of results obtained by both reference and molecular methods wascalculated with DAG_Stat software (Mackinnon 2000) providing sensitivityspecificity Positive and Negative Agreement (PA and NA) indices The k statisticwas used to estimate concordance (Landis and Koch 1977)
Results
Development of a molecular method for the detection of P aeruginosa
Specificity and sensitivity of Real-Time PCR
The specificity of the Real-Time PCR assay was confirmed over a panel of 22bacterial strains of species likely to be present in water environments and four ofP aeruginosa (Table 1) Assay sensitivity showed a detection limit of 10 ge with anamplification efficiency of 105 (R2 099453) (Figure 1)
Artificially contaminated pool water samples
The application of the entire molecular procedure on pool water samples artificiallycontaminated with 10ndash103 cfu100 ml made it possible to assess the sensitivity of the
Figure 1 Real-Time PCR sensitivity and efficiency Bacterial DNA dilutions (106-1 genomeequivalents ge) of the target species were amplified as described in the Methods section Ctvalues were plotted against DNA concentrations and the slope of the linear curve was used foramplification efficiency calculation Efficiency data with respective correlation coefficients aresummarised in the inset
64 G Amagliani et al
Dow
nloa
ded
by [
Bib
liote
ca U
nive
rsita
ria]
[G
iulia
Am
aglia
ni]
at 0
717
19
Janu
ary
2012
developed protocol Column-based extraction proved to be more efficient thanmagnetic nanoparticles for the isolation of bacterial DNA from water samples witha sensitivity of 10 cfu and 103 cfu respectively Ct values were as follows Qiagen kit0 cfu100 ml tested negative 10 cfu100 ml Ct 3565 102 cfu100 ml Ct 3278103 cfu100 ml Ct 2872 Merck magnetic particles 0 cfu100 ml 10 cfu100 ml and102 cfu100 ml tested negative 103 cfu100 ml Ct 3741
Analysis of recreational water samples
One out of 17 pool water samples and 2027 inlet water samples tested positive forP aeruginosa when analysed using the reference method Contamination titersranged between 1 and 45 cfu per 100 ml A high bacterial concentration above 100cfu was detected on two occasions When the same samples were analysed usingthe molecular method positivity rates of 417 and 2027 in pool and inlet waterrespectively were found (Table 2)
Statistical analysis
Test reliability was estimated by statistical analysis of result accordance With regardto the standard method the molecular assay showed a sensitivity of 9048 and aspecificity of 7826 PA was 844 (95 CI 0730ndash0958) NA 837 (95 CI0718ndash0956) Cohenrsquos Kappa index was 06831 which indicates lsquolsquosubstantialagreementrsquorsquo according to Landis and Koch (1977) classification
RAPD fingerprinting
The efficacy of the RAPD typing scheme was shown with bacterial strains of thesame species but different origins These included two additional isolates that hadoriginally been recovered from other swimming pools (environmental isolates 1 and2) one collection strain ATCC 10145 and one strain used as quality control strainby the ASUR (CQXVIII) (Table 1) As shown in Figure 2 panel B RAPDfingerprinting allowed us to differentiate all these distinct strains of P aeruginosaWhen all the P aeruginosa isolated from water samples were tested using the sameprotocol they were found to possess identical RAPD fingerprint profiles (Figure 2panel A)
Discussion
P aeruginosa is the second most common cause of recreational disease outbreaks(Craun et al 2005) Ear infection and otitis externa caused by this microorganism inswimming pools are probably more common than reported (Guida et al 2009)P aeruginosa presence in recreational water has been investigated by few authors(Barben et al 2005 Papadopoulou et al 2008 Guida et al 2009 Nikaeen et al2009) and additional data have been provided by Mena and Gerba (2009) Howevernone of these studies made use of a PCR-based method for the detection of thisspecies
More recently Villarreal et al (2010) described culture-independent techniquesapplied to drinking water surveillance Perkins et al (2009) to shower water andLavenir et al (2007) to environmental water while the use of PCR for pathogen
International Journal of Environmental Health Research 65
Dow
nloa
ded
by [
Bib
liote
ca U
nive
rsita
ria]
[G
iulia
Am
aglia
ni]
at 0
717
19
Janu
ary
2012
Table 2 Comparison of results obtained through reference and molecular methods forP aeruginosa detection in pool and inlet water
aColony count after membrane growth on Pseudomonas Agar Base CN (see Materials and methods)
66 G Amagliani et al
Dow
nloa
ded
by [
Bib
liote
ca U
nive
rsita
ria]
[G
iulia
Am
aglia
ni]
at 0
717
19
Janu
ary
2012
detection in swimming pools has been limited to the identification of adenoviruses(Heerden et al 2005) enteric protozoa (Fournier et al 2002) and free-living amoebae(Gianinazzi et al 2009) To the best of our knowledge the application of a molecularassay for the detection of P aeruginosa in recreational water has yet to be reported
The aim of this study was the development of a new molecular method forP aeruginosa identification in recreational water and the evaluation of itsperformance in the monitoring of a swimming pool in comparison with thereference culture method (UNI EN ISO 162662008) In order to make such acomparison inlet and pool water samples were collected from a local swimming pooland examined by both systems
Results of microbiological analysis indicated higher positivity rates in inlet water(74) compared to pool water (6) with higher levels of P aeruginosa (147 vs1 cfu100 ml mean values) in accordance with values reported by Guida et al(2009) These results may be explained by the presence of biofilm in the inlet waterpipeline and are supported by RAPD typing of isolates which indicate theirepidemiological correlation A longer period of contact with chlorine and thedilution process may have led to lower levels of P aeruginosa in pool water (Guidaet al 2009) Based on criteria provided by Italian Guidelines (Italian Republic 2003)fixing acceptability limits of 1 cfu100 ml in pool water and 0 cfu100 ml of inletwater in this study only the samples of pool water proved to be in compliance
lsquolsquoSubstantial agreementrsquorsquo (Landis and Koch 1977) was found between thedeveloped molecular method and the reference protocol The higher sensitivity of themolecular approach compared to the reference method in testing pool water samplescould be ascribed to the greater starting volume used for the analysis Moreoverbacteria in the pool water may have been in a subvital state due to chlorine watertreatment and able to recover culturability after a short enrichment in liquid
Figure 2 Discrimination of P aeruginosa by RAPD typing The ability of RAPDfingerprinting with primer 272 to cluster identical isolates (Panel A) and differentiatedistinct isolates within the P aeruginosa species (Panel B) is shown M 100 bp Ladder PlusFermentas Lanes 1ndash25 P aeruginosa isolated from recreational water 26 P aeruginosaenvironmental isolate 1 27 P aeruginosa environmental isolate 2 28 CQXVIII 29P aeruginosa ATCC 10145
International Journal of Environmental Health Research 67
Dow
nloa
ded
by [
Bib
liote
ca U
nive
rsita
ria]
[G
iulia
Am
aglia
ni]
at 0
717
19
Janu
ary
2012
medium thus providing resuscitation of injured or stressed cells However it iscommonly accepted that culture-dependent methods can fail in the identification ofbacteria in VBNC state (Oliver 2005)
It has been reported that the examination of colony morphology after membranegrowth on Cetrimide agar is not conclusive for P aeruginosa identification andconfirmation tests are needed for the correct identification of presumptive positivecolonies (Casanovas-Massana et al 2010) The need for such tests was illustrated bythe interlaboratory proficiency test carried out by the Italian National Health Service(Bonadonna and Ottaviani 2007) owing to the high rate (64) of false positiveresults obtained Conversely the Real-Time PCR assay specificity previously testedby Anuj et al (2009) on 101 target and non-target isolates and confirmed with 26further target and non-target strains in the present study proved to be very highConsidering that the assay has been successfully applied for the screening ofsuspected positive colonies on Pseudomonas Agar Base CN it may constitute aviable alternative to conventional time-consuming confirmation tests with a briefReal-Time PCR assay
A limitation for the broad application of molecular assays for testingenvironmental samples stems from the presence of substances of environmentalorigin such as humic acids and other organic molecules well known as PCRinhibitors (Wilson 1997) Hence the development of a DNA isolation system able toeffectively remove such contaminating molecules is a key step In a first attemptDNA direct isolation from filter membrane was carried out although the sensitivityobtained was not sufficient to be in compliance with National standards provided forrecreational water (not shown) After introducing a short (6 h) enrichment step twodifferent DNA extraction strategies were compared silica-based spin columns andmagnetic nanoparticles However the magnetic procedure for DNA isolation wasnot able to eliminate water-derived PCR inhibitors or did not provide sufficientrecovery especially with low bacterial concentrations On the other hand thecolumn devices proved effective and appropriate in this application
The employment of Real-Time PCR for the detection of P aeruginosa in wateroffers high specificity and sensitivity levels with a reduction of analysis time Inaddition this technique has the potential to screen large numbers of environmentalsamples limiting the application of the conventional culture techniques only toPCR-positive samples Hence the method could be proposed as part of a self-monitoring plan for swimming pool facilities to evaluate the quality of water usedfor recreational purposes improving surveillance and early warning systemsAlthough the use of PCR-based methods has not been recognised by AuthoritiesEU Directive 9883EC (EU 1998) allows the application of molecular diagnosis if itsequivalence with the reference protocol is demonstrated However a validationprogramme is needed to assess the level of accordance of the described system withthe reference method in order to verify the equivalence of results required byEuropean legislation
Acknowledgements
The authors would like to thank Dr G Cappuccini Director of the Department of Preventionand Head of the Hygiene and Public Health Service ASUR ZT 2 Urbino the personnel at theswimming pool facility of the Faculty of Health and Physical Exercise University of UrbinolsquolsquoCarlo Borsquorsquo for their collaboration and Professor Timothy Bloom of the University ofUrbino for a critical reading of the manuscript
68 G Amagliani et al
Dow
nloa
ded
by [
Bib
liote
ca U
nive
rsita
ria]
[G
iulia
Am
aglia
ni]
at 0
717
19
Janu
ary
2012
References
Anuj SN Whiley DM Kidd TJ Bell SC Wainwright CE Nissen MD Sloots TP 2009Identification of Pseudomonas aeruginosa by a duplex real-time polymerase chain reactionassay targeting the ecfX and the gyrB genes Diagn Microbiol Infect Dis 63 127ndash131
Barben J Hafen G Schmid J 2005 Pseudomonas aeruginosa in public swimming pools andbathroom water of patients with cystic fibrosis J Cystic Fibros 4227ndash231
Bonadonna L Ottaviani M 2007 Reference analytical methods for water intended forhuman consumption according to the Italian Legislative Decree 312001 Microbiologicalmethods Rapporti ISTISAN 075 Rome Istituto Superiore di Sanita (National HealthService)
Casanovas-Massana A Lucena F Blanch AR 2010 Identification of Pseudomonas aeruginosain water-bottling plants on the basis of procedures included in ISO 162662006J Microbiol Meth 811ndash5
Craun GF Calderon RL Craun MF 2005 Outbreaks associated with recreational water inthe United States Int J Environ Health Res 15243ndash262
European Union (EU) 1998 Council Directive 9883EC of 3 November 1998 on the qualityof water intended for human consumption
Fournier S Dubrou S Liguory O Gaussin F Santillana-Hayat M Sarfati C Molina JMDerouin F 2002 Detection of microsporidia cryptosporidia and giardia in swimmingpools a one-year prospective study FEMS Immunol Med Microbiol 33209ndash213
Gianinazzi C Schild M Wuthrich F Muller N Schurch N Gottstein B 2009 Potentiallyhuman pathogenic Acanthamoeba isolated from a heated indoor swimming pool inSwitzerland Exp Parasitol 121180ndash186
Goeres DM Palys T Sandel BB Geiger J 2004 Evaluation of disinfectant efficacy againstbiofilm and suspended bacteria in a laboratory swimming pool model Water Res383103ndash3109
Guida M Galle F Mattei ML Anastasi D Liguori G 2009 Microbiological quality of thewater of recreational and rehabilitation pools a 2-year survey in Naples Italy PublicHealth 123448ndash451
Heerden J Ehlers MM Grabow WOK 2005 Detection and risk assessment of adenovirusesin swimming pool water J Appl Microbiol 991256ndash1264
Italian Republic 2001 Legislative Decree No 31 2 February implementing Directive 9883EC on the quality of water intended for human consumption Official Gazette No 52 of3rd March 2001 Rome
Italian Republic 2002 Legislative Decree No 27 2 February amending and supplementingLegislative Decree No 31 of 2 February 2001 implementing Directive 9883EC on thequality of water intended for human consumption Official Gazette No 58 of 9th of March2002 Rome
Italian Republic 2003 Permanent Council State-Regions and Autonomous ProvincesAccordo Stato ndash Regioni e Province Autonome 16-1-2003 Official Gazette No 51 of 3rdof March 2003 Rome
Landis JR Koch GG 1977 The measurement of observer agreement for categorical dataBiometrics 33159ndash174
Lanotte P Watt S Mereghetti L Dartiguelongue N Rastegar-Lari A Goudeau A QuentinR 2004 Genetic features of Pseudomonas aeruginosa isolates from cystic fibrosis patientscompared with those of isolates from other origins J Med Microbiol 5373ndash81
Lavenir R Jocktane D Laurent F Nazaret S Cournoyer B 2007 Improved reliability ofPseudomonas aeruginosa PCR detection by the use of the species-specific ecfX gene targetJ Microbiol Meth 7020ndash29
Leoni E Legnani P Guberti E Masotti A 1999 Risk of infection associated withmicrobiological quality of public swimming pools in Bologna Italy Public Health113227ndash232
Mackinnon A 2000 A spreadsheet for the calculation of comprehensive statistics for theassessment of diagnostic tests and inter-rater agreement Comput Biol Med 30127ndash134
Mena KD Gerba CP 2009 Risk assessment of Pseudomonas aeruginosa in water RevEnviron Contam Toxicol 20171ndash115
Moore JE Heaney N Millar BC Crowe M Elborn JS 2002 Incidence of Pseudomonasaeruginosa in recreational and hydrotherapy pools Commun Dis Public Health 523ndash26
International Journal of Environmental Health Research 69
Dow
nloa
ded
by [
Bib
liote
ca U
nive
rsita
ria]
[G
iulia
Am
aglia
ni]
at 0
717
19
Janu
ary
2012
Nikaeen M Hatamzadeh M Vahid Dastjerdi M Hassanzadeh A 2009 Predictive indicatorsof the safety of swimming pool waters Water Sci Technol 603101ndash3107
Oliver JD 2005 The viable but nonculturable state in bacteria J Microbiol 4393ndash100Papadopoulou C Economou V Sakkas H Gousia P Giannakopoulos X Dontorou C
Filioussis G Gessouli H Karanis P Leveidiotou S 2008 Microbiological quality ofindoor and outdoor swimming pools in Greece investigation of the antibiotic resistanceof the bacterial isolates Int J Hyg Environ Health 211385ndash397
Perkins SD Mayfield J Fraser V Angenent LT 2009 Potentially pathogenic bacteria inshower water and air of a stem cell transplant unit Appl Environ Microbiol 755363ndash5372
Pond K 2005 Water recreation and disease Plausibility of associated infections acute effectssequelae and mortality London WHO IWA Publishing
UNI EN ISO 162662008 ndash 18092008 ndashWater quality ndash Detection and enumeration ofPseudomonas aeruginosa ndash Method by membrane filtration
Villarreal JV Schwartz T Obst U 2010 Culture-independent techniques applied to foodindustry water surveillance ndash a case study Int J Food Microbiol 141(Suppl 1)S147ndash55
Wang MC Liu CY Shiao AS Wang T 2005 Ear problems in swimmers J Chin Med Assoc68347ndash352
World Health Organisation (WHO) 2006 Guidelines for safe recreational water environ-ments Vol 2 Swimming pools and similar environments Geneva WHO Press
Wilson IG 1997 Inhibition and facilitation of nucleic acid amplification Appl EnvironMicrobiol 633741ndash3751
70 G Amagliani et al
Dow
nloa
ded
by [
Bib
liote
ca U
nive
rsita
ria]
[G
iulia
Am
aglia
ni]
at 0
717
19
Janu
ary
2012
were recorded at the moment of sampling (PoolTester Lovibond DortmundGermany)
Water analysis with standard method
Pseudomonas aeruginosa was isolated and characterised from water specimensaccording to the official reference method (UNI EN ISO 162662008) 100 ml waterwas filtered through a 045 mm gridded cellulose ester membrane of 47 mm diameter(Millipore Billerica MA USA) the membrane was placed on Pseudomonas AgarBase (Oxoid Basingstoke UK) with Pseudomonas CN Selective Supplement(Oxoid) and 10 ml glycerol l71 and incubated at 36+ 28C for 44+ 4 h Coloniesthat clearly showed pyocyanin production (blue-green fluorescent colonies) wereconsidered positive for P aeruginosa counted and expressed as cfu per 100 ml Allcolonies were confirmed in accordance with the reference method
Molecular characterisation of P aeruginosa isolates
Each bacterial isolate obtained on Pseudomonas CN agar was also tested for speciesconfirmation by amplification of a 528 bp specific fragment of the ecfX gene(GenBank Accession No AE004091 nt 1409949-1410476) as described by Laveniret al (2007) with the HotStarTaq Plus DNA Polymerase kit (Qiagen HildenGermany) and primers ECF1 (50-ATGGATGAGCGCTTCCGTG-30) -ECF2(50-TCATCCTTCGCCTCCCTG-30)
DNA was extracted from each isolate using DNeasy Blood and Tissue Kit(Qiagen) and its concentration was estimated through Quant-iT dsDNA HS AssayKit on Qubit fluorometer (both from Invitrogen Paisley UK) RAPD (randomamplification of polymorphic DNA) typing was conducted according to Lanotteet al (2004) with 272 primer (50-AGCGGGCCAA-30) PCR products were run on15 agarose gel with 100 bp Ladder Plus (MBI Fermentas Burlington Canada)and analysed on a Gel Doc 2000 apparatus using Quantity One QuantitationSoftware (Bio-Rad) In order to test the discriminatory power of RAPD typing with272 primer four additional P aeruginosa strains (Table 1) were used along withisolates obtained from water samples
Molecular detection of P aeruginosa in water
Specificity and sensitivity of Real-Time PCR
The Real-Time PCR assay was performed according to Anuj et al (2009) using Hot-Rescue Real-Time PCR Kit FP (Diatheva Fano Italy) with 5 mmol l71 MgCl204 mmol l71 each primer ecfXF (50-CGCATGCCTATCAGGCGTT-30) ecfXR(50-GAACTGCCCAGGTGCTTGC-30) 016 mmol l71 probe ecf-X-TM (50-FAM-ATGGCGAGTTGCTGCGCTTCCT-BHQ1-30) and 08 units DNA polymerase ina final reaction volume of 25 ml The amplification was carried out in a Rotor-Gene3000 (Corbett Research Sydney Australia) with the following thermal protocoldenaturation at 958C 10 min 50 cycles at 958C 15 s and 608C 1 min The expectedproduct size was 63 bp
Assay specificity was tested on a panel of 22 strains of non-target species and fourstrains of P aeruginosa (Table 1) The sensitivity was determined with serial dilutionsof P aeruginosa ATCC 10145 DNA extracted and quantified as above calculating
62 G Amagliani et al
Dow
nloa
ded
by [
Bib
liote
ca U
nive
rsita
ria]
[G
iulia
Am
aglia
ni]
at 0
717
19
Janu
ary
2012
genomic units based on bacterial genome size at httpwwwncbinlmgovgenomeslprokscgi DNA was diluted in dH2O and amplified in the range 106-1 genomicequivalents (ge) per PCR in triplicate Efficiency was calculated by plotting Ct(threshold cycle) values against log of genome copy number and using the slope ofthe resulting curve in the following equation
Efficiency frac14 101=slope 1
Artificially contaminated pool water samples
Preliminary tests to assess method sensitivity were carried out on artificiallycontaminated pool water samples Neutralised pool water was contaminated withvarious concentrations of P aeruginosa as follows P aeruginosa ATCC 10145 wasgrown in Brain Heart Infusion (BHI Oxoid) at 378C to an A600 of 018corresponding to 108 cfu ml71 The bacterial suspension was serially diluted and1-ml aliquots used for water contamination in the range 10ndash103 cfu100 ml Bacterialtitre was confirmed by plating 100 ml of each dilution on Pseudomonas AgarBase CN Water samples were filtered as described above and each membrane wasincubated for culture-enrichment in 20 ml of Pseudomonas Selective Brothsupplemented with 1 glycerol (Biolife Milan Italy) at 378C in agitation After
Table 1 Bacterial strains used for Real-Time PCR specificity test and corresponding results
International Journal of Environmental Health Research 63
Dow
nloa
ded
by [
Bib
liote
ca U
nive
rsita
ria]
[G
iulia
Am
aglia
ni]
at 0
717
19
Janu
ary
2012
6 h membranes were removed and the cultures were centrifuged at 6000 g for 20min Total DNA was extracted from the bacterial pellets either through a column-based kit (DNeasy Blood and Tissue Kit) or by magnetic extraction (MagPrep HSMerck Darmstadt Germany) with 1 mg nanoparticles per sample according tomanufacturer protocol under mild acidic conditions and 5 ml of each sample wasused for the Real-Time PCR assay
Analysis of field samples
The same 44 water samples analysed with the standard method were also examinedwith the molecular protocol starting from 1-l aliquots filtration culture enrichmentcolumn-based DNA extraction and Real-Time PCR were carried out as describedfor artificially contaminated water samples
Statistical analysis
Accordance of results obtained by both reference and molecular methods wascalculated with DAG_Stat software (Mackinnon 2000) providing sensitivityspecificity Positive and Negative Agreement (PA and NA) indices The k statisticwas used to estimate concordance (Landis and Koch 1977)
Results
Development of a molecular method for the detection of P aeruginosa
Specificity and sensitivity of Real-Time PCR
The specificity of the Real-Time PCR assay was confirmed over a panel of 22bacterial strains of species likely to be present in water environments and four ofP aeruginosa (Table 1) Assay sensitivity showed a detection limit of 10 ge with anamplification efficiency of 105 (R2 099453) (Figure 1)
Artificially contaminated pool water samples
The application of the entire molecular procedure on pool water samples artificiallycontaminated with 10ndash103 cfu100 ml made it possible to assess the sensitivity of the
Figure 1 Real-Time PCR sensitivity and efficiency Bacterial DNA dilutions (106-1 genomeequivalents ge) of the target species were amplified as described in the Methods section Ctvalues were plotted against DNA concentrations and the slope of the linear curve was used foramplification efficiency calculation Efficiency data with respective correlation coefficients aresummarised in the inset
64 G Amagliani et al
Dow
nloa
ded
by [
Bib
liote
ca U
nive
rsita
ria]
[G
iulia
Am
aglia
ni]
at 0
717
19
Janu
ary
2012
developed protocol Column-based extraction proved to be more efficient thanmagnetic nanoparticles for the isolation of bacterial DNA from water samples witha sensitivity of 10 cfu and 103 cfu respectively Ct values were as follows Qiagen kit0 cfu100 ml tested negative 10 cfu100 ml Ct 3565 102 cfu100 ml Ct 3278103 cfu100 ml Ct 2872 Merck magnetic particles 0 cfu100 ml 10 cfu100 ml and102 cfu100 ml tested negative 103 cfu100 ml Ct 3741
Analysis of recreational water samples
One out of 17 pool water samples and 2027 inlet water samples tested positive forP aeruginosa when analysed using the reference method Contamination titersranged between 1 and 45 cfu per 100 ml A high bacterial concentration above 100cfu was detected on two occasions When the same samples were analysed usingthe molecular method positivity rates of 417 and 2027 in pool and inlet waterrespectively were found (Table 2)
Statistical analysis
Test reliability was estimated by statistical analysis of result accordance With regardto the standard method the molecular assay showed a sensitivity of 9048 and aspecificity of 7826 PA was 844 (95 CI 0730ndash0958) NA 837 (95 CI0718ndash0956) Cohenrsquos Kappa index was 06831 which indicates lsquolsquosubstantialagreementrsquorsquo according to Landis and Koch (1977) classification
RAPD fingerprinting
The efficacy of the RAPD typing scheme was shown with bacterial strains of thesame species but different origins These included two additional isolates that hadoriginally been recovered from other swimming pools (environmental isolates 1 and2) one collection strain ATCC 10145 and one strain used as quality control strainby the ASUR (CQXVIII) (Table 1) As shown in Figure 2 panel B RAPDfingerprinting allowed us to differentiate all these distinct strains of P aeruginosaWhen all the P aeruginosa isolated from water samples were tested using the sameprotocol they were found to possess identical RAPD fingerprint profiles (Figure 2panel A)
Discussion
P aeruginosa is the second most common cause of recreational disease outbreaks(Craun et al 2005) Ear infection and otitis externa caused by this microorganism inswimming pools are probably more common than reported (Guida et al 2009)P aeruginosa presence in recreational water has been investigated by few authors(Barben et al 2005 Papadopoulou et al 2008 Guida et al 2009 Nikaeen et al2009) and additional data have been provided by Mena and Gerba (2009) Howevernone of these studies made use of a PCR-based method for the detection of thisspecies
More recently Villarreal et al (2010) described culture-independent techniquesapplied to drinking water surveillance Perkins et al (2009) to shower water andLavenir et al (2007) to environmental water while the use of PCR for pathogen
International Journal of Environmental Health Research 65
Dow
nloa
ded
by [
Bib
liote
ca U
nive
rsita
ria]
[G
iulia
Am
aglia
ni]
at 0
717
19
Janu
ary
2012
Table 2 Comparison of results obtained through reference and molecular methods forP aeruginosa detection in pool and inlet water
aColony count after membrane growth on Pseudomonas Agar Base CN (see Materials and methods)
66 G Amagliani et al
Dow
nloa
ded
by [
Bib
liote
ca U
nive
rsita
ria]
[G
iulia
Am
aglia
ni]
at 0
717
19
Janu
ary
2012
detection in swimming pools has been limited to the identification of adenoviruses(Heerden et al 2005) enteric protozoa (Fournier et al 2002) and free-living amoebae(Gianinazzi et al 2009) To the best of our knowledge the application of a molecularassay for the detection of P aeruginosa in recreational water has yet to be reported
The aim of this study was the development of a new molecular method forP aeruginosa identification in recreational water and the evaluation of itsperformance in the monitoring of a swimming pool in comparison with thereference culture method (UNI EN ISO 162662008) In order to make such acomparison inlet and pool water samples were collected from a local swimming pooland examined by both systems
Results of microbiological analysis indicated higher positivity rates in inlet water(74) compared to pool water (6) with higher levels of P aeruginosa (147 vs1 cfu100 ml mean values) in accordance with values reported by Guida et al(2009) These results may be explained by the presence of biofilm in the inlet waterpipeline and are supported by RAPD typing of isolates which indicate theirepidemiological correlation A longer period of contact with chlorine and thedilution process may have led to lower levels of P aeruginosa in pool water (Guidaet al 2009) Based on criteria provided by Italian Guidelines (Italian Republic 2003)fixing acceptability limits of 1 cfu100 ml in pool water and 0 cfu100 ml of inletwater in this study only the samples of pool water proved to be in compliance
lsquolsquoSubstantial agreementrsquorsquo (Landis and Koch 1977) was found between thedeveloped molecular method and the reference protocol The higher sensitivity of themolecular approach compared to the reference method in testing pool water samplescould be ascribed to the greater starting volume used for the analysis Moreoverbacteria in the pool water may have been in a subvital state due to chlorine watertreatment and able to recover culturability after a short enrichment in liquid
Figure 2 Discrimination of P aeruginosa by RAPD typing The ability of RAPDfingerprinting with primer 272 to cluster identical isolates (Panel A) and differentiatedistinct isolates within the P aeruginosa species (Panel B) is shown M 100 bp Ladder PlusFermentas Lanes 1ndash25 P aeruginosa isolated from recreational water 26 P aeruginosaenvironmental isolate 1 27 P aeruginosa environmental isolate 2 28 CQXVIII 29P aeruginosa ATCC 10145
International Journal of Environmental Health Research 67
Dow
nloa
ded
by [
Bib
liote
ca U
nive
rsita
ria]
[G
iulia
Am
aglia
ni]
at 0
717
19
Janu
ary
2012
medium thus providing resuscitation of injured or stressed cells However it iscommonly accepted that culture-dependent methods can fail in the identification ofbacteria in VBNC state (Oliver 2005)
It has been reported that the examination of colony morphology after membranegrowth on Cetrimide agar is not conclusive for P aeruginosa identification andconfirmation tests are needed for the correct identification of presumptive positivecolonies (Casanovas-Massana et al 2010) The need for such tests was illustrated bythe interlaboratory proficiency test carried out by the Italian National Health Service(Bonadonna and Ottaviani 2007) owing to the high rate (64) of false positiveresults obtained Conversely the Real-Time PCR assay specificity previously testedby Anuj et al (2009) on 101 target and non-target isolates and confirmed with 26further target and non-target strains in the present study proved to be very highConsidering that the assay has been successfully applied for the screening ofsuspected positive colonies on Pseudomonas Agar Base CN it may constitute aviable alternative to conventional time-consuming confirmation tests with a briefReal-Time PCR assay
A limitation for the broad application of molecular assays for testingenvironmental samples stems from the presence of substances of environmentalorigin such as humic acids and other organic molecules well known as PCRinhibitors (Wilson 1997) Hence the development of a DNA isolation system able toeffectively remove such contaminating molecules is a key step In a first attemptDNA direct isolation from filter membrane was carried out although the sensitivityobtained was not sufficient to be in compliance with National standards provided forrecreational water (not shown) After introducing a short (6 h) enrichment step twodifferent DNA extraction strategies were compared silica-based spin columns andmagnetic nanoparticles However the magnetic procedure for DNA isolation wasnot able to eliminate water-derived PCR inhibitors or did not provide sufficientrecovery especially with low bacterial concentrations On the other hand thecolumn devices proved effective and appropriate in this application
The employment of Real-Time PCR for the detection of P aeruginosa in wateroffers high specificity and sensitivity levels with a reduction of analysis time Inaddition this technique has the potential to screen large numbers of environmentalsamples limiting the application of the conventional culture techniques only toPCR-positive samples Hence the method could be proposed as part of a self-monitoring plan for swimming pool facilities to evaluate the quality of water usedfor recreational purposes improving surveillance and early warning systemsAlthough the use of PCR-based methods has not been recognised by AuthoritiesEU Directive 9883EC (EU 1998) allows the application of molecular diagnosis if itsequivalence with the reference protocol is demonstrated However a validationprogramme is needed to assess the level of accordance of the described system withthe reference method in order to verify the equivalence of results required byEuropean legislation
Acknowledgements
The authors would like to thank Dr G Cappuccini Director of the Department of Preventionand Head of the Hygiene and Public Health Service ASUR ZT 2 Urbino the personnel at theswimming pool facility of the Faculty of Health and Physical Exercise University of UrbinolsquolsquoCarlo Borsquorsquo for their collaboration and Professor Timothy Bloom of the University ofUrbino for a critical reading of the manuscript
68 G Amagliani et al
Dow
nloa
ded
by [
Bib
liote
ca U
nive
rsita
ria]
[G
iulia
Am
aglia
ni]
at 0
717
19
Janu
ary
2012
References
Anuj SN Whiley DM Kidd TJ Bell SC Wainwright CE Nissen MD Sloots TP 2009Identification of Pseudomonas aeruginosa by a duplex real-time polymerase chain reactionassay targeting the ecfX and the gyrB genes Diagn Microbiol Infect Dis 63 127ndash131
Barben J Hafen G Schmid J 2005 Pseudomonas aeruginosa in public swimming pools andbathroom water of patients with cystic fibrosis J Cystic Fibros 4227ndash231
Bonadonna L Ottaviani M 2007 Reference analytical methods for water intended forhuman consumption according to the Italian Legislative Decree 312001 Microbiologicalmethods Rapporti ISTISAN 075 Rome Istituto Superiore di Sanita (National HealthService)
Casanovas-Massana A Lucena F Blanch AR 2010 Identification of Pseudomonas aeruginosain water-bottling plants on the basis of procedures included in ISO 162662006J Microbiol Meth 811ndash5
Craun GF Calderon RL Craun MF 2005 Outbreaks associated with recreational water inthe United States Int J Environ Health Res 15243ndash262
European Union (EU) 1998 Council Directive 9883EC of 3 November 1998 on the qualityof water intended for human consumption
Fournier S Dubrou S Liguory O Gaussin F Santillana-Hayat M Sarfati C Molina JMDerouin F 2002 Detection of microsporidia cryptosporidia and giardia in swimmingpools a one-year prospective study FEMS Immunol Med Microbiol 33209ndash213
Gianinazzi C Schild M Wuthrich F Muller N Schurch N Gottstein B 2009 Potentiallyhuman pathogenic Acanthamoeba isolated from a heated indoor swimming pool inSwitzerland Exp Parasitol 121180ndash186
Goeres DM Palys T Sandel BB Geiger J 2004 Evaluation of disinfectant efficacy againstbiofilm and suspended bacteria in a laboratory swimming pool model Water Res383103ndash3109
Guida M Galle F Mattei ML Anastasi D Liguori G 2009 Microbiological quality of thewater of recreational and rehabilitation pools a 2-year survey in Naples Italy PublicHealth 123448ndash451
Heerden J Ehlers MM Grabow WOK 2005 Detection and risk assessment of adenovirusesin swimming pool water J Appl Microbiol 991256ndash1264
Italian Republic 2001 Legislative Decree No 31 2 February implementing Directive 9883EC on the quality of water intended for human consumption Official Gazette No 52 of3rd March 2001 Rome
Italian Republic 2002 Legislative Decree No 27 2 February amending and supplementingLegislative Decree No 31 of 2 February 2001 implementing Directive 9883EC on thequality of water intended for human consumption Official Gazette No 58 of 9th of March2002 Rome
Italian Republic 2003 Permanent Council State-Regions and Autonomous ProvincesAccordo Stato ndash Regioni e Province Autonome 16-1-2003 Official Gazette No 51 of 3rdof March 2003 Rome
Landis JR Koch GG 1977 The measurement of observer agreement for categorical dataBiometrics 33159ndash174
Lanotte P Watt S Mereghetti L Dartiguelongue N Rastegar-Lari A Goudeau A QuentinR 2004 Genetic features of Pseudomonas aeruginosa isolates from cystic fibrosis patientscompared with those of isolates from other origins J Med Microbiol 5373ndash81
Lavenir R Jocktane D Laurent F Nazaret S Cournoyer B 2007 Improved reliability ofPseudomonas aeruginosa PCR detection by the use of the species-specific ecfX gene targetJ Microbiol Meth 7020ndash29
Leoni E Legnani P Guberti E Masotti A 1999 Risk of infection associated withmicrobiological quality of public swimming pools in Bologna Italy Public Health113227ndash232
Mackinnon A 2000 A spreadsheet for the calculation of comprehensive statistics for theassessment of diagnostic tests and inter-rater agreement Comput Biol Med 30127ndash134
Mena KD Gerba CP 2009 Risk assessment of Pseudomonas aeruginosa in water RevEnviron Contam Toxicol 20171ndash115
Moore JE Heaney N Millar BC Crowe M Elborn JS 2002 Incidence of Pseudomonasaeruginosa in recreational and hydrotherapy pools Commun Dis Public Health 523ndash26
International Journal of Environmental Health Research 69
Dow
nloa
ded
by [
Bib
liote
ca U
nive
rsita
ria]
[G
iulia
Am
aglia
ni]
at 0
717
19
Janu
ary
2012
Nikaeen M Hatamzadeh M Vahid Dastjerdi M Hassanzadeh A 2009 Predictive indicatorsof the safety of swimming pool waters Water Sci Technol 603101ndash3107
Oliver JD 2005 The viable but nonculturable state in bacteria J Microbiol 4393ndash100Papadopoulou C Economou V Sakkas H Gousia P Giannakopoulos X Dontorou C
Filioussis G Gessouli H Karanis P Leveidiotou S 2008 Microbiological quality ofindoor and outdoor swimming pools in Greece investigation of the antibiotic resistanceof the bacterial isolates Int J Hyg Environ Health 211385ndash397
Perkins SD Mayfield J Fraser V Angenent LT 2009 Potentially pathogenic bacteria inshower water and air of a stem cell transplant unit Appl Environ Microbiol 755363ndash5372
Pond K 2005 Water recreation and disease Plausibility of associated infections acute effectssequelae and mortality London WHO IWA Publishing
UNI EN ISO 162662008 ndash 18092008 ndashWater quality ndash Detection and enumeration ofPseudomonas aeruginosa ndash Method by membrane filtration
Villarreal JV Schwartz T Obst U 2010 Culture-independent techniques applied to foodindustry water surveillance ndash a case study Int J Food Microbiol 141(Suppl 1)S147ndash55
Wang MC Liu CY Shiao AS Wang T 2005 Ear problems in swimmers J Chin Med Assoc68347ndash352
World Health Organisation (WHO) 2006 Guidelines for safe recreational water environ-ments Vol 2 Swimming pools and similar environments Geneva WHO Press
Wilson IG 1997 Inhibition and facilitation of nucleic acid amplification Appl EnvironMicrobiol 633741ndash3751
70 G Amagliani et al
Dow
nloa
ded
by [
Bib
liote
ca U
nive
rsita
ria]
[G
iulia
Am
aglia
ni]
at 0
717
19
Janu
ary
2012
genomic units based on bacterial genome size at httpwwwncbinlmgovgenomeslprokscgi DNA was diluted in dH2O and amplified in the range 106-1 genomicequivalents (ge) per PCR in triplicate Efficiency was calculated by plotting Ct(threshold cycle) values against log of genome copy number and using the slope ofthe resulting curve in the following equation
Efficiency frac14 101=slope 1
Artificially contaminated pool water samples
Preliminary tests to assess method sensitivity were carried out on artificiallycontaminated pool water samples Neutralised pool water was contaminated withvarious concentrations of P aeruginosa as follows P aeruginosa ATCC 10145 wasgrown in Brain Heart Infusion (BHI Oxoid) at 378C to an A600 of 018corresponding to 108 cfu ml71 The bacterial suspension was serially diluted and1-ml aliquots used for water contamination in the range 10ndash103 cfu100 ml Bacterialtitre was confirmed by plating 100 ml of each dilution on Pseudomonas AgarBase CN Water samples were filtered as described above and each membrane wasincubated for culture-enrichment in 20 ml of Pseudomonas Selective Brothsupplemented with 1 glycerol (Biolife Milan Italy) at 378C in agitation After
Table 1 Bacterial strains used for Real-Time PCR specificity test and corresponding results
International Journal of Environmental Health Research 63
Dow
nloa
ded
by [
Bib
liote
ca U
nive
rsita
ria]
[G
iulia
Am
aglia
ni]
at 0
717
19
Janu
ary
2012
6 h membranes were removed and the cultures were centrifuged at 6000 g for 20min Total DNA was extracted from the bacterial pellets either through a column-based kit (DNeasy Blood and Tissue Kit) or by magnetic extraction (MagPrep HSMerck Darmstadt Germany) with 1 mg nanoparticles per sample according tomanufacturer protocol under mild acidic conditions and 5 ml of each sample wasused for the Real-Time PCR assay
Analysis of field samples
The same 44 water samples analysed with the standard method were also examinedwith the molecular protocol starting from 1-l aliquots filtration culture enrichmentcolumn-based DNA extraction and Real-Time PCR were carried out as describedfor artificially contaminated water samples
Statistical analysis
Accordance of results obtained by both reference and molecular methods wascalculated with DAG_Stat software (Mackinnon 2000) providing sensitivityspecificity Positive and Negative Agreement (PA and NA) indices The k statisticwas used to estimate concordance (Landis and Koch 1977)
Results
Development of a molecular method for the detection of P aeruginosa
Specificity and sensitivity of Real-Time PCR
The specificity of the Real-Time PCR assay was confirmed over a panel of 22bacterial strains of species likely to be present in water environments and four ofP aeruginosa (Table 1) Assay sensitivity showed a detection limit of 10 ge with anamplification efficiency of 105 (R2 099453) (Figure 1)
Artificially contaminated pool water samples
The application of the entire molecular procedure on pool water samples artificiallycontaminated with 10ndash103 cfu100 ml made it possible to assess the sensitivity of the
Figure 1 Real-Time PCR sensitivity and efficiency Bacterial DNA dilutions (106-1 genomeequivalents ge) of the target species were amplified as described in the Methods section Ctvalues were plotted against DNA concentrations and the slope of the linear curve was used foramplification efficiency calculation Efficiency data with respective correlation coefficients aresummarised in the inset
64 G Amagliani et al
Dow
nloa
ded
by [
Bib
liote
ca U
nive
rsita
ria]
[G
iulia
Am
aglia
ni]
at 0
717
19
Janu
ary
2012
developed protocol Column-based extraction proved to be more efficient thanmagnetic nanoparticles for the isolation of bacterial DNA from water samples witha sensitivity of 10 cfu and 103 cfu respectively Ct values were as follows Qiagen kit0 cfu100 ml tested negative 10 cfu100 ml Ct 3565 102 cfu100 ml Ct 3278103 cfu100 ml Ct 2872 Merck magnetic particles 0 cfu100 ml 10 cfu100 ml and102 cfu100 ml tested negative 103 cfu100 ml Ct 3741
Analysis of recreational water samples
One out of 17 pool water samples and 2027 inlet water samples tested positive forP aeruginosa when analysed using the reference method Contamination titersranged between 1 and 45 cfu per 100 ml A high bacterial concentration above 100cfu was detected on two occasions When the same samples were analysed usingthe molecular method positivity rates of 417 and 2027 in pool and inlet waterrespectively were found (Table 2)
Statistical analysis
Test reliability was estimated by statistical analysis of result accordance With regardto the standard method the molecular assay showed a sensitivity of 9048 and aspecificity of 7826 PA was 844 (95 CI 0730ndash0958) NA 837 (95 CI0718ndash0956) Cohenrsquos Kappa index was 06831 which indicates lsquolsquosubstantialagreementrsquorsquo according to Landis and Koch (1977) classification
RAPD fingerprinting
The efficacy of the RAPD typing scheme was shown with bacterial strains of thesame species but different origins These included two additional isolates that hadoriginally been recovered from other swimming pools (environmental isolates 1 and2) one collection strain ATCC 10145 and one strain used as quality control strainby the ASUR (CQXVIII) (Table 1) As shown in Figure 2 panel B RAPDfingerprinting allowed us to differentiate all these distinct strains of P aeruginosaWhen all the P aeruginosa isolated from water samples were tested using the sameprotocol they were found to possess identical RAPD fingerprint profiles (Figure 2panel A)
Discussion
P aeruginosa is the second most common cause of recreational disease outbreaks(Craun et al 2005) Ear infection and otitis externa caused by this microorganism inswimming pools are probably more common than reported (Guida et al 2009)P aeruginosa presence in recreational water has been investigated by few authors(Barben et al 2005 Papadopoulou et al 2008 Guida et al 2009 Nikaeen et al2009) and additional data have been provided by Mena and Gerba (2009) Howevernone of these studies made use of a PCR-based method for the detection of thisspecies
More recently Villarreal et al (2010) described culture-independent techniquesapplied to drinking water surveillance Perkins et al (2009) to shower water andLavenir et al (2007) to environmental water while the use of PCR for pathogen
International Journal of Environmental Health Research 65
Dow
nloa
ded
by [
Bib
liote
ca U
nive
rsita
ria]
[G
iulia
Am
aglia
ni]
at 0
717
19
Janu
ary
2012
Table 2 Comparison of results obtained through reference and molecular methods forP aeruginosa detection in pool and inlet water
aColony count after membrane growth on Pseudomonas Agar Base CN (see Materials and methods)
66 G Amagliani et al
Dow
nloa
ded
by [
Bib
liote
ca U
nive
rsita
ria]
[G
iulia
Am
aglia
ni]
at 0
717
19
Janu
ary
2012
detection in swimming pools has been limited to the identification of adenoviruses(Heerden et al 2005) enteric protozoa (Fournier et al 2002) and free-living amoebae(Gianinazzi et al 2009) To the best of our knowledge the application of a molecularassay for the detection of P aeruginosa in recreational water has yet to be reported
The aim of this study was the development of a new molecular method forP aeruginosa identification in recreational water and the evaluation of itsperformance in the monitoring of a swimming pool in comparison with thereference culture method (UNI EN ISO 162662008) In order to make such acomparison inlet and pool water samples were collected from a local swimming pooland examined by both systems
Results of microbiological analysis indicated higher positivity rates in inlet water(74) compared to pool water (6) with higher levels of P aeruginosa (147 vs1 cfu100 ml mean values) in accordance with values reported by Guida et al(2009) These results may be explained by the presence of biofilm in the inlet waterpipeline and are supported by RAPD typing of isolates which indicate theirepidemiological correlation A longer period of contact with chlorine and thedilution process may have led to lower levels of P aeruginosa in pool water (Guidaet al 2009) Based on criteria provided by Italian Guidelines (Italian Republic 2003)fixing acceptability limits of 1 cfu100 ml in pool water and 0 cfu100 ml of inletwater in this study only the samples of pool water proved to be in compliance
lsquolsquoSubstantial agreementrsquorsquo (Landis and Koch 1977) was found between thedeveloped molecular method and the reference protocol The higher sensitivity of themolecular approach compared to the reference method in testing pool water samplescould be ascribed to the greater starting volume used for the analysis Moreoverbacteria in the pool water may have been in a subvital state due to chlorine watertreatment and able to recover culturability after a short enrichment in liquid
Figure 2 Discrimination of P aeruginosa by RAPD typing The ability of RAPDfingerprinting with primer 272 to cluster identical isolates (Panel A) and differentiatedistinct isolates within the P aeruginosa species (Panel B) is shown M 100 bp Ladder PlusFermentas Lanes 1ndash25 P aeruginosa isolated from recreational water 26 P aeruginosaenvironmental isolate 1 27 P aeruginosa environmental isolate 2 28 CQXVIII 29P aeruginosa ATCC 10145
International Journal of Environmental Health Research 67
Dow
nloa
ded
by [
Bib
liote
ca U
nive
rsita
ria]
[G
iulia
Am
aglia
ni]
at 0
717
19
Janu
ary
2012
medium thus providing resuscitation of injured or stressed cells However it iscommonly accepted that culture-dependent methods can fail in the identification ofbacteria in VBNC state (Oliver 2005)
It has been reported that the examination of colony morphology after membranegrowth on Cetrimide agar is not conclusive for P aeruginosa identification andconfirmation tests are needed for the correct identification of presumptive positivecolonies (Casanovas-Massana et al 2010) The need for such tests was illustrated bythe interlaboratory proficiency test carried out by the Italian National Health Service(Bonadonna and Ottaviani 2007) owing to the high rate (64) of false positiveresults obtained Conversely the Real-Time PCR assay specificity previously testedby Anuj et al (2009) on 101 target and non-target isolates and confirmed with 26further target and non-target strains in the present study proved to be very highConsidering that the assay has been successfully applied for the screening ofsuspected positive colonies on Pseudomonas Agar Base CN it may constitute aviable alternative to conventional time-consuming confirmation tests with a briefReal-Time PCR assay
A limitation for the broad application of molecular assays for testingenvironmental samples stems from the presence of substances of environmentalorigin such as humic acids and other organic molecules well known as PCRinhibitors (Wilson 1997) Hence the development of a DNA isolation system able toeffectively remove such contaminating molecules is a key step In a first attemptDNA direct isolation from filter membrane was carried out although the sensitivityobtained was not sufficient to be in compliance with National standards provided forrecreational water (not shown) After introducing a short (6 h) enrichment step twodifferent DNA extraction strategies were compared silica-based spin columns andmagnetic nanoparticles However the magnetic procedure for DNA isolation wasnot able to eliminate water-derived PCR inhibitors or did not provide sufficientrecovery especially with low bacterial concentrations On the other hand thecolumn devices proved effective and appropriate in this application
The employment of Real-Time PCR for the detection of P aeruginosa in wateroffers high specificity and sensitivity levels with a reduction of analysis time Inaddition this technique has the potential to screen large numbers of environmentalsamples limiting the application of the conventional culture techniques only toPCR-positive samples Hence the method could be proposed as part of a self-monitoring plan for swimming pool facilities to evaluate the quality of water usedfor recreational purposes improving surveillance and early warning systemsAlthough the use of PCR-based methods has not been recognised by AuthoritiesEU Directive 9883EC (EU 1998) allows the application of molecular diagnosis if itsequivalence with the reference protocol is demonstrated However a validationprogramme is needed to assess the level of accordance of the described system withthe reference method in order to verify the equivalence of results required byEuropean legislation
Acknowledgements
The authors would like to thank Dr G Cappuccini Director of the Department of Preventionand Head of the Hygiene and Public Health Service ASUR ZT 2 Urbino the personnel at theswimming pool facility of the Faculty of Health and Physical Exercise University of UrbinolsquolsquoCarlo Borsquorsquo for their collaboration and Professor Timothy Bloom of the University ofUrbino for a critical reading of the manuscript
68 G Amagliani et al
Dow
nloa
ded
by [
Bib
liote
ca U
nive
rsita
ria]
[G
iulia
Am
aglia
ni]
at 0
717
19
Janu
ary
2012
References
Anuj SN Whiley DM Kidd TJ Bell SC Wainwright CE Nissen MD Sloots TP 2009Identification of Pseudomonas aeruginosa by a duplex real-time polymerase chain reactionassay targeting the ecfX and the gyrB genes Diagn Microbiol Infect Dis 63 127ndash131
Barben J Hafen G Schmid J 2005 Pseudomonas aeruginosa in public swimming pools andbathroom water of patients with cystic fibrosis J Cystic Fibros 4227ndash231
Bonadonna L Ottaviani M 2007 Reference analytical methods for water intended forhuman consumption according to the Italian Legislative Decree 312001 Microbiologicalmethods Rapporti ISTISAN 075 Rome Istituto Superiore di Sanita (National HealthService)
Casanovas-Massana A Lucena F Blanch AR 2010 Identification of Pseudomonas aeruginosain water-bottling plants on the basis of procedures included in ISO 162662006J Microbiol Meth 811ndash5
Craun GF Calderon RL Craun MF 2005 Outbreaks associated with recreational water inthe United States Int J Environ Health Res 15243ndash262
European Union (EU) 1998 Council Directive 9883EC of 3 November 1998 on the qualityof water intended for human consumption
Fournier S Dubrou S Liguory O Gaussin F Santillana-Hayat M Sarfati C Molina JMDerouin F 2002 Detection of microsporidia cryptosporidia and giardia in swimmingpools a one-year prospective study FEMS Immunol Med Microbiol 33209ndash213
Gianinazzi C Schild M Wuthrich F Muller N Schurch N Gottstein B 2009 Potentiallyhuman pathogenic Acanthamoeba isolated from a heated indoor swimming pool inSwitzerland Exp Parasitol 121180ndash186
Goeres DM Palys T Sandel BB Geiger J 2004 Evaluation of disinfectant efficacy againstbiofilm and suspended bacteria in a laboratory swimming pool model Water Res383103ndash3109
Guida M Galle F Mattei ML Anastasi D Liguori G 2009 Microbiological quality of thewater of recreational and rehabilitation pools a 2-year survey in Naples Italy PublicHealth 123448ndash451
Heerden J Ehlers MM Grabow WOK 2005 Detection and risk assessment of adenovirusesin swimming pool water J Appl Microbiol 991256ndash1264
Italian Republic 2001 Legislative Decree No 31 2 February implementing Directive 9883EC on the quality of water intended for human consumption Official Gazette No 52 of3rd March 2001 Rome
Italian Republic 2002 Legislative Decree No 27 2 February amending and supplementingLegislative Decree No 31 of 2 February 2001 implementing Directive 9883EC on thequality of water intended for human consumption Official Gazette No 58 of 9th of March2002 Rome
Italian Republic 2003 Permanent Council State-Regions and Autonomous ProvincesAccordo Stato ndash Regioni e Province Autonome 16-1-2003 Official Gazette No 51 of 3rdof March 2003 Rome
Landis JR Koch GG 1977 The measurement of observer agreement for categorical dataBiometrics 33159ndash174
Lanotte P Watt S Mereghetti L Dartiguelongue N Rastegar-Lari A Goudeau A QuentinR 2004 Genetic features of Pseudomonas aeruginosa isolates from cystic fibrosis patientscompared with those of isolates from other origins J Med Microbiol 5373ndash81
Lavenir R Jocktane D Laurent F Nazaret S Cournoyer B 2007 Improved reliability ofPseudomonas aeruginosa PCR detection by the use of the species-specific ecfX gene targetJ Microbiol Meth 7020ndash29
Leoni E Legnani P Guberti E Masotti A 1999 Risk of infection associated withmicrobiological quality of public swimming pools in Bologna Italy Public Health113227ndash232
Mackinnon A 2000 A spreadsheet for the calculation of comprehensive statistics for theassessment of diagnostic tests and inter-rater agreement Comput Biol Med 30127ndash134
Mena KD Gerba CP 2009 Risk assessment of Pseudomonas aeruginosa in water RevEnviron Contam Toxicol 20171ndash115
Moore JE Heaney N Millar BC Crowe M Elborn JS 2002 Incidence of Pseudomonasaeruginosa in recreational and hydrotherapy pools Commun Dis Public Health 523ndash26
International Journal of Environmental Health Research 69
Dow
nloa
ded
by [
Bib
liote
ca U
nive
rsita
ria]
[G
iulia
Am
aglia
ni]
at 0
717
19
Janu
ary
2012
Nikaeen M Hatamzadeh M Vahid Dastjerdi M Hassanzadeh A 2009 Predictive indicatorsof the safety of swimming pool waters Water Sci Technol 603101ndash3107
Oliver JD 2005 The viable but nonculturable state in bacteria J Microbiol 4393ndash100Papadopoulou C Economou V Sakkas H Gousia P Giannakopoulos X Dontorou C
Filioussis G Gessouli H Karanis P Leveidiotou S 2008 Microbiological quality ofindoor and outdoor swimming pools in Greece investigation of the antibiotic resistanceof the bacterial isolates Int J Hyg Environ Health 211385ndash397
Perkins SD Mayfield J Fraser V Angenent LT 2009 Potentially pathogenic bacteria inshower water and air of a stem cell transplant unit Appl Environ Microbiol 755363ndash5372
Pond K 2005 Water recreation and disease Plausibility of associated infections acute effectssequelae and mortality London WHO IWA Publishing
UNI EN ISO 162662008 ndash 18092008 ndashWater quality ndash Detection and enumeration ofPseudomonas aeruginosa ndash Method by membrane filtration
Villarreal JV Schwartz T Obst U 2010 Culture-independent techniques applied to foodindustry water surveillance ndash a case study Int J Food Microbiol 141(Suppl 1)S147ndash55
Wang MC Liu CY Shiao AS Wang T 2005 Ear problems in swimmers J Chin Med Assoc68347ndash352
World Health Organisation (WHO) 2006 Guidelines for safe recreational water environ-ments Vol 2 Swimming pools and similar environments Geneva WHO Press
Wilson IG 1997 Inhibition and facilitation of nucleic acid amplification Appl EnvironMicrobiol 633741ndash3751
70 G Amagliani et al
Dow
nloa
ded
by [
Bib
liote
ca U
nive
rsita
ria]
[G
iulia
Am
aglia
ni]
at 0
717
19
Janu
ary
2012
6 h membranes were removed and the cultures were centrifuged at 6000 g for 20min Total DNA was extracted from the bacterial pellets either through a column-based kit (DNeasy Blood and Tissue Kit) or by magnetic extraction (MagPrep HSMerck Darmstadt Germany) with 1 mg nanoparticles per sample according tomanufacturer protocol under mild acidic conditions and 5 ml of each sample wasused for the Real-Time PCR assay
Analysis of field samples
The same 44 water samples analysed with the standard method were also examinedwith the molecular protocol starting from 1-l aliquots filtration culture enrichmentcolumn-based DNA extraction and Real-Time PCR were carried out as describedfor artificially contaminated water samples
Statistical analysis
Accordance of results obtained by both reference and molecular methods wascalculated with DAG_Stat software (Mackinnon 2000) providing sensitivityspecificity Positive and Negative Agreement (PA and NA) indices The k statisticwas used to estimate concordance (Landis and Koch 1977)
Results
Development of a molecular method for the detection of P aeruginosa
Specificity and sensitivity of Real-Time PCR
The specificity of the Real-Time PCR assay was confirmed over a panel of 22bacterial strains of species likely to be present in water environments and four ofP aeruginosa (Table 1) Assay sensitivity showed a detection limit of 10 ge with anamplification efficiency of 105 (R2 099453) (Figure 1)
Artificially contaminated pool water samples
The application of the entire molecular procedure on pool water samples artificiallycontaminated with 10ndash103 cfu100 ml made it possible to assess the sensitivity of the
Figure 1 Real-Time PCR sensitivity and efficiency Bacterial DNA dilutions (106-1 genomeequivalents ge) of the target species were amplified as described in the Methods section Ctvalues were plotted against DNA concentrations and the slope of the linear curve was used foramplification efficiency calculation Efficiency data with respective correlation coefficients aresummarised in the inset
64 G Amagliani et al
Dow
nloa
ded
by [
Bib
liote
ca U
nive
rsita
ria]
[G
iulia
Am
aglia
ni]
at 0
717
19
Janu
ary
2012
developed protocol Column-based extraction proved to be more efficient thanmagnetic nanoparticles for the isolation of bacterial DNA from water samples witha sensitivity of 10 cfu and 103 cfu respectively Ct values were as follows Qiagen kit0 cfu100 ml tested negative 10 cfu100 ml Ct 3565 102 cfu100 ml Ct 3278103 cfu100 ml Ct 2872 Merck magnetic particles 0 cfu100 ml 10 cfu100 ml and102 cfu100 ml tested negative 103 cfu100 ml Ct 3741
Analysis of recreational water samples
One out of 17 pool water samples and 2027 inlet water samples tested positive forP aeruginosa when analysed using the reference method Contamination titersranged between 1 and 45 cfu per 100 ml A high bacterial concentration above 100cfu was detected on two occasions When the same samples were analysed usingthe molecular method positivity rates of 417 and 2027 in pool and inlet waterrespectively were found (Table 2)
Statistical analysis
Test reliability was estimated by statistical analysis of result accordance With regardto the standard method the molecular assay showed a sensitivity of 9048 and aspecificity of 7826 PA was 844 (95 CI 0730ndash0958) NA 837 (95 CI0718ndash0956) Cohenrsquos Kappa index was 06831 which indicates lsquolsquosubstantialagreementrsquorsquo according to Landis and Koch (1977) classification
RAPD fingerprinting
The efficacy of the RAPD typing scheme was shown with bacterial strains of thesame species but different origins These included two additional isolates that hadoriginally been recovered from other swimming pools (environmental isolates 1 and2) one collection strain ATCC 10145 and one strain used as quality control strainby the ASUR (CQXVIII) (Table 1) As shown in Figure 2 panel B RAPDfingerprinting allowed us to differentiate all these distinct strains of P aeruginosaWhen all the P aeruginosa isolated from water samples were tested using the sameprotocol they were found to possess identical RAPD fingerprint profiles (Figure 2panel A)
Discussion
P aeruginosa is the second most common cause of recreational disease outbreaks(Craun et al 2005) Ear infection and otitis externa caused by this microorganism inswimming pools are probably more common than reported (Guida et al 2009)P aeruginosa presence in recreational water has been investigated by few authors(Barben et al 2005 Papadopoulou et al 2008 Guida et al 2009 Nikaeen et al2009) and additional data have been provided by Mena and Gerba (2009) Howevernone of these studies made use of a PCR-based method for the detection of thisspecies
More recently Villarreal et al (2010) described culture-independent techniquesapplied to drinking water surveillance Perkins et al (2009) to shower water andLavenir et al (2007) to environmental water while the use of PCR for pathogen
International Journal of Environmental Health Research 65
Dow
nloa
ded
by [
Bib
liote
ca U
nive
rsita
ria]
[G
iulia
Am
aglia
ni]
at 0
717
19
Janu
ary
2012
Table 2 Comparison of results obtained through reference and molecular methods forP aeruginosa detection in pool and inlet water
aColony count after membrane growth on Pseudomonas Agar Base CN (see Materials and methods)
66 G Amagliani et al
Dow
nloa
ded
by [
Bib
liote
ca U
nive
rsita
ria]
[G
iulia
Am
aglia
ni]
at 0
717
19
Janu
ary
2012
detection in swimming pools has been limited to the identification of adenoviruses(Heerden et al 2005) enteric protozoa (Fournier et al 2002) and free-living amoebae(Gianinazzi et al 2009) To the best of our knowledge the application of a molecularassay for the detection of P aeruginosa in recreational water has yet to be reported
The aim of this study was the development of a new molecular method forP aeruginosa identification in recreational water and the evaluation of itsperformance in the monitoring of a swimming pool in comparison with thereference culture method (UNI EN ISO 162662008) In order to make such acomparison inlet and pool water samples were collected from a local swimming pooland examined by both systems
Results of microbiological analysis indicated higher positivity rates in inlet water(74) compared to pool water (6) with higher levels of P aeruginosa (147 vs1 cfu100 ml mean values) in accordance with values reported by Guida et al(2009) These results may be explained by the presence of biofilm in the inlet waterpipeline and are supported by RAPD typing of isolates which indicate theirepidemiological correlation A longer period of contact with chlorine and thedilution process may have led to lower levels of P aeruginosa in pool water (Guidaet al 2009) Based on criteria provided by Italian Guidelines (Italian Republic 2003)fixing acceptability limits of 1 cfu100 ml in pool water and 0 cfu100 ml of inletwater in this study only the samples of pool water proved to be in compliance
lsquolsquoSubstantial agreementrsquorsquo (Landis and Koch 1977) was found between thedeveloped molecular method and the reference protocol The higher sensitivity of themolecular approach compared to the reference method in testing pool water samplescould be ascribed to the greater starting volume used for the analysis Moreoverbacteria in the pool water may have been in a subvital state due to chlorine watertreatment and able to recover culturability after a short enrichment in liquid
Figure 2 Discrimination of P aeruginosa by RAPD typing The ability of RAPDfingerprinting with primer 272 to cluster identical isolates (Panel A) and differentiatedistinct isolates within the P aeruginosa species (Panel B) is shown M 100 bp Ladder PlusFermentas Lanes 1ndash25 P aeruginosa isolated from recreational water 26 P aeruginosaenvironmental isolate 1 27 P aeruginosa environmental isolate 2 28 CQXVIII 29P aeruginosa ATCC 10145
International Journal of Environmental Health Research 67
Dow
nloa
ded
by [
Bib
liote
ca U
nive
rsita
ria]
[G
iulia
Am
aglia
ni]
at 0
717
19
Janu
ary
2012
medium thus providing resuscitation of injured or stressed cells However it iscommonly accepted that culture-dependent methods can fail in the identification ofbacteria in VBNC state (Oliver 2005)
It has been reported that the examination of colony morphology after membranegrowth on Cetrimide agar is not conclusive for P aeruginosa identification andconfirmation tests are needed for the correct identification of presumptive positivecolonies (Casanovas-Massana et al 2010) The need for such tests was illustrated bythe interlaboratory proficiency test carried out by the Italian National Health Service(Bonadonna and Ottaviani 2007) owing to the high rate (64) of false positiveresults obtained Conversely the Real-Time PCR assay specificity previously testedby Anuj et al (2009) on 101 target and non-target isolates and confirmed with 26further target and non-target strains in the present study proved to be very highConsidering that the assay has been successfully applied for the screening ofsuspected positive colonies on Pseudomonas Agar Base CN it may constitute aviable alternative to conventional time-consuming confirmation tests with a briefReal-Time PCR assay
A limitation for the broad application of molecular assays for testingenvironmental samples stems from the presence of substances of environmentalorigin such as humic acids and other organic molecules well known as PCRinhibitors (Wilson 1997) Hence the development of a DNA isolation system able toeffectively remove such contaminating molecules is a key step In a first attemptDNA direct isolation from filter membrane was carried out although the sensitivityobtained was not sufficient to be in compliance with National standards provided forrecreational water (not shown) After introducing a short (6 h) enrichment step twodifferent DNA extraction strategies were compared silica-based spin columns andmagnetic nanoparticles However the magnetic procedure for DNA isolation wasnot able to eliminate water-derived PCR inhibitors or did not provide sufficientrecovery especially with low bacterial concentrations On the other hand thecolumn devices proved effective and appropriate in this application
The employment of Real-Time PCR for the detection of P aeruginosa in wateroffers high specificity and sensitivity levels with a reduction of analysis time Inaddition this technique has the potential to screen large numbers of environmentalsamples limiting the application of the conventional culture techniques only toPCR-positive samples Hence the method could be proposed as part of a self-monitoring plan for swimming pool facilities to evaluate the quality of water usedfor recreational purposes improving surveillance and early warning systemsAlthough the use of PCR-based methods has not been recognised by AuthoritiesEU Directive 9883EC (EU 1998) allows the application of molecular diagnosis if itsequivalence with the reference protocol is demonstrated However a validationprogramme is needed to assess the level of accordance of the described system withthe reference method in order to verify the equivalence of results required byEuropean legislation
Acknowledgements
The authors would like to thank Dr G Cappuccini Director of the Department of Preventionand Head of the Hygiene and Public Health Service ASUR ZT 2 Urbino the personnel at theswimming pool facility of the Faculty of Health and Physical Exercise University of UrbinolsquolsquoCarlo Borsquorsquo for their collaboration and Professor Timothy Bloom of the University ofUrbino for a critical reading of the manuscript
68 G Amagliani et al
Dow
nloa
ded
by [
Bib
liote
ca U
nive
rsita
ria]
[G
iulia
Am
aglia
ni]
at 0
717
19
Janu
ary
2012
References
Anuj SN Whiley DM Kidd TJ Bell SC Wainwright CE Nissen MD Sloots TP 2009Identification of Pseudomonas aeruginosa by a duplex real-time polymerase chain reactionassay targeting the ecfX and the gyrB genes Diagn Microbiol Infect Dis 63 127ndash131
Barben J Hafen G Schmid J 2005 Pseudomonas aeruginosa in public swimming pools andbathroom water of patients with cystic fibrosis J Cystic Fibros 4227ndash231
Bonadonna L Ottaviani M 2007 Reference analytical methods for water intended forhuman consumption according to the Italian Legislative Decree 312001 Microbiologicalmethods Rapporti ISTISAN 075 Rome Istituto Superiore di Sanita (National HealthService)
Casanovas-Massana A Lucena F Blanch AR 2010 Identification of Pseudomonas aeruginosain water-bottling plants on the basis of procedures included in ISO 162662006J Microbiol Meth 811ndash5
Craun GF Calderon RL Craun MF 2005 Outbreaks associated with recreational water inthe United States Int J Environ Health Res 15243ndash262
European Union (EU) 1998 Council Directive 9883EC of 3 November 1998 on the qualityof water intended for human consumption
Fournier S Dubrou S Liguory O Gaussin F Santillana-Hayat M Sarfati C Molina JMDerouin F 2002 Detection of microsporidia cryptosporidia and giardia in swimmingpools a one-year prospective study FEMS Immunol Med Microbiol 33209ndash213
Gianinazzi C Schild M Wuthrich F Muller N Schurch N Gottstein B 2009 Potentiallyhuman pathogenic Acanthamoeba isolated from a heated indoor swimming pool inSwitzerland Exp Parasitol 121180ndash186
Goeres DM Palys T Sandel BB Geiger J 2004 Evaluation of disinfectant efficacy againstbiofilm and suspended bacteria in a laboratory swimming pool model Water Res383103ndash3109
Guida M Galle F Mattei ML Anastasi D Liguori G 2009 Microbiological quality of thewater of recreational and rehabilitation pools a 2-year survey in Naples Italy PublicHealth 123448ndash451
Heerden J Ehlers MM Grabow WOK 2005 Detection and risk assessment of adenovirusesin swimming pool water J Appl Microbiol 991256ndash1264
Italian Republic 2001 Legislative Decree No 31 2 February implementing Directive 9883EC on the quality of water intended for human consumption Official Gazette No 52 of3rd March 2001 Rome
Italian Republic 2002 Legislative Decree No 27 2 February amending and supplementingLegislative Decree No 31 of 2 February 2001 implementing Directive 9883EC on thequality of water intended for human consumption Official Gazette No 58 of 9th of March2002 Rome
Italian Republic 2003 Permanent Council State-Regions and Autonomous ProvincesAccordo Stato ndash Regioni e Province Autonome 16-1-2003 Official Gazette No 51 of 3rdof March 2003 Rome
Landis JR Koch GG 1977 The measurement of observer agreement for categorical dataBiometrics 33159ndash174
Lanotte P Watt S Mereghetti L Dartiguelongue N Rastegar-Lari A Goudeau A QuentinR 2004 Genetic features of Pseudomonas aeruginosa isolates from cystic fibrosis patientscompared with those of isolates from other origins J Med Microbiol 5373ndash81
Lavenir R Jocktane D Laurent F Nazaret S Cournoyer B 2007 Improved reliability ofPseudomonas aeruginosa PCR detection by the use of the species-specific ecfX gene targetJ Microbiol Meth 7020ndash29
Leoni E Legnani P Guberti E Masotti A 1999 Risk of infection associated withmicrobiological quality of public swimming pools in Bologna Italy Public Health113227ndash232
Mackinnon A 2000 A spreadsheet for the calculation of comprehensive statistics for theassessment of diagnostic tests and inter-rater agreement Comput Biol Med 30127ndash134
Mena KD Gerba CP 2009 Risk assessment of Pseudomonas aeruginosa in water RevEnviron Contam Toxicol 20171ndash115
Moore JE Heaney N Millar BC Crowe M Elborn JS 2002 Incidence of Pseudomonasaeruginosa in recreational and hydrotherapy pools Commun Dis Public Health 523ndash26
International Journal of Environmental Health Research 69
Dow
nloa
ded
by [
Bib
liote
ca U
nive
rsita
ria]
[G
iulia
Am
aglia
ni]
at 0
717
19
Janu
ary
2012
Nikaeen M Hatamzadeh M Vahid Dastjerdi M Hassanzadeh A 2009 Predictive indicatorsof the safety of swimming pool waters Water Sci Technol 603101ndash3107
Oliver JD 2005 The viable but nonculturable state in bacteria J Microbiol 4393ndash100Papadopoulou C Economou V Sakkas H Gousia P Giannakopoulos X Dontorou C
Filioussis G Gessouli H Karanis P Leveidiotou S 2008 Microbiological quality ofindoor and outdoor swimming pools in Greece investigation of the antibiotic resistanceof the bacterial isolates Int J Hyg Environ Health 211385ndash397
Perkins SD Mayfield J Fraser V Angenent LT 2009 Potentially pathogenic bacteria inshower water and air of a stem cell transplant unit Appl Environ Microbiol 755363ndash5372
Pond K 2005 Water recreation and disease Plausibility of associated infections acute effectssequelae and mortality London WHO IWA Publishing
UNI EN ISO 162662008 ndash 18092008 ndashWater quality ndash Detection and enumeration ofPseudomonas aeruginosa ndash Method by membrane filtration
Villarreal JV Schwartz T Obst U 2010 Culture-independent techniques applied to foodindustry water surveillance ndash a case study Int J Food Microbiol 141(Suppl 1)S147ndash55
Wang MC Liu CY Shiao AS Wang T 2005 Ear problems in swimmers J Chin Med Assoc68347ndash352
World Health Organisation (WHO) 2006 Guidelines for safe recreational water environ-ments Vol 2 Swimming pools and similar environments Geneva WHO Press
Wilson IG 1997 Inhibition and facilitation of nucleic acid amplification Appl EnvironMicrobiol 633741ndash3751
70 G Amagliani et al
Dow
nloa
ded
by [
Bib
liote
ca U
nive
rsita
ria]
[G
iulia
Am
aglia
ni]
at 0
717
19
Janu
ary
2012
developed protocol Column-based extraction proved to be more efficient thanmagnetic nanoparticles for the isolation of bacterial DNA from water samples witha sensitivity of 10 cfu and 103 cfu respectively Ct values were as follows Qiagen kit0 cfu100 ml tested negative 10 cfu100 ml Ct 3565 102 cfu100 ml Ct 3278103 cfu100 ml Ct 2872 Merck magnetic particles 0 cfu100 ml 10 cfu100 ml and102 cfu100 ml tested negative 103 cfu100 ml Ct 3741
Analysis of recreational water samples
One out of 17 pool water samples and 2027 inlet water samples tested positive forP aeruginosa when analysed using the reference method Contamination titersranged between 1 and 45 cfu per 100 ml A high bacterial concentration above 100cfu was detected on two occasions When the same samples were analysed usingthe molecular method positivity rates of 417 and 2027 in pool and inlet waterrespectively were found (Table 2)
Statistical analysis
Test reliability was estimated by statistical analysis of result accordance With regardto the standard method the molecular assay showed a sensitivity of 9048 and aspecificity of 7826 PA was 844 (95 CI 0730ndash0958) NA 837 (95 CI0718ndash0956) Cohenrsquos Kappa index was 06831 which indicates lsquolsquosubstantialagreementrsquorsquo according to Landis and Koch (1977) classification
RAPD fingerprinting
The efficacy of the RAPD typing scheme was shown with bacterial strains of thesame species but different origins These included two additional isolates that hadoriginally been recovered from other swimming pools (environmental isolates 1 and2) one collection strain ATCC 10145 and one strain used as quality control strainby the ASUR (CQXVIII) (Table 1) As shown in Figure 2 panel B RAPDfingerprinting allowed us to differentiate all these distinct strains of P aeruginosaWhen all the P aeruginosa isolated from water samples were tested using the sameprotocol they were found to possess identical RAPD fingerprint profiles (Figure 2panel A)
Discussion
P aeruginosa is the second most common cause of recreational disease outbreaks(Craun et al 2005) Ear infection and otitis externa caused by this microorganism inswimming pools are probably more common than reported (Guida et al 2009)P aeruginosa presence in recreational water has been investigated by few authors(Barben et al 2005 Papadopoulou et al 2008 Guida et al 2009 Nikaeen et al2009) and additional data have been provided by Mena and Gerba (2009) Howevernone of these studies made use of a PCR-based method for the detection of thisspecies
More recently Villarreal et al (2010) described culture-independent techniquesapplied to drinking water surveillance Perkins et al (2009) to shower water andLavenir et al (2007) to environmental water while the use of PCR for pathogen
International Journal of Environmental Health Research 65
Dow
nloa
ded
by [
Bib
liote
ca U
nive
rsita
ria]
[G
iulia
Am
aglia
ni]
at 0
717
19
Janu
ary
2012
Table 2 Comparison of results obtained through reference and molecular methods forP aeruginosa detection in pool and inlet water
aColony count after membrane growth on Pseudomonas Agar Base CN (see Materials and methods)
66 G Amagliani et al
Dow
nloa
ded
by [
Bib
liote
ca U
nive
rsita
ria]
[G
iulia
Am
aglia
ni]
at 0
717
19
Janu
ary
2012
detection in swimming pools has been limited to the identification of adenoviruses(Heerden et al 2005) enteric protozoa (Fournier et al 2002) and free-living amoebae(Gianinazzi et al 2009) To the best of our knowledge the application of a molecularassay for the detection of P aeruginosa in recreational water has yet to be reported
The aim of this study was the development of a new molecular method forP aeruginosa identification in recreational water and the evaluation of itsperformance in the monitoring of a swimming pool in comparison with thereference culture method (UNI EN ISO 162662008) In order to make such acomparison inlet and pool water samples were collected from a local swimming pooland examined by both systems
Results of microbiological analysis indicated higher positivity rates in inlet water(74) compared to pool water (6) with higher levels of P aeruginosa (147 vs1 cfu100 ml mean values) in accordance with values reported by Guida et al(2009) These results may be explained by the presence of biofilm in the inlet waterpipeline and are supported by RAPD typing of isolates which indicate theirepidemiological correlation A longer period of contact with chlorine and thedilution process may have led to lower levels of P aeruginosa in pool water (Guidaet al 2009) Based on criteria provided by Italian Guidelines (Italian Republic 2003)fixing acceptability limits of 1 cfu100 ml in pool water and 0 cfu100 ml of inletwater in this study only the samples of pool water proved to be in compliance
lsquolsquoSubstantial agreementrsquorsquo (Landis and Koch 1977) was found between thedeveloped molecular method and the reference protocol The higher sensitivity of themolecular approach compared to the reference method in testing pool water samplescould be ascribed to the greater starting volume used for the analysis Moreoverbacteria in the pool water may have been in a subvital state due to chlorine watertreatment and able to recover culturability after a short enrichment in liquid
Figure 2 Discrimination of P aeruginosa by RAPD typing The ability of RAPDfingerprinting with primer 272 to cluster identical isolates (Panel A) and differentiatedistinct isolates within the P aeruginosa species (Panel B) is shown M 100 bp Ladder PlusFermentas Lanes 1ndash25 P aeruginosa isolated from recreational water 26 P aeruginosaenvironmental isolate 1 27 P aeruginosa environmental isolate 2 28 CQXVIII 29P aeruginosa ATCC 10145
International Journal of Environmental Health Research 67
Dow
nloa
ded
by [
Bib
liote
ca U
nive
rsita
ria]
[G
iulia
Am
aglia
ni]
at 0
717
19
Janu
ary
2012
medium thus providing resuscitation of injured or stressed cells However it iscommonly accepted that culture-dependent methods can fail in the identification ofbacteria in VBNC state (Oliver 2005)
It has been reported that the examination of colony morphology after membranegrowth on Cetrimide agar is not conclusive for P aeruginosa identification andconfirmation tests are needed for the correct identification of presumptive positivecolonies (Casanovas-Massana et al 2010) The need for such tests was illustrated bythe interlaboratory proficiency test carried out by the Italian National Health Service(Bonadonna and Ottaviani 2007) owing to the high rate (64) of false positiveresults obtained Conversely the Real-Time PCR assay specificity previously testedby Anuj et al (2009) on 101 target and non-target isolates and confirmed with 26further target and non-target strains in the present study proved to be very highConsidering that the assay has been successfully applied for the screening ofsuspected positive colonies on Pseudomonas Agar Base CN it may constitute aviable alternative to conventional time-consuming confirmation tests with a briefReal-Time PCR assay
A limitation for the broad application of molecular assays for testingenvironmental samples stems from the presence of substances of environmentalorigin such as humic acids and other organic molecules well known as PCRinhibitors (Wilson 1997) Hence the development of a DNA isolation system able toeffectively remove such contaminating molecules is a key step In a first attemptDNA direct isolation from filter membrane was carried out although the sensitivityobtained was not sufficient to be in compliance with National standards provided forrecreational water (not shown) After introducing a short (6 h) enrichment step twodifferent DNA extraction strategies were compared silica-based spin columns andmagnetic nanoparticles However the magnetic procedure for DNA isolation wasnot able to eliminate water-derived PCR inhibitors or did not provide sufficientrecovery especially with low bacterial concentrations On the other hand thecolumn devices proved effective and appropriate in this application
The employment of Real-Time PCR for the detection of P aeruginosa in wateroffers high specificity and sensitivity levels with a reduction of analysis time Inaddition this technique has the potential to screen large numbers of environmentalsamples limiting the application of the conventional culture techniques only toPCR-positive samples Hence the method could be proposed as part of a self-monitoring plan for swimming pool facilities to evaluate the quality of water usedfor recreational purposes improving surveillance and early warning systemsAlthough the use of PCR-based methods has not been recognised by AuthoritiesEU Directive 9883EC (EU 1998) allows the application of molecular diagnosis if itsequivalence with the reference protocol is demonstrated However a validationprogramme is needed to assess the level of accordance of the described system withthe reference method in order to verify the equivalence of results required byEuropean legislation
Acknowledgements
The authors would like to thank Dr G Cappuccini Director of the Department of Preventionand Head of the Hygiene and Public Health Service ASUR ZT 2 Urbino the personnel at theswimming pool facility of the Faculty of Health and Physical Exercise University of UrbinolsquolsquoCarlo Borsquorsquo for their collaboration and Professor Timothy Bloom of the University ofUrbino for a critical reading of the manuscript
68 G Amagliani et al
Dow
nloa
ded
by [
Bib
liote
ca U
nive
rsita
ria]
[G
iulia
Am
aglia
ni]
at 0
717
19
Janu
ary
2012
References
Anuj SN Whiley DM Kidd TJ Bell SC Wainwright CE Nissen MD Sloots TP 2009Identification of Pseudomonas aeruginosa by a duplex real-time polymerase chain reactionassay targeting the ecfX and the gyrB genes Diagn Microbiol Infect Dis 63 127ndash131
Barben J Hafen G Schmid J 2005 Pseudomonas aeruginosa in public swimming pools andbathroom water of patients with cystic fibrosis J Cystic Fibros 4227ndash231
Bonadonna L Ottaviani M 2007 Reference analytical methods for water intended forhuman consumption according to the Italian Legislative Decree 312001 Microbiologicalmethods Rapporti ISTISAN 075 Rome Istituto Superiore di Sanita (National HealthService)
Casanovas-Massana A Lucena F Blanch AR 2010 Identification of Pseudomonas aeruginosain water-bottling plants on the basis of procedures included in ISO 162662006J Microbiol Meth 811ndash5
Craun GF Calderon RL Craun MF 2005 Outbreaks associated with recreational water inthe United States Int J Environ Health Res 15243ndash262
European Union (EU) 1998 Council Directive 9883EC of 3 November 1998 on the qualityof water intended for human consumption
Fournier S Dubrou S Liguory O Gaussin F Santillana-Hayat M Sarfati C Molina JMDerouin F 2002 Detection of microsporidia cryptosporidia and giardia in swimmingpools a one-year prospective study FEMS Immunol Med Microbiol 33209ndash213
Gianinazzi C Schild M Wuthrich F Muller N Schurch N Gottstein B 2009 Potentiallyhuman pathogenic Acanthamoeba isolated from a heated indoor swimming pool inSwitzerland Exp Parasitol 121180ndash186
Goeres DM Palys T Sandel BB Geiger J 2004 Evaluation of disinfectant efficacy againstbiofilm and suspended bacteria in a laboratory swimming pool model Water Res383103ndash3109
Guida M Galle F Mattei ML Anastasi D Liguori G 2009 Microbiological quality of thewater of recreational and rehabilitation pools a 2-year survey in Naples Italy PublicHealth 123448ndash451
Heerden J Ehlers MM Grabow WOK 2005 Detection and risk assessment of adenovirusesin swimming pool water J Appl Microbiol 991256ndash1264
Italian Republic 2001 Legislative Decree No 31 2 February implementing Directive 9883EC on the quality of water intended for human consumption Official Gazette No 52 of3rd March 2001 Rome
Italian Republic 2002 Legislative Decree No 27 2 February amending and supplementingLegislative Decree No 31 of 2 February 2001 implementing Directive 9883EC on thequality of water intended for human consumption Official Gazette No 58 of 9th of March2002 Rome
Italian Republic 2003 Permanent Council State-Regions and Autonomous ProvincesAccordo Stato ndash Regioni e Province Autonome 16-1-2003 Official Gazette No 51 of 3rdof March 2003 Rome
Landis JR Koch GG 1977 The measurement of observer agreement for categorical dataBiometrics 33159ndash174
Lanotte P Watt S Mereghetti L Dartiguelongue N Rastegar-Lari A Goudeau A QuentinR 2004 Genetic features of Pseudomonas aeruginosa isolates from cystic fibrosis patientscompared with those of isolates from other origins J Med Microbiol 5373ndash81
Lavenir R Jocktane D Laurent F Nazaret S Cournoyer B 2007 Improved reliability ofPseudomonas aeruginosa PCR detection by the use of the species-specific ecfX gene targetJ Microbiol Meth 7020ndash29
Leoni E Legnani P Guberti E Masotti A 1999 Risk of infection associated withmicrobiological quality of public swimming pools in Bologna Italy Public Health113227ndash232
Mackinnon A 2000 A spreadsheet for the calculation of comprehensive statistics for theassessment of diagnostic tests and inter-rater agreement Comput Biol Med 30127ndash134
Mena KD Gerba CP 2009 Risk assessment of Pseudomonas aeruginosa in water RevEnviron Contam Toxicol 20171ndash115
Moore JE Heaney N Millar BC Crowe M Elborn JS 2002 Incidence of Pseudomonasaeruginosa in recreational and hydrotherapy pools Commun Dis Public Health 523ndash26
International Journal of Environmental Health Research 69
Dow
nloa
ded
by [
Bib
liote
ca U
nive
rsita
ria]
[G
iulia
Am
aglia
ni]
at 0
717
19
Janu
ary
2012
Nikaeen M Hatamzadeh M Vahid Dastjerdi M Hassanzadeh A 2009 Predictive indicatorsof the safety of swimming pool waters Water Sci Technol 603101ndash3107
Oliver JD 2005 The viable but nonculturable state in bacteria J Microbiol 4393ndash100Papadopoulou C Economou V Sakkas H Gousia P Giannakopoulos X Dontorou C
Filioussis G Gessouli H Karanis P Leveidiotou S 2008 Microbiological quality ofindoor and outdoor swimming pools in Greece investigation of the antibiotic resistanceof the bacterial isolates Int J Hyg Environ Health 211385ndash397
Perkins SD Mayfield J Fraser V Angenent LT 2009 Potentially pathogenic bacteria inshower water and air of a stem cell transplant unit Appl Environ Microbiol 755363ndash5372
Pond K 2005 Water recreation and disease Plausibility of associated infections acute effectssequelae and mortality London WHO IWA Publishing
UNI EN ISO 162662008 ndash 18092008 ndashWater quality ndash Detection and enumeration ofPseudomonas aeruginosa ndash Method by membrane filtration
Villarreal JV Schwartz T Obst U 2010 Culture-independent techniques applied to foodindustry water surveillance ndash a case study Int J Food Microbiol 141(Suppl 1)S147ndash55
Wang MC Liu CY Shiao AS Wang T 2005 Ear problems in swimmers J Chin Med Assoc68347ndash352
World Health Organisation (WHO) 2006 Guidelines for safe recreational water environ-ments Vol 2 Swimming pools and similar environments Geneva WHO Press
Wilson IG 1997 Inhibition and facilitation of nucleic acid amplification Appl EnvironMicrobiol 633741ndash3751
70 G Amagliani et al
Dow
nloa
ded
by [
Bib
liote
ca U
nive
rsita
ria]
[G
iulia
Am
aglia
ni]
at 0
717
19
Janu
ary
2012
Table 2 Comparison of results obtained through reference and molecular methods forP aeruginosa detection in pool and inlet water
aColony count after membrane growth on Pseudomonas Agar Base CN (see Materials and methods)
66 G Amagliani et al
Dow
nloa
ded
by [
Bib
liote
ca U
nive
rsita
ria]
[G
iulia
Am
aglia
ni]
at 0
717
19
Janu
ary
2012
detection in swimming pools has been limited to the identification of adenoviruses(Heerden et al 2005) enteric protozoa (Fournier et al 2002) and free-living amoebae(Gianinazzi et al 2009) To the best of our knowledge the application of a molecularassay for the detection of P aeruginosa in recreational water has yet to be reported
The aim of this study was the development of a new molecular method forP aeruginosa identification in recreational water and the evaluation of itsperformance in the monitoring of a swimming pool in comparison with thereference culture method (UNI EN ISO 162662008) In order to make such acomparison inlet and pool water samples were collected from a local swimming pooland examined by both systems
Results of microbiological analysis indicated higher positivity rates in inlet water(74) compared to pool water (6) with higher levels of P aeruginosa (147 vs1 cfu100 ml mean values) in accordance with values reported by Guida et al(2009) These results may be explained by the presence of biofilm in the inlet waterpipeline and are supported by RAPD typing of isolates which indicate theirepidemiological correlation A longer period of contact with chlorine and thedilution process may have led to lower levels of P aeruginosa in pool water (Guidaet al 2009) Based on criteria provided by Italian Guidelines (Italian Republic 2003)fixing acceptability limits of 1 cfu100 ml in pool water and 0 cfu100 ml of inletwater in this study only the samples of pool water proved to be in compliance
lsquolsquoSubstantial agreementrsquorsquo (Landis and Koch 1977) was found between thedeveloped molecular method and the reference protocol The higher sensitivity of themolecular approach compared to the reference method in testing pool water samplescould be ascribed to the greater starting volume used for the analysis Moreoverbacteria in the pool water may have been in a subvital state due to chlorine watertreatment and able to recover culturability after a short enrichment in liquid
Figure 2 Discrimination of P aeruginosa by RAPD typing The ability of RAPDfingerprinting with primer 272 to cluster identical isolates (Panel A) and differentiatedistinct isolates within the P aeruginosa species (Panel B) is shown M 100 bp Ladder PlusFermentas Lanes 1ndash25 P aeruginosa isolated from recreational water 26 P aeruginosaenvironmental isolate 1 27 P aeruginosa environmental isolate 2 28 CQXVIII 29P aeruginosa ATCC 10145
International Journal of Environmental Health Research 67
Dow
nloa
ded
by [
Bib
liote
ca U
nive
rsita
ria]
[G
iulia
Am
aglia
ni]
at 0
717
19
Janu
ary
2012
medium thus providing resuscitation of injured or stressed cells However it iscommonly accepted that culture-dependent methods can fail in the identification ofbacteria in VBNC state (Oliver 2005)
It has been reported that the examination of colony morphology after membranegrowth on Cetrimide agar is not conclusive for P aeruginosa identification andconfirmation tests are needed for the correct identification of presumptive positivecolonies (Casanovas-Massana et al 2010) The need for such tests was illustrated bythe interlaboratory proficiency test carried out by the Italian National Health Service(Bonadonna and Ottaviani 2007) owing to the high rate (64) of false positiveresults obtained Conversely the Real-Time PCR assay specificity previously testedby Anuj et al (2009) on 101 target and non-target isolates and confirmed with 26further target and non-target strains in the present study proved to be very highConsidering that the assay has been successfully applied for the screening ofsuspected positive colonies on Pseudomonas Agar Base CN it may constitute aviable alternative to conventional time-consuming confirmation tests with a briefReal-Time PCR assay
A limitation for the broad application of molecular assays for testingenvironmental samples stems from the presence of substances of environmentalorigin such as humic acids and other organic molecules well known as PCRinhibitors (Wilson 1997) Hence the development of a DNA isolation system able toeffectively remove such contaminating molecules is a key step In a first attemptDNA direct isolation from filter membrane was carried out although the sensitivityobtained was not sufficient to be in compliance with National standards provided forrecreational water (not shown) After introducing a short (6 h) enrichment step twodifferent DNA extraction strategies were compared silica-based spin columns andmagnetic nanoparticles However the magnetic procedure for DNA isolation wasnot able to eliminate water-derived PCR inhibitors or did not provide sufficientrecovery especially with low bacterial concentrations On the other hand thecolumn devices proved effective and appropriate in this application
The employment of Real-Time PCR for the detection of P aeruginosa in wateroffers high specificity and sensitivity levels with a reduction of analysis time Inaddition this technique has the potential to screen large numbers of environmentalsamples limiting the application of the conventional culture techniques only toPCR-positive samples Hence the method could be proposed as part of a self-monitoring plan for swimming pool facilities to evaluate the quality of water usedfor recreational purposes improving surveillance and early warning systemsAlthough the use of PCR-based methods has not been recognised by AuthoritiesEU Directive 9883EC (EU 1998) allows the application of molecular diagnosis if itsequivalence with the reference protocol is demonstrated However a validationprogramme is needed to assess the level of accordance of the described system withthe reference method in order to verify the equivalence of results required byEuropean legislation
Acknowledgements
The authors would like to thank Dr G Cappuccini Director of the Department of Preventionand Head of the Hygiene and Public Health Service ASUR ZT 2 Urbino the personnel at theswimming pool facility of the Faculty of Health and Physical Exercise University of UrbinolsquolsquoCarlo Borsquorsquo for their collaboration and Professor Timothy Bloom of the University ofUrbino for a critical reading of the manuscript
68 G Amagliani et al
Dow
nloa
ded
by [
Bib
liote
ca U
nive
rsita
ria]
[G
iulia
Am
aglia
ni]
at 0
717
19
Janu
ary
2012
References
Anuj SN Whiley DM Kidd TJ Bell SC Wainwright CE Nissen MD Sloots TP 2009Identification of Pseudomonas aeruginosa by a duplex real-time polymerase chain reactionassay targeting the ecfX and the gyrB genes Diagn Microbiol Infect Dis 63 127ndash131
Barben J Hafen G Schmid J 2005 Pseudomonas aeruginosa in public swimming pools andbathroom water of patients with cystic fibrosis J Cystic Fibros 4227ndash231
Bonadonna L Ottaviani M 2007 Reference analytical methods for water intended forhuman consumption according to the Italian Legislative Decree 312001 Microbiologicalmethods Rapporti ISTISAN 075 Rome Istituto Superiore di Sanita (National HealthService)
Casanovas-Massana A Lucena F Blanch AR 2010 Identification of Pseudomonas aeruginosain water-bottling plants on the basis of procedures included in ISO 162662006J Microbiol Meth 811ndash5
Craun GF Calderon RL Craun MF 2005 Outbreaks associated with recreational water inthe United States Int J Environ Health Res 15243ndash262
European Union (EU) 1998 Council Directive 9883EC of 3 November 1998 on the qualityof water intended for human consumption
Fournier S Dubrou S Liguory O Gaussin F Santillana-Hayat M Sarfati C Molina JMDerouin F 2002 Detection of microsporidia cryptosporidia and giardia in swimmingpools a one-year prospective study FEMS Immunol Med Microbiol 33209ndash213
Gianinazzi C Schild M Wuthrich F Muller N Schurch N Gottstein B 2009 Potentiallyhuman pathogenic Acanthamoeba isolated from a heated indoor swimming pool inSwitzerland Exp Parasitol 121180ndash186
Goeres DM Palys T Sandel BB Geiger J 2004 Evaluation of disinfectant efficacy againstbiofilm and suspended bacteria in a laboratory swimming pool model Water Res383103ndash3109
Guida M Galle F Mattei ML Anastasi D Liguori G 2009 Microbiological quality of thewater of recreational and rehabilitation pools a 2-year survey in Naples Italy PublicHealth 123448ndash451
Heerden J Ehlers MM Grabow WOK 2005 Detection and risk assessment of adenovirusesin swimming pool water J Appl Microbiol 991256ndash1264
Italian Republic 2001 Legislative Decree No 31 2 February implementing Directive 9883EC on the quality of water intended for human consumption Official Gazette No 52 of3rd March 2001 Rome
Italian Republic 2002 Legislative Decree No 27 2 February amending and supplementingLegislative Decree No 31 of 2 February 2001 implementing Directive 9883EC on thequality of water intended for human consumption Official Gazette No 58 of 9th of March2002 Rome
Italian Republic 2003 Permanent Council State-Regions and Autonomous ProvincesAccordo Stato ndash Regioni e Province Autonome 16-1-2003 Official Gazette No 51 of 3rdof March 2003 Rome
Landis JR Koch GG 1977 The measurement of observer agreement for categorical dataBiometrics 33159ndash174
Lanotte P Watt S Mereghetti L Dartiguelongue N Rastegar-Lari A Goudeau A QuentinR 2004 Genetic features of Pseudomonas aeruginosa isolates from cystic fibrosis patientscompared with those of isolates from other origins J Med Microbiol 5373ndash81
Lavenir R Jocktane D Laurent F Nazaret S Cournoyer B 2007 Improved reliability ofPseudomonas aeruginosa PCR detection by the use of the species-specific ecfX gene targetJ Microbiol Meth 7020ndash29
Leoni E Legnani P Guberti E Masotti A 1999 Risk of infection associated withmicrobiological quality of public swimming pools in Bologna Italy Public Health113227ndash232
Mackinnon A 2000 A spreadsheet for the calculation of comprehensive statistics for theassessment of diagnostic tests and inter-rater agreement Comput Biol Med 30127ndash134
Mena KD Gerba CP 2009 Risk assessment of Pseudomonas aeruginosa in water RevEnviron Contam Toxicol 20171ndash115
Moore JE Heaney N Millar BC Crowe M Elborn JS 2002 Incidence of Pseudomonasaeruginosa in recreational and hydrotherapy pools Commun Dis Public Health 523ndash26
International Journal of Environmental Health Research 69
Dow
nloa
ded
by [
Bib
liote
ca U
nive
rsita
ria]
[G
iulia
Am
aglia
ni]
at 0
717
19
Janu
ary
2012
Nikaeen M Hatamzadeh M Vahid Dastjerdi M Hassanzadeh A 2009 Predictive indicatorsof the safety of swimming pool waters Water Sci Technol 603101ndash3107
Oliver JD 2005 The viable but nonculturable state in bacteria J Microbiol 4393ndash100Papadopoulou C Economou V Sakkas H Gousia P Giannakopoulos X Dontorou C
Filioussis G Gessouli H Karanis P Leveidiotou S 2008 Microbiological quality ofindoor and outdoor swimming pools in Greece investigation of the antibiotic resistanceof the bacterial isolates Int J Hyg Environ Health 211385ndash397
Perkins SD Mayfield J Fraser V Angenent LT 2009 Potentially pathogenic bacteria inshower water and air of a stem cell transplant unit Appl Environ Microbiol 755363ndash5372
Pond K 2005 Water recreation and disease Plausibility of associated infections acute effectssequelae and mortality London WHO IWA Publishing
UNI EN ISO 162662008 ndash 18092008 ndashWater quality ndash Detection and enumeration ofPseudomonas aeruginosa ndash Method by membrane filtration
Villarreal JV Schwartz T Obst U 2010 Culture-independent techniques applied to foodindustry water surveillance ndash a case study Int J Food Microbiol 141(Suppl 1)S147ndash55
Wang MC Liu CY Shiao AS Wang T 2005 Ear problems in swimmers J Chin Med Assoc68347ndash352
World Health Organisation (WHO) 2006 Guidelines for safe recreational water environ-ments Vol 2 Swimming pools and similar environments Geneva WHO Press
Wilson IG 1997 Inhibition and facilitation of nucleic acid amplification Appl EnvironMicrobiol 633741ndash3751
70 G Amagliani et al
Dow
nloa
ded
by [
Bib
liote
ca U
nive
rsita
ria]
[G
iulia
Am
aglia
ni]
at 0
717
19
Janu
ary
2012
detection in swimming pools has been limited to the identification of adenoviruses(Heerden et al 2005) enteric protozoa (Fournier et al 2002) and free-living amoebae(Gianinazzi et al 2009) To the best of our knowledge the application of a molecularassay for the detection of P aeruginosa in recreational water has yet to be reported
The aim of this study was the development of a new molecular method forP aeruginosa identification in recreational water and the evaluation of itsperformance in the monitoring of a swimming pool in comparison with thereference culture method (UNI EN ISO 162662008) In order to make such acomparison inlet and pool water samples were collected from a local swimming pooland examined by both systems
Results of microbiological analysis indicated higher positivity rates in inlet water(74) compared to pool water (6) with higher levels of P aeruginosa (147 vs1 cfu100 ml mean values) in accordance with values reported by Guida et al(2009) These results may be explained by the presence of biofilm in the inlet waterpipeline and are supported by RAPD typing of isolates which indicate theirepidemiological correlation A longer period of contact with chlorine and thedilution process may have led to lower levels of P aeruginosa in pool water (Guidaet al 2009) Based on criteria provided by Italian Guidelines (Italian Republic 2003)fixing acceptability limits of 1 cfu100 ml in pool water and 0 cfu100 ml of inletwater in this study only the samples of pool water proved to be in compliance
lsquolsquoSubstantial agreementrsquorsquo (Landis and Koch 1977) was found between thedeveloped molecular method and the reference protocol The higher sensitivity of themolecular approach compared to the reference method in testing pool water samplescould be ascribed to the greater starting volume used for the analysis Moreoverbacteria in the pool water may have been in a subvital state due to chlorine watertreatment and able to recover culturability after a short enrichment in liquid
Figure 2 Discrimination of P aeruginosa by RAPD typing The ability of RAPDfingerprinting with primer 272 to cluster identical isolates (Panel A) and differentiatedistinct isolates within the P aeruginosa species (Panel B) is shown M 100 bp Ladder PlusFermentas Lanes 1ndash25 P aeruginosa isolated from recreational water 26 P aeruginosaenvironmental isolate 1 27 P aeruginosa environmental isolate 2 28 CQXVIII 29P aeruginosa ATCC 10145
International Journal of Environmental Health Research 67
Dow
nloa
ded
by [
Bib
liote
ca U
nive
rsita
ria]
[G
iulia
Am
aglia
ni]
at 0
717
19
Janu
ary
2012
medium thus providing resuscitation of injured or stressed cells However it iscommonly accepted that culture-dependent methods can fail in the identification ofbacteria in VBNC state (Oliver 2005)
It has been reported that the examination of colony morphology after membranegrowth on Cetrimide agar is not conclusive for P aeruginosa identification andconfirmation tests are needed for the correct identification of presumptive positivecolonies (Casanovas-Massana et al 2010) The need for such tests was illustrated bythe interlaboratory proficiency test carried out by the Italian National Health Service(Bonadonna and Ottaviani 2007) owing to the high rate (64) of false positiveresults obtained Conversely the Real-Time PCR assay specificity previously testedby Anuj et al (2009) on 101 target and non-target isolates and confirmed with 26further target and non-target strains in the present study proved to be very highConsidering that the assay has been successfully applied for the screening ofsuspected positive colonies on Pseudomonas Agar Base CN it may constitute aviable alternative to conventional time-consuming confirmation tests with a briefReal-Time PCR assay
A limitation for the broad application of molecular assays for testingenvironmental samples stems from the presence of substances of environmentalorigin such as humic acids and other organic molecules well known as PCRinhibitors (Wilson 1997) Hence the development of a DNA isolation system able toeffectively remove such contaminating molecules is a key step In a first attemptDNA direct isolation from filter membrane was carried out although the sensitivityobtained was not sufficient to be in compliance with National standards provided forrecreational water (not shown) After introducing a short (6 h) enrichment step twodifferent DNA extraction strategies were compared silica-based spin columns andmagnetic nanoparticles However the magnetic procedure for DNA isolation wasnot able to eliminate water-derived PCR inhibitors or did not provide sufficientrecovery especially with low bacterial concentrations On the other hand thecolumn devices proved effective and appropriate in this application
The employment of Real-Time PCR for the detection of P aeruginosa in wateroffers high specificity and sensitivity levels with a reduction of analysis time Inaddition this technique has the potential to screen large numbers of environmentalsamples limiting the application of the conventional culture techniques only toPCR-positive samples Hence the method could be proposed as part of a self-monitoring plan for swimming pool facilities to evaluate the quality of water usedfor recreational purposes improving surveillance and early warning systemsAlthough the use of PCR-based methods has not been recognised by AuthoritiesEU Directive 9883EC (EU 1998) allows the application of molecular diagnosis if itsequivalence with the reference protocol is demonstrated However a validationprogramme is needed to assess the level of accordance of the described system withthe reference method in order to verify the equivalence of results required byEuropean legislation
Acknowledgements
The authors would like to thank Dr G Cappuccini Director of the Department of Preventionand Head of the Hygiene and Public Health Service ASUR ZT 2 Urbino the personnel at theswimming pool facility of the Faculty of Health and Physical Exercise University of UrbinolsquolsquoCarlo Borsquorsquo for their collaboration and Professor Timothy Bloom of the University ofUrbino for a critical reading of the manuscript
68 G Amagliani et al
Dow
nloa
ded
by [
Bib
liote
ca U
nive
rsita
ria]
[G
iulia
Am
aglia
ni]
at 0
717
19
Janu
ary
2012
References
Anuj SN Whiley DM Kidd TJ Bell SC Wainwright CE Nissen MD Sloots TP 2009Identification of Pseudomonas aeruginosa by a duplex real-time polymerase chain reactionassay targeting the ecfX and the gyrB genes Diagn Microbiol Infect Dis 63 127ndash131
Barben J Hafen G Schmid J 2005 Pseudomonas aeruginosa in public swimming pools andbathroom water of patients with cystic fibrosis J Cystic Fibros 4227ndash231
Bonadonna L Ottaviani M 2007 Reference analytical methods for water intended forhuman consumption according to the Italian Legislative Decree 312001 Microbiologicalmethods Rapporti ISTISAN 075 Rome Istituto Superiore di Sanita (National HealthService)
Casanovas-Massana A Lucena F Blanch AR 2010 Identification of Pseudomonas aeruginosain water-bottling plants on the basis of procedures included in ISO 162662006J Microbiol Meth 811ndash5
Craun GF Calderon RL Craun MF 2005 Outbreaks associated with recreational water inthe United States Int J Environ Health Res 15243ndash262
European Union (EU) 1998 Council Directive 9883EC of 3 November 1998 on the qualityof water intended for human consumption
Fournier S Dubrou S Liguory O Gaussin F Santillana-Hayat M Sarfati C Molina JMDerouin F 2002 Detection of microsporidia cryptosporidia and giardia in swimmingpools a one-year prospective study FEMS Immunol Med Microbiol 33209ndash213
Gianinazzi C Schild M Wuthrich F Muller N Schurch N Gottstein B 2009 Potentiallyhuman pathogenic Acanthamoeba isolated from a heated indoor swimming pool inSwitzerland Exp Parasitol 121180ndash186
Goeres DM Palys T Sandel BB Geiger J 2004 Evaluation of disinfectant efficacy againstbiofilm and suspended bacteria in a laboratory swimming pool model Water Res383103ndash3109
Guida M Galle F Mattei ML Anastasi D Liguori G 2009 Microbiological quality of thewater of recreational and rehabilitation pools a 2-year survey in Naples Italy PublicHealth 123448ndash451
Heerden J Ehlers MM Grabow WOK 2005 Detection and risk assessment of adenovirusesin swimming pool water J Appl Microbiol 991256ndash1264
Italian Republic 2001 Legislative Decree No 31 2 February implementing Directive 9883EC on the quality of water intended for human consumption Official Gazette No 52 of3rd March 2001 Rome
Italian Republic 2002 Legislative Decree No 27 2 February amending and supplementingLegislative Decree No 31 of 2 February 2001 implementing Directive 9883EC on thequality of water intended for human consumption Official Gazette No 58 of 9th of March2002 Rome
Italian Republic 2003 Permanent Council State-Regions and Autonomous ProvincesAccordo Stato ndash Regioni e Province Autonome 16-1-2003 Official Gazette No 51 of 3rdof March 2003 Rome
Landis JR Koch GG 1977 The measurement of observer agreement for categorical dataBiometrics 33159ndash174
Lanotte P Watt S Mereghetti L Dartiguelongue N Rastegar-Lari A Goudeau A QuentinR 2004 Genetic features of Pseudomonas aeruginosa isolates from cystic fibrosis patientscompared with those of isolates from other origins J Med Microbiol 5373ndash81
Lavenir R Jocktane D Laurent F Nazaret S Cournoyer B 2007 Improved reliability ofPseudomonas aeruginosa PCR detection by the use of the species-specific ecfX gene targetJ Microbiol Meth 7020ndash29
Leoni E Legnani P Guberti E Masotti A 1999 Risk of infection associated withmicrobiological quality of public swimming pools in Bologna Italy Public Health113227ndash232
Mackinnon A 2000 A spreadsheet for the calculation of comprehensive statistics for theassessment of diagnostic tests and inter-rater agreement Comput Biol Med 30127ndash134
Mena KD Gerba CP 2009 Risk assessment of Pseudomonas aeruginosa in water RevEnviron Contam Toxicol 20171ndash115
Moore JE Heaney N Millar BC Crowe M Elborn JS 2002 Incidence of Pseudomonasaeruginosa in recreational and hydrotherapy pools Commun Dis Public Health 523ndash26
International Journal of Environmental Health Research 69
Dow
nloa
ded
by [
Bib
liote
ca U
nive
rsita
ria]
[G
iulia
Am
aglia
ni]
at 0
717
19
Janu
ary
2012
Nikaeen M Hatamzadeh M Vahid Dastjerdi M Hassanzadeh A 2009 Predictive indicatorsof the safety of swimming pool waters Water Sci Technol 603101ndash3107
Oliver JD 2005 The viable but nonculturable state in bacteria J Microbiol 4393ndash100Papadopoulou C Economou V Sakkas H Gousia P Giannakopoulos X Dontorou C
Filioussis G Gessouli H Karanis P Leveidiotou S 2008 Microbiological quality ofindoor and outdoor swimming pools in Greece investigation of the antibiotic resistanceof the bacterial isolates Int J Hyg Environ Health 211385ndash397
Perkins SD Mayfield J Fraser V Angenent LT 2009 Potentially pathogenic bacteria inshower water and air of a stem cell transplant unit Appl Environ Microbiol 755363ndash5372
Pond K 2005 Water recreation and disease Plausibility of associated infections acute effectssequelae and mortality London WHO IWA Publishing
UNI EN ISO 162662008 ndash 18092008 ndashWater quality ndash Detection and enumeration ofPseudomonas aeruginosa ndash Method by membrane filtration
Villarreal JV Schwartz T Obst U 2010 Culture-independent techniques applied to foodindustry water surveillance ndash a case study Int J Food Microbiol 141(Suppl 1)S147ndash55
Wang MC Liu CY Shiao AS Wang T 2005 Ear problems in swimmers J Chin Med Assoc68347ndash352
World Health Organisation (WHO) 2006 Guidelines for safe recreational water environ-ments Vol 2 Swimming pools and similar environments Geneva WHO Press
Wilson IG 1997 Inhibition and facilitation of nucleic acid amplification Appl EnvironMicrobiol 633741ndash3751
70 G Amagliani et al
Dow
nloa
ded
by [
Bib
liote
ca U
nive
rsita
ria]
[G
iulia
Am
aglia
ni]
at 0
717
19
Janu
ary
2012
medium thus providing resuscitation of injured or stressed cells However it iscommonly accepted that culture-dependent methods can fail in the identification ofbacteria in VBNC state (Oliver 2005)
It has been reported that the examination of colony morphology after membranegrowth on Cetrimide agar is not conclusive for P aeruginosa identification andconfirmation tests are needed for the correct identification of presumptive positivecolonies (Casanovas-Massana et al 2010) The need for such tests was illustrated bythe interlaboratory proficiency test carried out by the Italian National Health Service(Bonadonna and Ottaviani 2007) owing to the high rate (64) of false positiveresults obtained Conversely the Real-Time PCR assay specificity previously testedby Anuj et al (2009) on 101 target and non-target isolates and confirmed with 26further target and non-target strains in the present study proved to be very highConsidering that the assay has been successfully applied for the screening ofsuspected positive colonies on Pseudomonas Agar Base CN it may constitute aviable alternative to conventional time-consuming confirmation tests with a briefReal-Time PCR assay
A limitation for the broad application of molecular assays for testingenvironmental samples stems from the presence of substances of environmentalorigin such as humic acids and other organic molecules well known as PCRinhibitors (Wilson 1997) Hence the development of a DNA isolation system able toeffectively remove such contaminating molecules is a key step In a first attemptDNA direct isolation from filter membrane was carried out although the sensitivityobtained was not sufficient to be in compliance with National standards provided forrecreational water (not shown) After introducing a short (6 h) enrichment step twodifferent DNA extraction strategies were compared silica-based spin columns andmagnetic nanoparticles However the magnetic procedure for DNA isolation wasnot able to eliminate water-derived PCR inhibitors or did not provide sufficientrecovery especially with low bacterial concentrations On the other hand thecolumn devices proved effective and appropriate in this application
The employment of Real-Time PCR for the detection of P aeruginosa in wateroffers high specificity and sensitivity levels with a reduction of analysis time Inaddition this technique has the potential to screen large numbers of environmentalsamples limiting the application of the conventional culture techniques only toPCR-positive samples Hence the method could be proposed as part of a self-monitoring plan for swimming pool facilities to evaluate the quality of water usedfor recreational purposes improving surveillance and early warning systemsAlthough the use of PCR-based methods has not been recognised by AuthoritiesEU Directive 9883EC (EU 1998) allows the application of molecular diagnosis if itsequivalence with the reference protocol is demonstrated However a validationprogramme is needed to assess the level of accordance of the described system withthe reference method in order to verify the equivalence of results required byEuropean legislation
Acknowledgements
The authors would like to thank Dr G Cappuccini Director of the Department of Preventionand Head of the Hygiene and Public Health Service ASUR ZT 2 Urbino the personnel at theswimming pool facility of the Faculty of Health and Physical Exercise University of UrbinolsquolsquoCarlo Borsquorsquo for their collaboration and Professor Timothy Bloom of the University ofUrbino for a critical reading of the manuscript
68 G Amagliani et al
Dow
nloa
ded
by [
Bib
liote
ca U
nive
rsita
ria]
[G
iulia
Am
aglia
ni]
at 0
717
19
Janu
ary
2012
References
Anuj SN Whiley DM Kidd TJ Bell SC Wainwright CE Nissen MD Sloots TP 2009Identification of Pseudomonas aeruginosa by a duplex real-time polymerase chain reactionassay targeting the ecfX and the gyrB genes Diagn Microbiol Infect Dis 63 127ndash131
Barben J Hafen G Schmid J 2005 Pseudomonas aeruginosa in public swimming pools andbathroom water of patients with cystic fibrosis J Cystic Fibros 4227ndash231
Bonadonna L Ottaviani M 2007 Reference analytical methods for water intended forhuman consumption according to the Italian Legislative Decree 312001 Microbiologicalmethods Rapporti ISTISAN 075 Rome Istituto Superiore di Sanita (National HealthService)
Casanovas-Massana A Lucena F Blanch AR 2010 Identification of Pseudomonas aeruginosain water-bottling plants on the basis of procedures included in ISO 162662006J Microbiol Meth 811ndash5
Craun GF Calderon RL Craun MF 2005 Outbreaks associated with recreational water inthe United States Int J Environ Health Res 15243ndash262
European Union (EU) 1998 Council Directive 9883EC of 3 November 1998 on the qualityof water intended for human consumption
Fournier S Dubrou S Liguory O Gaussin F Santillana-Hayat M Sarfati C Molina JMDerouin F 2002 Detection of microsporidia cryptosporidia and giardia in swimmingpools a one-year prospective study FEMS Immunol Med Microbiol 33209ndash213
Gianinazzi C Schild M Wuthrich F Muller N Schurch N Gottstein B 2009 Potentiallyhuman pathogenic Acanthamoeba isolated from a heated indoor swimming pool inSwitzerland Exp Parasitol 121180ndash186
Goeres DM Palys T Sandel BB Geiger J 2004 Evaluation of disinfectant efficacy againstbiofilm and suspended bacteria in a laboratory swimming pool model Water Res383103ndash3109
Guida M Galle F Mattei ML Anastasi D Liguori G 2009 Microbiological quality of thewater of recreational and rehabilitation pools a 2-year survey in Naples Italy PublicHealth 123448ndash451
Heerden J Ehlers MM Grabow WOK 2005 Detection and risk assessment of adenovirusesin swimming pool water J Appl Microbiol 991256ndash1264
Italian Republic 2001 Legislative Decree No 31 2 February implementing Directive 9883EC on the quality of water intended for human consumption Official Gazette No 52 of3rd March 2001 Rome
Italian Republic 2002 Legislative Decree No 27 2 February amending and supplementingLegislative Decree No 31 of 2 February 2001 implementing Directive 9883EC on thequality of water intended for human consumption Official Gazette No 58 of 9th of March2002 Rome
Italian Republic 2003 Permanent Council State-Regions and Autonomous ProvincesAccordo Stato ndash Regioni e Province Autonome 16-1-2003 Official Gazette No 51 of 3rdof March 2003 Rome
Landis JR Koch GG 1977 The measurement of observer agreement for categorical dataBiometrics 33159ndash174
Lanotte P Watt S Mereghetti L Dartiguelongue N Rastegar-Lari A Goudeau A QuentinR 2004 Genetic features of Pseudomonas aeruginosa isolates from cystic fibrosis patientscompared with those of isolates from other origins J Med Microbiol 5373ndash81
Lavenir R Jocktane D Laurent F Nazaret S Cournoyer B 2007 Improved reliability ofPseudomonas aeruginosa PCR detection by the use of the species-specific ecfX gene targetJ Microbiol Meth 7020ndash29
Leoni E Legnani P Guberti E Masotti A 1999 Risk of infection associated withmicrobiological quality of public swimming pools in Bologna Italy Public Health113227ndash232
Mackinnon A 2000 A spreadsheet for the calculation of comprehensive statistics for theassessment of diagnostic tests and inter-rater agreement Comput Biol Med 30127ndash134
Mena KD Gerba CP 2009 Risk assessment of Pseudomonas aeruginosa in water RevEnviron Contam Toxicol 20171ndash115
Moore JE Heaney N Millar BC Crowe M Elborn JS 2002 Incidence of Pseudomonasaeruginosa in recreational and hydrotherapy pools Commun Dis Public Health 523ndash26
International Journal of Environmental Health Research 69
Dow
nloa
ded
by [
Bib
liote
ca U
nive
rsita
ria]
[G
iulia
Am
aglia
ni]
at 0
717
19
Janu
ary
2012
Nikaeen M Hatamzadeh M Vahid Dastjerdi M Hassanzadeh A 2009 Predictive indicatorsof the safety of swimming pool waters Water Sci Technol 603101ndash3107
Oliver JD 2005 The viable but nonculturable state in bacteria J Microbiol 4393ndash100Papadopoulou C Economou V Sakkas H Gousia P Giannakopoulos X Dontorou C
Filioussis G Gessouli H Karanis P Leveidiotou S 2008 Microbiological quality ofindoor and outdoor swimming pools in Greece investigation of the antibiotic resistanceof the bacterial isolates Int J Hyg Environ Health 211385ndash397
Perkins SD Mayfield J Fraser V Angenent LT 2009 Potentially pathogenic bacteria inshower water and air of a stem cell transplant unit Appl Environ Microbiol 755363ndash5372
Pond K 2005 Water recreation and disease Plausibility of associated infections acute effectssequelae and mortality London WHO IWA Publishing
UNI EN ISO 162662008 ndash 18092008 ndashWater quality ndash Detection and enumeration ofPseudomonas aeruginosa ndash Method by membrane filtration
Villarreal JV Schwartz T Obst U 2010 Culture-independent techniques applied to foodindustry water surveillance ndash a case study Int J Food Microbiol 141(Suppl 1)S147ndash55
Wang MC Liu CY Shiao AS Wang T 2005 Ear problems in swimmers J Chin Med Assoc68347ndash352
World Health Organisation (WHO) 2006 Guidelines for safe recreational water environ-ments Vol 2 Swimming pools and similar environments Geneva WHO Press
Wilson IG 1997 Inhibition and facilitation of nucleic acid amplification Appl EnvironMicrobiol 633741ndash3751
70 G Amagliani et al
Dow
nloa
ded
by [
Bib
liote
ca U
nive
rsita
ria]
[G
iulia
Am
aglia
ni]
at 0
717
19
Janu
ary
2012
References
Anuj SN Whiley DM Kidd TJ Bell SC Wainwright CE Nissen MD Sloots TP 2009Identification of Pseudomonas aeruginosa by a duplex real-time polymerase chain reactionassay targeting the ecfX and the gyrB genes Diagn Microbiol Infect Dis 63 127ndash131
Barben J Hafen G Schmid J 2005 Pseudomonas aeruginosa in public swimming pools andbathroom water of patients with cystic fibrosis J Cystic Fibros 4227ndash231
Bonadonna L Ottaviani M 2007 Reference analytical methods for water intended forhuman consumption according to the Italian Legislative Decree 312001 Microbiologicalmethods Rapporti ISTISAN 075 Rome Istituto Superiore di Sanita (National HealthService)
Casanovas-Massana A Lucena F Blanch AR 2010 Identification of Pseudomonas aeruginosain water-bottling plants on the basis of procedures included in ISO 162662006J Microbiol Meth 811ndash5
Craun GF Calderon RL Craun MF 2005 Outbreaks associated with recreational water inthe United States Int J Environ Health Res 15243ndash262
European Union (EU) 1998 Council Directive 9883EC of 3 November 1998 on the qualityof water intended for human consumption
Fournier S Dubrou S Liguory O Gaussin F Santillana-Hayat M Sarfati C Molina JMDerouin F 2002 Detection of microsporidia cryptosporidia and giardia in swimmingpools a one-year prospective study FEMS Immunol Med Microbiol 33209ndash213
Gianinazzi C Schild M Wuthrich F Muller N Schurch N Gottstein B 2009 Potentiallyhuman pathogenic Acanthamoeba isolated from a heated indoor swimming pool inSwitzerland Exp Parasitol 121180ndash186
Goeres DM Palys T Sandel BB Geiger J 2004 Evaluation of disinfectant efficacy againstbiofilm and suspended bacteria in a laboratory swimming pool model Water Res383103ndash3109
Guida M Galle F Mattei ML Anastasi D Liguori G 2009 Microbiological quality of thewater of recreational and rehabilitation pools a 2-year survey in Naples Italy PublicHealth 123448ndash451
Heerden J Ehlers MM Grabow WOK 2005 Detection and risk assessment of adenovirusesin swimming pool water J Appl Microbiol 991256ndash1264
Italian Republic 2001 Legislative Decree No 31 2 February implementing Directive 9883EC on the quality of water intended for human consumption Official Gazette No 52 of3rd March 2001 Rome
Italian Republic 2002 Legislative Decree No 27 2 February amending and supplementingLegislative Decree No 31 of 2 February 2001 implementing Directive 9883EC on thequality of water intended for human consumption Official Gazette No 58 of 9th of March2002 Rome
Italian Republic 2003 Permanent Council State-Regions and Autonomous ProvincesAccordo Stato ndash Regioni e Province Autonome 16-1-2003 Official Gazette No 51 of 3rdof March 2003 Rome
Landis JR Koch GG 1977 The measurement of observer agreement for categorical dataBiometrics 33159ndash174
Lanotte P Watt S Mereghetti L Dartiguelongue N Rastegar-Lari A Goudeau A QuentinR 2004 Genetic features of Pseudomonas aeruginosa isolates from cystic fibrosis patientscompared with those of isolates from other origins J Med Microbiol 5373ndash81
Lavenir R Jocktane D Laurent F Nazaret S Cournoyer B 2007 Improved reliability ofPseudomonas aeruginosa PCR detection by the use of the species-specific ecfX gene targetJ Microbiol Meth 7020ndash29
Leoni E Legnani P Guberti E Masotti A 1999 Risk of infection associated withmicrobiological quality of public swimming pools in Bologna Italy Public Health113227ndash232
Mackinnon A 2000 A spreadsheet for the calculation of comprehensive statistics for theassessment of diagnostic tests and inter-rater agreement Comput Biol Med 30127ndash134
Mena KD Gerba CP 2009 Risk assessment of Pseudomonas aeruginosa in water RevEnviron Contam Toxicol 20171ndash115
Moore JE Heaney N Millar BC Crowe M Elborn JS 2002 Incidence of Pseudomonasaeruginosa in recreational and hydrotherapy pools Commun Dis Public Health 523ndash26
International Journal of Environmental Health Research 69
Dow
nloa
ded
by [
Bib
liote
ca U
nive
rsita
ria]
[G
iulia
Am
aglia
ni]
at 0
717
19
Janu
ary
2012
Nikaeen M Hatamzadeh M Vahid Dastjerdi M Hassanzadeh A 2009 Predictive indicatorsof the safety of swimming pool waters Water Sci Technol 603101ndash3107
Oliver JD 2005 The viable but nonculturable state in bacteria J Microbiol 4393ndash100Papadopoulou C Economou V Sakkas H Gousia P Giannakopoulos X Dontorou C
Filioussis G Gessouli H Karanis P Leveidiotou S 2008 Microbiological quality ofindoor and outdoor swimming pools in Greece investigation of the antibiotic resistanceof the bacterial isolates Int J Hyg Environ Health 211385ndash397
Perkins SD Mayfield J Fraser V Angenent LT 2009 Potentially pathogenic bacteria inshower water and air of a stem cell transplant unit Appl Environ Microbiol 755363ndash5372
Pond K 2005 Water recreation and disease Plausibility of associated infections acute effectssequelae and mortality London WHO IWA Publishing
UNI EN ISO 162662008 ndash 18092008 ndashWater quality ndash Detection and enumeration ofPseudomonas aeruginosa ndash Method by membrane filtration
Villarreal JV Schwartz T Obst U 2010 Culture-independent techniques applied to foodindustry water surveillance ndash a case study Int J Food Microbiol 141(Suppl 1)S147ndash55
Wang MC Liu CY Shiao AS Wang T 2005 Ear problems in swimmers J Chin Med Assoc68347ndash352
World Health Organisation (WHO) 2006 Guidelines for safe recreational water environ-ments Vol 2 Swimming pools and similar environments Geneva WHO Press
Wilson IG 1997 Inhibition and facilitation of nucleic acid amplification Appl EnvironMicrobiol 633741ndash3751
70 G Amagliani et al
Dow
nloa
ded
by [
Bib
liote
ca U
nive
rsita
ria]
[G
iulia
Am
aglia
ni]
at 0
717
19
Janu
ary
2012
Nikaeen M Hatamzadeh M Vahid Dastjerdi M Hassanzadeh A 2009 Predictive indicatorsof the safety of swimming pool waters Water Sci Technol 603101ndash3107
Oliver JD 2005 The viable but nonculturable state in bacteria J Microbiol 4393ndash100Papadopoulou C Economou V Sakkas H Gousia P Giannakopoulos X Dontorou C
Filioussis G Gessouli H Karanis P Leveidiotou S 2008 Microbiological quality ofindoor and outdoor swimming pools in Greece investigation of the antibiotic resistanceof the bacterial isolates Int J Hyg Environ Health 211385ndash397
Perkins SD Mayfield J Fraser V Angenent LT 2009 Potentially pathogenic bacteria inshower water and air of a stem cell transplant unit Appl Environ Microbiol 755363ndash5372
Pond K 2005 Water recreation and disease Plausibility of associated infections acute effectssequelae and mortality London WHO IWA Publishing
UNI EN ISO 162662008 ndash 18092008 ndashWater quality ndash Detection and enumeration ofPseudomonas aeruginosa ndash Method by membrane filtration
Villarreal JV Schwartz T Obst U 2010 Culture-independent techniques applied to foodindustry water surveillance ndash a case study Int J Food Microbiol 141(Suppl 1)S147ndash55
Wang MC Liu CY Shiao AS Wang T 2005 Ear problems in swimmers J Chin Med Assoc68347ndash352
World Health Organisation (WHO) 2006 Guidelines for safe recreational water environ-ments Vol 2 Swimming pools and similar environments Geneva WHO Press
Wilson IG 1997 Inhibition and facilitation of nucleic acid amplification Appl EnvironMicrobiol 633741ndash3751
