Occurrence of virulence genes among Vibrio choleraeand Vibrio parahaemolyticus strains from treated wastewaters

Sadok Khouadja & Elisabetta Suffredini &Besma Baccouche & Luciana Croci & Amina Bakhrouf

Received: 15 September 2013 /Accepted: 30 June 2014# Springer International Publishing Switzerland 2014

Abstract Pathogenic Vibrio species are an importantcause of foodborne illnesses. The aim of this study wasto describe the occurrence of potentially pathogenic Vib-rio species in the final effluents of a wastewater treatmentplant and the risk that they may pose to public health.During the 1-year monitoring, a total of 43 Vibrio strainswere isolated: 23 Vibrio alginolyticus, 1 Vibrio cholerae,4 Vibrio vulnificus, and 15 Vibrio parahaemolyticus. ThePCR investigation of V. parahaemolyticus andV. cholerae virulence genes (tlh, trh, tdh, toxR, toxS,toxRS, toxT, zot, ctxAB, tcp, ace, vpi, nanH) revealedthe presence of some of these genes in a significantnumber of strains. Intraspecies variability and geneticrelationships among the environmental isolates were an-alyzed by random amplified polymorphic DNA-PCR(RAPD-PCR). We report the results of the first isolationand characterization of an environmentalV. cholerae non-O1 non-O139 and of a toxigenic V. parahaemolyticusstrain in Tunisia. We suggest that non-pathogenic Vibriomight represent a marine reservoir of virulence genes thatcan be transmitted between strains by horizontal transfer.

Keywords Vibrio parahaemolyticus . Vibrio cholerae .

Virulence genes . PCR

Introduction

The genus Vibrio includes more than 135 species, most-ly marine in origin, and its taxonomy is continuouslyupdated due to the addition of new species (Dawyndtet al. 2005; Thompson et al. 2009). Vibrio cholerae,Vibrio parahaemolyticus, and Vibrio vulnificus are seri-ous human pathogens. Cholera is a severe disease main-ly in developing countries as a result of poor watersupplies and sanitation, and the main route of contami-nation is via water and food (Thompson et al. 2004).Pathogenicity is associated mainly to O1 and O139serotypes of V. cholerae (Sechi et al. 2000; Gil et al.2004), while some V. cholerae non-O1 non-O139 sero-types can cause septicemia or skin lesions in associationwith diarrhea (Farina et al. 2000). V. cholerae is non-invasive, affecting the small intestine through adhesionto the epithelium and production of an enterotoxin,cholera toxin (CT) (Reidl and Klose 2002). Severalvirulence genes within the ToxR regulon are involvedin cholera disease. In addition to the essential role of CTin cholera, the toxin-coregulated pilus (TCP), encodedby the tcpA to tcpF genes, is pivotal for the colonizationof the intestine epithelium. TCP helps in microcolonyformation on the epithelial surface. Other colonizationfactors include mannose-fucose hemagglutinin, regula-tory proteins (ToxR/ToxS and ToxT), outer membraneporins, biotin and purine biosynthetic genes, the O

Environ Monit AssessDOI 10.1007/s10661-014-3900-9

S. Khouadja (*) : B. Baccouche :A. BakhroufLaboratoire d’Analyse, Traitement et Valorisation desPolluants de l’Environnement et des Produits, Département deMicrobiologie, Faculté de Pharmacie,Rue Avicenne, 5000 Monastir, Tunisiae-mail: khouadja_sadok@yahoo.fr

E. Suffredini : L. CrociReparto Adempimenti Comunitari e Sanità Pubblica,Dipartimento di Sanità Pubblica Veterinaria e SicurezzaAlimentare, Istituto Superiore di Sanità,Rome, Italy

antigen of the lipopolysaccharide, and accessory colo-nization factors (Reidl and Klose 2002; Thompson et al.2004).

A number of Vibrio species, other than V. cholerae,can cause disease in human mainly by the ingestion ofcontaminated water. V. parahaemolyticus, Vibriomimicus, and V. vulnificus are food-poisoning bacteriawhich are normal inhabitants of estuarine and marineenvironments. V. parahaemolyticus causes gastroenteri-tis in which the hemolysins, thermostable direct hemo-lysin (TDH), and/or TDH-related hemolysin (TRH)have been considered to play a crucial role (Nishibuchiet al. 1996). Among halophilic Vibrio, Vibrioalginolyticus (Zanetti et al. 2000), Vibrio fluvialis(Ahmed et al. 2004), and Vibrio metschnikovii are alsopathogenic for humans, while V. alginolyticus also rep-resents a pathogen for fishes and other marine animals(Khouadja et al. 2012; Ripabelli et al. 2003). Besides, inlight of heightened human dependence on marine envi-ronments for fisheries, aquaculture, waste disposal, andrecreation, the potential for pathogen emergence fromocean ecosystems remains a cause for concern. Assess-ment of water and wastewater is very crucial to safe-guard public health and the environment (Okoh andIgbinosa 2010). Water contaminated by effluents fromvarious sources is associated with heavy disease burden,and this could influence the current shorter life expec-tancy in the developing countries when compared to thatin developed nations (WHO 2002). Vibrio spp. arewidely distributed in effluent environments associatedwith domestic sewage, and their presence and abun-dance are important for the control of epidemiologicaland ecological site quality. In this study, we investigatedthe presence of fiveV. parahaemolyticus and V. choleraevirulence genes in 43 Vibrio strains, obtained from theeffluent of a wastewater treatment plant.

Materials and methods

Wastewater samples

The area of study included a wastewater treatment plantlocated on the eastern littoral of Tunisia, near to the cityof Mahdia (latitude 35° 30′ 0″ N, longitude 11° 3′ 36″E). The plant, which has a design capacity of 1,500 m3/day, receives domestic sewage and some light industrialwastewater, as well as runoff water, and treatment isbased on the activated sludge system. The final effluent

is released into a lagoon and then to the sea. Samplingwas carried out with a monthly frequency during theyear 2010 in three different points: (A) the final effluentin wastewater treatment plant, (B) the lagoon after thewastewater treatment plant, and (C) the surroundings ofthe contact point between the lagoon and the sea.

Isolation of Vibrio strains

For each point, a 200-mL water sample was collected ina sterilized container. The samples were filtered throughpolycarbonate membranes (0.2-μm pore size). Mem-branes were incubated in 5 mL of APW (1 % peptoneand 1 % NaCl, w/v; pH 8.6) for 6 h at 37±1 °C. Theenriched samples were streaked onto TCBS agar plates(Oxoid) and incubated overnight at 37±1 °C. Yellowand green colonies were randomly selected andsubcultured on Tryptic soy agar (TSA, Difco, Spain)supplemented with 1 % NaCl. The isolated bacteriawere frozen at −80 °C with 20 % (v/v) glycerol forfurther analysis.

Phenotypic characterization of bacterial strains

All strains were subjected to standard morphological,physiological, and biochemical plate and tube tests byusing the procedures described in Bergey’s Manual ofSystematic Bacteriology (Alsina and Blanch 1994). Theisolates were then identified by API 20 E (API SystemSA, France). Isolates identified as V. cholerae by mor-phological and biochemical features were taken fromplates, emulsified in saline, and mixed with an equalvolume of V. cholerae antiserum (Difco, USA) and wereexamined for antigen-antibody interaction.

Molecular identification

All isolates identified as Vibrio by biochemical testswere grown overnight at 37 °C on Tryptic soy agarsupplemented with 1 % NaCl. Chromosomal DNAwas extracted for each strain using the Wizard genomicDNA purification kit (Promega), and biochemical iden-tification was confirmed by means of previously pub-lished polymerase chain reaction (PCR) protocolstargeting toxR gene for the identification ofV. parahaemolyticus (Kim et al. 1999), the outer mem-brane protein (ompW) gene for V. cholerae (Nandi et al.2000), the collagenase gene for V. alginolyticus (DiPinto et al. 2005), and vvh gene for V. vulnificus

Environ Monit Assess

(Brauns et al. 1991). The primers used are summarizedin Table 1, while PCR conditions were those describedin the reference papers. V. parahaemolyticus ATCC17802, V. cholerae N16961, V. alginolyticus ATCC17749, and V. vulnificus ATCC 33149 were used aspositive controls for species identification andEcherichia coli ATCC 35218 as a negative control.

Detection of pathogenicity genes

PCR for the detection of pathogenicity genes was per-formed, by distinct assays, on all strains independentlyfrom species identification; primers used for the detec-tion of these genes are summarized in Table 2.

For the detection of toxR, toxS, toxT, toxRS, zot,nanH, ctxA, ace, and vpi genes, amplification reactionswere performed in a 25-μL reaction mixture containing3 μL of genomic DNA, 5 μL of 5× green GoTaq buffer,200 μM of each dNTP, 25 μM of each primer, and 1 Uof GO Taq DNA polymerase (all the reagents weresupplied by Promega, USA). The mixtures were incu-bated for 5 min at 94 °C, followed by 35 cycles ofamplifications. Each cycle of denaturation consisted ofa step of denaturation at 94 °C for 1 min, annealing for1 min, and primer extension for 2 min at 72 °C, followedby a final extension at 72 °C for 10 min. The annealingtemperatures were 58 °C for toxR, toxS, toxT, and toxRS,60 °C for zot, nanH, and ctxA, and 62 °C for ace and vpi(Colombo et al. 1994; Folgosa et al. 2001; Kim et al.1999; Koch et al. 1993).

For the detection of tlh, tdh, and trh genes, PCRreactions were performed in a total volume of 25 μLcontaining 2.5 μL of DNA sample, 2.5 μL of 10×buffer, 2.5 μL of 25 mMMgCl2, 200 μMof each dNTP,1 μM of each primer (Table 2), and 1.25 U of Taq DNApolymerase (all the reagents were supplied by Promega,USA). PCR reactions were carried out with the

following reaction conditions: denaturation at 94 °Cfor 3 min, followed by 30 cycles of denaturation at94 °C for 1 min, annealing at 58 °C for 1 min, andprimer extension at 72 °C for 1 min. A final extensionwas performed at 72 °C for 5 min (Bej et al. 1999).

For all assays, positive strains were confirmed threet ime s w i t h i ndependen t l y r ep e a t ed PCR .V. p a r a h a e m o l y t i c u s AT C C 1 7 8 0 2 ,V. parahaemolyticus ATCC 43996, and V. choleraeN16961 were used as positive control and E. coliATCC35218 as a negative control.

Gel electrophoresis

Five microliters of the PCR products was analyzedusing a 1 % agarose gel stained with ethidium bromide(0.5 μg/mL), visualized under UV transilluminator, andphotographed using Gel Doc XR apparatus (Bio-Rad,USA). The HyperLadder II (Promega, USA) was usedas a DNA molecular weight marker.

Detection of caseinase and haemolytic activity

The caseinase activity of the different strains was exam-ined using the single diffusion method with 1 % (w/v)agar gel containing 1.5 % (w/v) skimmed milk (Iyeret al. 2000). In brief, a straight line in the center of theagar gel plate was inoculated with each test strain. Afterincubation for up to 72 h at 37 °C, the formation of aclear zone caused by protein degradation is considered apositive test (Zanetti et al. 2000).

Hemolysin potency was evaluated using a modifica-tion technique of the plate assay previously described(Quindos et al. 1994). In brief, 10 μL of suspension(108 CFU/mL) was spotted onto Sheep Blood Agar,prepared by mixing 70 mL fresh sheep blood with1,000 mL TSA supplemented with 3 % NaCl

Table 1 PCR primers used for species identification

Species Target Primers Amplicon size (bp) References

V. parahaemolyticus toxR 5′- GTCTTCTGACGCAATCGTTG-3′ 366 Kim et al. (1999)5′- ATACGAGTGGTTGCTGTCATG-3′

V. alginolyticus collagenase 5′- CGAGTACAGTCACTTGAAAGCC-3′ 737 Di Pinto et al. (2005)5′- CACAACAGAACTCGCGTTACC-3′

V. cholerae ompW 5′- CACCAAGAAGGTGACTTTATTGTG-3′ 587 Nandi et al. (2000)5′- GAACTTATAACCACCCGCG-3′

V. vulnificus vvh 5′- CGCCGCTCACTGGGGCAGTGGCTG-3′ 387 Brauns et al. (1991)5′- CCAGCCGTTAACCGAACCACCCGC-3′

Environ Monit Assess

(Khouadja et al. 2012). The plates were incubated at37 °C for 24 h. Positive hemolytic potencywas recordedby the presence of a distinct translucent halo around theinoculum area. The lysis zones and colony diameterswere measured, and the ratio (equal to or larger than 1)was used as a hemolytic index to represent the intensityof hemolysin production by the tested strains. All theexperiments were repeated three times.

RAPD-PCR fingerprinting of the isolates

random amplified polymorphic DNA-PCR (RAPD-PCR)of the isolates was performed with the following reactionmixture: 2.0μL of Taq reaction buffer (100mMTris–HClpH 8.3, 500 mM KCl, 20 mM MgCl2, and 0.001 %gelatin), 0.125 mM dNTPs (Promega, USA), 1 U of TaqDNA polymerase (Promega, USA), 30 pM of each primer(P5: 5′-AACGCGCAAC-3′ and P6: 5′-CCCGTCAGCA-3′), and 40 ng of the DNA template in a total reactionvolume of 20 μL. Cycling conditions included an initial

denaturation cycle at 94 °C for 2 min, followed by 45cycles of denaturation at 94 °C for 1 min, primer anneal-ing at 40 °C for 1 min, and primer extension at 72 °C for2 min; amplification was concluded by a final extensionstep at 72 °C for 5 min. The ramp time from primerannealing to primer extension temperature was set at 30 s.

RAPD pat terns were analyzed using theBioNumerics software v. 7.0 (AppliedMaths, Belgium).Clustering was performed using the unweighted pairgroup method (UPGMA) and the Dice correlation coef-ficient with a position tolerance of 1.5 % and an 80 %similarity cutoff.

Results

Biochemical and molecular identification

Among all the isolates, 32 were identified as Vibriospecies by the API 20 E system, while PCR assay

Table 2 PCR primers used for virulence genes investigation

Target Primers Amplicon size References

Tlh 5′-AAAGCGGATTATGCAGAAGCACTG-3′ 450 pb Bej et al. (1999)5′-GCTACT TTCTAGCATTTTCTCTGC-3′

Tdh 5′-GTAAAGGTCTCTGACTTTTGGAC-3′ 269 bp Bej et al. (1999)5′-TGGAATAGAACCTTCATCTTCACC-3′

Trh 5′-TTGGCTTCGATATTTTCAGTATCT-3′ 500 bp Bej et al. (1999)5′-CATAACAAACATATGCCCATTTCCG-3′

toxR 5′-TGGTTTGGCGTGAGCAAGGTTT-3′ 595 pb Sechi et al. (2000)5′-GGTTATTTTGTCCGCCAGTGG-3′

toxS 5′-CCACTGGCGGACAAAATAACC-3′ 640 pb Sechi et al. (2000)5′-AACAGTACCGTAGAACCGTGA-3′

toxT 5′-TTGCTTGGTTAGTTATGAGAT-3′ 581 pb Sechi et al. (2000)5′-TTGCAAACCCAGACTGATAT-3′

toxRS 5′-GAGTCATATTGGTACTTAAATT-3′ 1397 pb Sechi et al. (2000)5′-AACAGTACCGTAGAACCGTGA-3′

Zot 5′-CGTCTCAGCATCAGTATCGAGTT-3′ 198 pb Colombo et al. (1994)5′-ATTTGGTCGCAGAGGATAGGCCT-3′

ctxAB 5′-TGAAATAAAGCAGTCAGGTG-3′ 779 pb Koch et al. (1993)5′-GGTATTCTGCACACAAATCAG-3′

Tcp 5′-TGTTGTTACGCTGGCTCAGC-3′ 465 pb Folgosa et al. (2001)5′-AAGTTGGCACTTCCTGGTGC-3′

Ace 5′-GCTTATGATGGACACCCTTTA-3′ 284 pb Colombo et al. (1994)5′-TTTGCCCTGCGAGCGTTAAAC-3′

Vpi 5′-GCAATTTAGGGGCGCGACGT-3′ 680 pb Sechi et al. (2000)5′-CCGCTCTTTCTTGATCTGGTAG-3′

nanH 5′-AAGCCTTACATTCGATGGAGAC-3′ 650 pb This study5′-TCGGCTGGATGTTGTCACG-3′

Environ Monit Assess

confirmed that 43 of the isolates belonged to the Vibriogenus. Detection of species target genes by PCR re-vealed the presence of V. alginolyticus (n=23),V. cholerae (n = 1), V. vulnif icus (n= 4), andV. parahaemolyticus (n=15). Eleven isolates (includingthe V. cholerae strain “S31”) gave concordant identifi-cation by both API 20 E and PCR, while three isolatesthat were V. parahaemolyticus and eight that wereV. alginolyticus according to PCR were identified re-spectively as Burkholderia gladioli and Aeromonashydrophila by the API 20 E. The V. cholerae S31 didnot agglutinate in either V. cholerae group O1 or groupO139 antiserum, therefore indicating a non-O1 non-O139 serotype.

Detection of pathogenicity genes

Results of virulence gene detection are summarized inTable 3. The operon toxR was found in fiveV. alginolyticus, in all V. parahaemolyticus, and in theV. cholerae strain S31. The expected toxS fragment wasamplified from the chromosome of the V. cholerae strainonly and from one strain of V. alginolyticus (S5). Fivestrains showed a positive amplification for the virulencepathogenic island (vpi) and the neuraminidase gene(nanH), while all strains tested were negative for theamplification of the zonula occludens toxin (zot), theace gene, the cholera toxin (ctxAB), and the toxincoregulated pilus (tcp). A non-specific amplicon wasproduced when ace primers were used to investigatethe homolog gene with one V. alginolyticus strain(S9). The tlh gene, encoding for the thermolabilehemolys in , was de t ec t ed in 16 s t r a in s o fV. parahaemolyticus and in 1 of V. alginolyticus,while the tdh gene (thermostable direct hemolysin)and trh gene (TDH-related hemolysin) were detectedin only 1 V. parahaemolyticus strain (S24) out of the43 Vibrio strains. None of the strains identified likeV. vulnificus showed the presence of any of the viru-lence genes tested in the study.

Caseinase and hemolytic activity

All V. parahaemolyticus strains showed weak hemo-lysis activity, except for strain S24 (tdh and trh pos-itive) that showed β-hemolysis (Table 3). Beta-hemolysis was evidenced also by all the strains withthe neuraminidase gene. Several strains showed bothcaseinase activity and hemolytic activity, in some

cases, as for the four V. vulnificus, despite the absenceof any other virulence gene.

RAPD-PCR analysis

RAPD-PCR of the three sets of strains (V. alginolyticus,V. parahaemolyticus, V. vulnificus) showed intraspecificdifferences in environmental strains. Thirty-five of the43 strains were typeable by RAPD, while 8 isolates wereuntypeable. After hierarchical clustering analysis withUPGMA, the patterns of the Tunisian isolates weregrouped into 16 clusters for the 20 typeableV. alginolyticus (Fig. 1), 9 clusters for the 13 typeableV. parahaemolyticus (Fig. 2), and 2 clusters for 2 type-able V. vulnificus isolates (Fig. 3), indicating a highgenetic diversity for V. parahaemolyticus andV. alginolyticus.

Most of the clusters included a single strain, but theclusters of V. alginolyticus with multiple strains in-cluded isolates from different periods and areas (e.g.,cluster with isolates S2 and S16 from sampling pointsB and A; S7 and S12 from sampling points B and C;isolates S37, S38, and S5 from sampling points A andC); in one case, isolates for different periods and areasshowed an identical profile (S14 and S36). InV. parahaemolyticus, instead, identical profiles weredetected only in isolates from the same area and thesame period (group S26, S27, and S30 and group S25,S28, and S29).

Discussion

According to epidemiologic investigation, fecally con-taminated water and food are the most common vehiclesfor cholera infection (Ceccarelli et al. 2006; Koch et al.1993). Cholera continues to be an important publichealth problem among poorer communities, particularlyin Africa which accounts for more than 90 % of theannual cholera cases notified to theWHO (WHO 2002).Only O1 and O139 serogroups are associated with ep-idemic cholera, but few cases of non-O1 and non-O139V. cholerae septicemia or skin lesions are associatedwith diarrhea, and in these cases, isolation of the organ-ism from stools has often been unsuccessful (Farinaet al. 2000). Although Vibrio species are autochthonousof the aquatic environment, the final effluents of waste-water treatment discharged into water sources add on tothe Vibrio population and also become a source of

Environ Monit Assess

Tab

le3

Resultsof

thetestsforpathogenicity

genespresence

Strains

Origin

Season

Species

tlhtdh

trh

toxR

toxS

toxT

toxR

Szot

ctxA

Btcp

ace

vpi

nanH

Caseinase

Hem

olisis

S1B

Winter

V.a

−−

−−

−−

−−

−−

−−

−−

−S2

BWinter

V.a

−−

−−

−−

−−

−−

−−

−−

−S3

BWinter

V.a

−−

−−

−−

−−

−−

−+

++

β

S4B

Winter

V.a

−−

−−

−−

−−

−−

−−

−−

−S5

CSp

ring

V.a

−−

−+

+−

−−

−−

−+

++

β

S6C

Spring

V.a

−−

−−

−−

−−

−−

−−

−−

−S7

BSp

ring

V.a

−−

−−

−−

−−

−−

−−

−−

−S8

BSp

ring

V.a

−−

−−

−−

−−

−−

−−

−−

−S9

ASu

mmer

V.a

−−

−+

−−

−−

−−

++

++

β

S10

ASu

mmer

V.a

+−

−+

−−

−−

−−

−−

−+

−S11

ASu

mmer

V.a

−−

−−

−−

−−

−−

−−

−−

−S1

2C

Summer

V.a

−−

−−

−−

−−

−−

−−

−−

−S1

3B

Summer

V.a

−−

−−

−−

−−

−−

−−

−−

−S1

4C

Summer

V.a

−−

−−

−−

−−

−−

−−

−−

−S1

5C

Summer

V.a

−−

−−

−−

−−

−−

−+

++

β

S16

ASu

mmer

V.a

−−

−−

−−

−−

−−

−−

−−

−S1

7C

Summer

V.a

−−

−−

−−

−−

−−

−−

−−

−S1

8C

Summer

V.a

−−

−−

−−

−−

−−

−−

−−

−S1

9C

Autum

nV.a

−−

−+

−−

+−

−−

−−

−+

α

S20

CAutum

nV.a

−−

−+

−−

+−

−−

−−

−+

α

S21

AWinter

V.p

+−

−+

−−

+−

−−

−−

−+

α

S22

CWinter

V.p

+−

−+

−−

+−

−−

−−

−+

α

S23

CWinter

V.p

+−

−+

−−

+−

−−

−−

−+

α

S24

BSp

ring

V.p

++

++

−−

+−

−−

−−

−+

β

S25

CSp

ring

V.p

+−

−+

−−

+−

−−

−−

−+

α

S26

CSp

ring

V.p

+−

−+

−−

+−

−−

−−

−+

α

S27

CSp

ring

V.p

+−

−+

−−

+−

−−

−−

−+

α

S28

CSp

ring

V.p

+−

−+

−−

+−

−−

−−

−+

α

S29

CSp

ring

V.p

+−

−+

−−

+−

−−

−−

−+

α

S30

CSp

ring

V.p

+−

−+

−−

+−

−−

−−

−+

α

S31

ASp

ring

V.c

−−

−+

+−

+−

−−

−+

++

β

S32

AWinter

V.V

−−

−−

−−

−−

−−

−−

−+

β

S33

AWinter

V.V

−−

−−

−−

−−

−−

−−

−+

β

Environ Monit Assess

nutrients which favor abundant growth and proliferationof the organism (Okoh and Igbinosa 2010). Severalauthors have emphasized that there is a broad con-sensus on the need to monitor the presence ofVibrio in the environment and to study their path-ogenicity potential in order to properly protect hu-man health. To this aim, PCR-based methods aresimple, specific, and helpful tools in the rapid iden-tification and characterization of Vibrio strains.Several of the strains that were isolated during ourstudy and that were analyzed with these techniquesare considered pathogenic to humans, and theirisolation in the effluents of a wastewater treatmentplant shows that pathogenic Vibrio can easily sur-vive the treatment processes applied to ruralwastewaters.

Recently, sporadic cases of non-O1 non-O139V. cholerae infections have been described inNorthern Africa and in Europe (Farina et al.2000). Non-toxigenic strains might be an importantreservoir of virulence genes. Some genes, includingctxA and tdh, may be horizontally transferred, lead-ing to new pathogenic strains (Nishibuchi et al.1996). Our results show that V. alginolyticus strainsoften possess homologs of the V. parahaemolyticusand V. cholerae virulence genes such as toxR, tlh,and VPI, which suggests that V. alginolyticus maybe an important reservoir of many known virulencegenes of other Vibrio species in the aquaticenvironment.

V. p a r a h a emo l y t i c u s a n d V. c h o l e r a epathogenicity-associated genes may be transferredto different Vibrio spp., and strains isolated from thecoastal waters suggest that the rise of a new patho-genic Vibrio from non-harmful species may be pos-sible in the aquatic environments. Although it hasbeen largely considered that environmental bacterialisolates lack those virulence genes which usually arefound in clinical strains, few recent studies indicatethat virulence genes, or their homologous, could alsobe present in strains from environmental sources andthat acquisition of such genes might have place in theaquatic environment (Sechi et al. 2000; Zanetti et al.2000). In this study, we have screened a collection ofVibrio strains isolated from final effluent wastewaterof an urban plant for the presence of virulence andfitness genes usually present in V. cholerae andV. parahaemolyticus pathogenic strains. None of thetes ted s t ra ins show the major V. choleraeT

able3

(contin

ued)

Strains

Origin

Season

Species

tlhtdh

trh

toxR

toxS

toxT

toxR

Szot

ctxA

Btcp

ace

vpi

nanH

Caseinase

Hem

olisis

S34

BWinter

V.V

−−

−−

−−

−−

−−

−−

−+

β

S35

ASp

ring

V.V

−−

−−

−−

−−

−−

−−

−+

β

S36

AAutum

nV.a

−−

−−

−−

−−

−−

−−

−−

−S3

7A

Autum

nV.a

−−

−−

−−

−−

−−

−−

−−

−S3

8A

Autum

nV.a

−−

−−

−−

−−

−−

−−

−−

−S3

9B

Summer

V.p

+−

−+

−−

−−

−−

−−

−+

−S4

0B

Summer

V.p

+−

−+

−−

−−

−−

−−

−+

−S4

1C

Summer

V.p

+−

−+

−−

−−

−−

−−

−+

−−S4

2C

Autum

nV.p

+−

−+

−−

−−

−−

−−

−+

α

S43

CAutum

nV.p

+−

−+

−−

−−

−−

−−

−+

α

%34.8

2.3

2.3

48.8

4.65

030.23

00

02.3

1211.6

62.7

53

V.a=V.alginolyticus,V.p

=V.parahaem

olyticus,V.c

=V.cholerae,V.V

=V.vulnificus,β=β-hem

olytic,α

=weakhemolysis;A

=finaleffluentinwastewatertreatm

entplant,B

=inthe

lagoon

justafterwastewater

treatm

entp

lant,and

C=finally

around

thecontactp

oint

betweenthelagoon

andthesea

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Fig. 2 RAPD dendrogram generated by BioNumerics software,showing the relationship of fingerprints for V. parahaemolyticusisolates; a final effluent in wastewater treatment plant, b lagoon

after the wastewater treatment plant, and c surroundings of thecontact point between the lagoon and the sea

Fig. 1 RAPD dendrogram generated by BioNumerics software,showing the relationship of fingerprints for V. alginolyticus iso-lates; a final effluent in wastewater treatment plant, b lagoon after

the wastewater treatment plant, and c surroundings of the contactpoint between the lagoon and the sea

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pathogenicity-associated genes (ctxAB, zot and ace),not even the V. cholerae S31 that we isolated. But, weobserved that in all the strains where we detect thepresence of the VPI gene, the neuraminidase gene(nanH) was also present. The gene nanH, consideredas a virulence-associated gene in V. cholerae, hasbeen detected in 11.6 % of our environmental strains.Several authors explain that the nanH gene is includ-ed in the V. cholerae pathogenicity island VPI-2(Jermyn and Boyd 2002). The possibility for a non-pathogenic Vibrio strain of acquiring an entire orpartial pathogenicity island is highly relevant, asVPI-2 is involved in bacterial virulence, and its rolein pathogenicity is supported by its detection in path-ogenic V. cholerae isolates and its absence in non-pathogenic strains (Jermyn and Boyd 2005). Thepresence of nanH on a pathogenicity island suggeststhat it was acquired by horizontal transfer.

While RAPD-PCR protocols are able to groupthe different Vibrio isolates into several broad cat-egories that seem to correlate fairly well withenvironmental sources, they do not permit finerdiscriminations that are needed for epidemiologicaland ecological studies. In the present study, nocorrelation was observed between the presence ofvirulence genes, origin, and different clusters givenby phylogenetic analysis, as the strains positive forthe virulence genes appeared in more than onecluster.

The V. cholerae strain that we isolated does notpresent any of the genes which are considered asvirulence factors. This is, to our knowledge, thefirst isolation and characterization of an environ-mental non-O1, non-O139 V. cholerae strains inTunisia. Non-O1 non-O139 V. cholerae has beenproven capable of causing human disease both involunteer studies and in isolation from patient(Chen et al. 2007). Many extracellular proteases

are suggested to play important roles in virulenceof Vibrio spp. Many Vibrio are pathogenic forhumans and/or marine vertebrates and invertebrates,with the virulence mechanisms reflecting the pres-ence of enterotoxin, hemolysin, cytotoxin, protease,lipase, phospholipase, siderophore, adhesive factor,and/or hemagglutinins (Zhang and Austin 2005). Aprotease was identified as the major virulence fac-tor, for example, in a clinical tdh- and trh-negativeV. parahaemolyticus isolate, and this enzymeshowed cytotoxic activity on CHO and Vero cells(Ottaviani et al. 2005). Environmental Vibrio can bepathogenic and can cause infections despite theabsence of tdh and trh genes or ctxAB, zot, andace genes. This study confirms the presence inenvironmental Vibrio of different virulence factorsbesides these toxins, whose contribution to theenteropathogenesis is still to be explained.

Conclusion

We report the first isolation and characterization ofenvironmental V. cholerae non-O1 non-O139 andtoxigenic V. parahaemolyticus in Tunisia. Non-toxigenic strains might be an important reservoir ofvirulence genes. Some genes, including ctxA and tdh,may be horizontally transferred, leading to new path-ogenic strains. Study of the pathogenic microfloraincluding Vibrionaceae family would give a betteridea about a possible circulation of these bacteriafrom the wastewater plants to the marine area and toestablish the clinical significance of these microor-ganisms. In this study, Vibrio strain does not presentany virulence factor known in epidemic strains, but itis necessary to monitor the various sources of waterdischarge in order to prevent the risks that they poseto public health.

Fig. 3 RAPD dendrogram generated by BioNumerics software,showing the relationship of fingerprints for V. vulnificus isolates; afinal effluent in wastewater treatment plant, b lagoon after the

wastewater treatment plant, and c surroundings of the contact pointbetween the lagoon and the sea

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Acknowledgments The authors thank Pr. Mauro M Colombofor assistance with reference strains.

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