Research ArticlePrevalence of Panton-Valentine LeukocidinGene among Community Acquired Staphylococcus aureusA Real-Time PCR Study
Amit Karmakar Debarati Jana Kunal Dutta Parimal Dua and Chandradipa Ghosh
Microbiology Laboratory Department of Human Physiology with Community Health Vidyasagar UniversityPaschim Medinipur West Bengal 721102 India
Correspondence should be addressed to Chandradipa Ghosh chandradipaghosh72gmailcom
Received 31 January 2018 Revised 10 July 2018 Accepted 5 August 2018 Published 2 September 2018
Academic Editor Alexander Rodriguez-Palacios
Copyright copy 2018 Amit Karmakar et alThis is an open access article distributed under the Creative Commons Attribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited
Panton-Valentine leukocidin (luk-pv) is a cytotoxin that causes leukocyte destruction and tissue necrosis The aim of this studywas to determine the prevalence of the pv1 mecA and nuc genes in Staphylococcus aureus isolates obtained from anterior naresand superficial infection sites of skin in a slum population of West Bengal India Expression level of pv1 gene was also analysedTwenty-two S aureus strains were isolated and phenotype and genotype specific examinations for S aureus isolates were carriedout Molecular identification was done by PCR using species-specific 16S rRNA primer pairs and finally 22 isolates were found to bepositive as S aureus The antibiotic responsiveness of all these isolates and the minimum inhibitory concentration (MIC) of MRSAisolates were determined using the broth dilution method with vancomycin Antibiogram analysis of isolated S aureus strains withrespect to different antimicrobial agents revealed antibiotic resistance ranging from 27 to 91 The results of MIC for vancomycinshowed 95 of strains to be VSSA and 5 to be VISA 68 isolates were resistant to methicillin All the isolates were subjected todetection of pv1 mecA and nuc genes and 9 68 and 27 were found to harbour pvl mecA and nuc genes respectively All theMRSA strains produced high to moderate levels of biofilm pvl gene expression was carried out in vitro by Real-Time PCRThe low998779Ct value (0493) was indicative of high expression of pvl in one S aureus strainThus detection of pvl gene in community acquiredS aureus indicates the emergence of pathogenic S aureus in community setup in the studied region The existing exploration isextremely imperative and informative for the high level multi-drug resistant S aureus infections inclusive of MRSA
1 Introduction
To date the major human pathogen Staphylococcus aureushas become a threat to our lives because of elaboration of sev-eral different virulence factors Panton-Valentine leukocidin(PVL) is one of the most important virulence factors of Saureus This beta pore forming cytotoxin is associated withtissue necrosis and also causes disruption of leukocyte mem-branes [1] The genetic material of bacteriophage has greatcontribution for producing PVL cytotoxin PVL carrying Saureus strains are more virulent and highly transmissiblestrains than PVL negative S aureus pvl gene that encodesPVL cytotoxin comprises two exoprotein subunits encodedby LukS-PV and LukF-PV [2 3] These two co-transcribedgenes act together as a subunit to form a pore by assemblingin the cell membranes of host immune cells particularly the
white blood cells monocytes and macrophages [4] PVLcarrying S aureus is responsible for different life-threateninginvasive diseases and also skin and soft tissue infectionsPVL-SA infected skin is red and inflamed with pus It canhave different other appearances like cellulitis abscessesboils folliculitis etc At first PVL carrying S aureus infectsskin and soft tissues but the infection gradually spreads tothe lung and disrupts the lung tissues causing hemorrhagicnecrotizing pneumonia one of the most lethal diseasescaused by S aureus [5]
In recent time there have been an overall increase inthe prevalence of pvl positive S aureus worldwide Butvariable rates of prevalence have been reported fromdifferentcountries ie 128 in China [6] 30 in Germany [7] 453in Japan and most remarkably 97 in USA [8]
HindawiJournal of PathogensVolume 2018 Article ID 4518541 8 pageshttpsdoiorg10115520184518541
2 Journal of Pathogens
Table 1 Source data of collected samples according to collection site sex and age groups of infected individual
Superficial infection site (n=5) Nasal swab (n=20)Male (n=3) Female (n=2) Male (n=7) Female (n=13)
le 30 years ge 31 years le 30 years ge 31 years le 30 years ge 31 year119904 le 30 year119904 ge 31 year1199042 1 1 1 4 3 8 5
∘
E
∘
E
∘
E
∘
E
∘
N∘
N
∘
N∘
N
N
W E
S
KM
Midnapore TownSlum Area
Paschim Medinipur
Midnapore Town
West Bengal
Paschim Medinipur
India
West BengalNepali para
Tantigeria
KabardangaRangamati
Arobindo nagar
Keranitola Golkua chak
Battala chak
Bus stand
Figure 1 Study region including different slum areas in Midnapore town (Lat 22∘ 2310158404157 N to 22∘ 261015840220210158401015840 N Long 87∘171015840236510158401015840 E to 87∘201015840 153510158401015840 E)
To date PVL has become most important and signifi-cant virulence factor of community acquired S aureus Theprevalence of pvl genes among MRSA isolates has not beenadequately reported from IndiaThis studywas undertaken toinvestigate the PVL prevalence and pvl expression level alongwith certain other virulentmarkers from state ofWest BengalIndia The study area is demonstrated in Figure 1
2 Materials and Methods
21 Sample Collection A total of 25 non-repeat communitystrains of S aureus were collected from human subjects ofdifferent slum areas at Midnapore town (Lat 22∘231015840415710158401015840 Nto 22∘261015840220210158401015840 N Long 87∘171015840236510158401015840 E to 87∘201015840153510158401015840 E) inthe state ofWest Bengal India fromNovember 2015 toMarch2016 The protocols in present study with human subjects
got ethical clearance from Institutional Review Board (IRV)who follows the Indian Council of Medical Research (ICMR)ethical guidelines for biomedical research on human subjectsBacterial isolates were categorised into three groups accord-ing to the site of collection age group and sex (Table 1) Forsample collection sterile swabs with rayon tips were usedfor moist wound it was used directly but for dry wound itwas moistened with sterile saline so that it could increase thechance of recovery of the organism from the infected sites
22 Isolation and Identification of S aureus All the sampleswere transferred into 2 gm Luria broth (one for each speci-men) and incubated at 37∘C in shaking incubator After 16-18hours all the samples were inoculated onto mannitol salt agarplate and incubated for 24-25 hours at 37∘C [14] All yellowpigmented colonies were inoculated into LB agar for culture
Journal of Pathogens 3
preservation at 4∘C The resulting growth from respectiveplates of media was examined for colony characteristic andmorphology Each culture underwent Gram staining andwas tested for production of catalase free coagulase yellowpigment and thermonuclease (TNase) according to methoddescribed by Lancette and Tatini [15]
23 DNA Extraction from Bacterial Culture Isolates Forextraction of DNA from bacterial culture the rapid boilingmethod was followed [14] In brief 4 to 5 single coloniesfrom overnight grown culture were inoculated in 200120583l ofsterile Milli-Q water and boiled at 99∘C for 10 minutesAfter centrifugation at 14000 rpm for 10min the supernatantwas collected in a sterile microcentrifuge tube gently Theresultant template DNA was stored at minus20∘C and 2 120583l of eachsample was used for PCR analysis
24 Molecular Identification through PCR Amplification of16S rRNA Gene Phenotypically isolated and confirmed Saureus isolates were further tested by PCR amplificationassay using primer pairs (Table 3) for species-specific 16SrRNA gene These primers amplified 228 bp fragment from16S rRNA gene of these isolated S aureus strains The PCRprocedure was carried out in a 25120583l PCR tube and each ofthe reaction mixtures contained 25120583l PCR buffer (10X) 1 120583l(1U120583l) of Taq DNA polymerase 2 120583l dNTPs (200mM each)1120583l of each primer (10 pmol120583l) 25120583l (10 ng120583l) of templateDNA and 1575120583l PCR grade water The amplifications tookplace by the process of PCR thermocycling in a thermalcycler (Eppendorf Germany) commencing with an initialdenaturation at 94∘C for 2minutes followed by 33 cycles eachconsisting of denaturation at 94∘C for 30 sec annealing at50∘C for 30 sec and polymerization at 72∘C for 45 sec witha final extension at 72∘C for 4minutes S aureusATCC 25923was used as positive control in this experiment
25 Biochemical Characterization of S aureus Strains Thebacterial strains which were confirmed as S aureus byspecies-specific 16S rRNA were further evaluated by differ-ent biochemical tests including mannitol fermentation andgrowth on high salt concentration gelatin hydrolysis ureahydrolysis protease activity on milk agar medium lipaseproduction on egg yolk agar medium (HiMedia Mumbai)and hydrolysis of esculin by standard method [16ndash19] andcongo red agar (CRA) test [20]
26 Biofilm Assay Biofilm assay was done in borosilicateglass tubes according to the method described by Watnicket al [21] Briefly the isolates were grown in 3ml of Luriabroth overnight Then 5120583l of the overnight culture was addedto 500120583l of sterile LB in test tubes to make a dilution of1100 Test tubes were kept standing statically for 24 hrs andafter that the test tubes were rinsed vigorously with distilledwater to remove all nonadherent cells Further 600120583l of 01(wv) crystal violet was added and incubated for 30 minutesTest tubes were then rinsed vigorously with distilled water1ml DMSO was added to it followed by vortexing Afterincubation for 10 minutes optical density was measured at
570nm Each assay was performed in triplicate The meanvalues of optical densities obtained were categorised intothree classes as suggested by Cha et al [22] such that thestrains with OD
570lt 02 02ge OD
570le10 and OD
570gt 10
were defined as biofilm nonformers and biofilm formers ofweak level and strong level respectively
27 Antibiotic Susceptibility Profile of Isolated S aureus Theantibiotic susceptibility pattern of S aureus isolates wasdone by disk diffusion method using commercial antibioticdisks procured from HiMedia Mumbai [23] The antibioticdisks used were as follows ampicillin (30120583g) nalidixicacid (30120583g) chloramphenicol (30120583g) streptomycin (10120583g)kanamycin (30120583g) cefoxitin (30120583g) novobiocin (30120583g) anderythromycin (10120583g) Antibiotic susceptibility of those iso-lates was evaluated according to the Clinical and LaboratoryStandard Institute (CLSI) [23]Minimum InhibitoryConcen-tration (MIC) of vancomycin was done by the broth dilutionmethod to distinguish VSSA from VRSA according to theClinical and Laboratory Standard Institute (CLSI 2015) [24]
28 Detection ofmecA nuc and pvl Gene Detection ofmecAnuc and pvl genes of S aureuswas done by PCR amplificationassay using gene-specific primer pairs (Table 3) The PCRprocedure was carried out in a 25120583l PCR tube and each ofthe reaction mixtures contained 25120583l PCR buffer (10X) 1 120583l(1U120583l) of Taq DNA polymerase 2 120583l dNTPs (200mM each)1 120583l of each primer (10 pmol120583l) 25120583l (10 ng120583l) of templateDNA and 1575120583l PCR grade water The amplifications tookplace by the process of PCR thermocycling in a thermalcycler (Eppendorf Germany) commencing with an initialdenaturation at 94∘C for 2 minutes followed by 33 cycleseach comprising 94∘C for 1 minute and annealing at 55∘C for45 sec 50∘C for 50 sec and 50∘C for 45 sec formecA nuc andpvl gene respectively and a common extension of 72∘C for45 sec with a final extension at 72∘C for 4 minutes A positivecontrol of ATCC 33591 ATCC 25923 and ATCC 49775 Saureus was used respectively in each of these experimentsThe PCR amplicons were visualized in agarose gel containingethidiumbromide (1 120583gml) under Gel DocXR system (Bio-Rad USA) and photographs were taken for image analysisFragments of DNA 147 bp 270 bp and 433 bp correspondedtomecA nuc and pvl gene respectively
29 Real-Time Polymerase Chain Reaction of pvl GeneTotal RNA was extracted using Pure Link RNA MiniKit (Invitrogen Carlsbad CA) and quantified using UV-Visspectrophotometer (Nano Drop 2000 Thermo Fisher) TotalRNA was then converted to cDNA by using Verso cDNAsynthesis kit (Thermo Scientific USA)The standard reactioncontained 1x Power SYBR Green PCR master mix (AppliedBiosystems) primers pairs (Table 3) and cDNA as templatestrand The temperature program for 40 cycles was set todenaturation at 94∘C for 1 minute and annealing at 55∘C for30 sec The reaction was conducted on Step One Plus 96-WellReal-Time PCR System (Applied Biosystems) The sampleswere analysed in triplicate and recA was used as endogenouscontrol for normalization [25]
4 Journal of Pathogens
Table 2 Biochemical identification and characterization of S aureus
TestsSource of the isolates Total
Superficial infection site Nasal swabNolowast () Nolowast () Nolowast ()
210 Statistical Analysis Data analysis and plotting of datawere done using Microsoft Excel Mean and standard error ofmean was also calculated for each quantitative variable usingMicrosoft Excel
3 Results
A total of 25 nonduplicate isolates of S aureus were obtainedfrom different slum areas in Midnapore town West BengalIndia and included in this study Samples were collectedfromnasal swab and superficial infection sites of the subjectsAmong 25 isolates 22 strains (88) were isolated usingselective MSA media and then these isolates were identifiedas S aureus by several phenotypic expressions by means ofdifferent biochemical tests (Table 2) All S aureus isolates inthis study were confirmed by PCR using the species-specificprimer pair (Table 3) In this study we found that 100 91and 23 isolates were positive for catalase coagulase andheat-stable nuclease respectivelyOf the isolated strains 6855 and 68 produced protease lipase and nonwhite pig-mented colonies respectively Rates of resistance of S aureus
ranged from 27 to 91 to different antibiotics Resistance of Saureus isolates to ampicillin (91) nalidixic acid (91) chlo-ramphenicol (36) streptomycin (27) kanamycin (55)cefoxitin (68) novobiocin (64) and erythromycin (82)was found S aureus isolates showing resistance to at leastone agent from three or more antimicrobial categories islabeled as multi-drug resistant Accordingly all S aureusstrains isolated in this study were found to be multi-drugresistant In the present study the prevalence rate of MRSAwas found to be 68 It was also found that 5 of theisolated S aureus were vancomycin intermediate and 95of the isolated S aureus were vancomycin sensitive None ofthe strains were found to be VRSA In general observationsslime-producing strains appear as black colonies whereasnon-slime-producing strains appear as red colonies in CRAplates Among the community acquired isolates 15 out of22 (68) S aureus strains appeared to be slime-producingafter 48 hrs of incubation at 37∘C Moreover in the presentstudy 7 (32) were found as strong biofilm forming and15 (68) isolates were moderate biofilm forming None ofthe isolates were biofilm-negative (Figure 2) Twenty-seven
Journal of Pathogens 5
02468
10121416
lt02 02-1 gt1Low Biofilm Former Moderate Biofilm Former High Biofilm Former
No of strains
Figure 2 Biofilm formation ability of community strains from slum area
228 bp
M 1 2 3 4 5 6 7 8 9 10 11 12 1413
(a)
270 bp
M 1 2 3 4 5 6 7
(b)
M 1 2 3 4 5 6 7
147 bp
1000 bp
500 bp
200 bp
(c)
M 1 2
433 bp
(d)
Figure 3 (a) Agarose gel electrophoresis pattern for identification of Staphylococcus aureus specific 16S rRNA gene M molecular weightmarker Lane 1-13 different clinical strains of Staphylococcus aureus Lane 14 negative control Escherichia coli (SM10 120582pir) (b) nuc geneM DNA molecular weight marker (100 bp DNA ladder) Lane 1 positive control Lanes 2 3 4 and 5 270 bp band obtained with DNAfrom clinical strains Lane 6 negative control (Staphylococcus epidermidis) (c) Agarose gel electrophoresis patterns showing PCR-amplifiedproducts for the mecA gene M DNA molecular weight marker (100 bp DNA ladder) Lane 1 positive control Lanes 2 3 4 5 and 6 147 bpband obtained with DNA from MRSA clinical strains Lane 7 negative control (Staphylococcus epidermidis) (d) Agarose gel electrophoresispatterns showing PCR-amplified products for the pvl gene Lanes 1 and 2 433 bp band obtained with DNA fromMRSA clinical strains
percent (27) strains were found to harbour nuc gene(Figure 3) mecA and pvl genes were detected in 17 out of22 (68) and 2 out of 22 isolates (9) respectively All thepvl harbouring isolates possessed mecA gene (Figure 3) Thelow 998779Ct value (0493) in qRT-PCR was indicative of highexpression of pvl in one S aureus strain (Figure 4)
4 Discussion
In the present study very high rates of resistance particu-larly towards penicillin and also for kanamycin cefoxitinnovobiocin and erythromycin were observed in the isolatedcommunity acquired S aureus strains In 2005 Kazakova
6 Journal of Pathogens
recA pvlGenes
23
235
24
245
25
255
26
265
27
CT M
ean
Figure 4 Comparative analysis of the cycle threshold by qRT-PCRassay Black bar represents the housekeeping gene recA and graybar represents pvl gene
et al [26] reported a community-associated MRSA (CA-MRSA) clone isolated from US football players with skinabscesses The strain was susceptible to most antimicrobialagents except 120573-lactams and macrolides Bhatta et al [27]reported the detection of mecA gene in 568 of the isolatesfrom clinical and community settings In this study outof twenty-two clinical isolates fifteen (68) isolates turnedout to be slime producer Jain and Agarwal [28] reporteda higher rate where 64 of staphylococcal intravascularisolates were biofilm producers by CRA method Zalipouret al [29] reported that forty-three (544) biofilm formingstrains were found in clinical specimens in Iran by CRAassay In the current study seven (32) isolates were strongbiofilm formers and fifteen (68) isolates were moderatebiofilm formers Mathur et al [30] reported that 578 ofstaphylococcal clinical isolates established biofilm-positivecharacteristics and 1447 and 394 exhibited high andmoderate biofilm production respectively in India Globalemergence of MRSA is serious public health problem andchallenge to clinicians Various factors devote to drug resis-tance and the pathogenicity of S aureus The first PVLpositive MRSA was noticed in the late 1990s and these strainsgot scattered worldwide in recent years [31] The role of PVLin boosting virulence of S aureus and their pathogenicityis being deliberated Panton-Valentine leukocidin rises thepathogenicity of S aureus by necrosis quickening apoptosisand damage of polymorphonuclear and mononuclear cellsthereby contributing to mortality and morbidity [32] PVL isgenerally used as a marker for community acquired MRSAliable for deep dermal infections including soft tissue [3334] However the worldwide scheme of PVL among MRSAisolates varies A lower prevalence of PVL has been reportedfrom other parts of world (5 in France 49 in UK81 in Saudi Arabia and 143 in Bangladesh) [32 35ndash37] reflecting the significant variation in prevalence of PVLamong geographical areas and communities Kaur et al [38]from India have reported overall 6285 prevalence of PVL
among MRSA and MSSA (MRSA 851 and MSSA 488)which delineates higher prevalence of PVL among MRSA incomparison to present findings Johnsson et al [39] detectedpvl gene in one isolate (1) from 65 patients with S aureusbacteraemia in two (219) isolates from 91 patients withcutaneous infections and in four (727) isolates from 55patients with respiratory tract infections Rostamzad et al[40] reported that out of thirty-two MRSA isolates thirteenisolates (4062)were positive for presence of the luk-pv genefrom hospitals isolates Higher prevalence of PVL amongchildren (lt14 years of age) was observed as compared toadults and old age group patients Similar observations weremade in another study from India [41] Epidemiological datasuggest that high virulence of community acquired MRSA isassociated with pvl gene but direct evidence of association ofPVL to pathogenesis remains limited [42]The low998779Ct value(0493) in qRT-PCR indicated high expression of pvl gene inS aureus strain in the present study However in previousstudy low expression of luk-pvwas reported [43] In S aureusexpression of toxins and others factors can be measured byusing Panton-Valentine leukocidin (PVL) expression in invivo condition [44] In addition expression of pvl gene isenhanced by sub-MIC values of 120573-lactam antibiotics [45]The studied population has a little health education andthey commonly use 120573-lactam antibiotics to treat any kindof infections this may be the reason of high expression ofpvl gene in in vitro condition Thus detection of pvl genein community isolated S aureus is indication of emergenceof pathogenic S aureus in community setup in the studiedregion
5 Conclusion
The current study reflects the elevated level of multi-drugresistant S aureus infections in community The prevalenceof the pvl gene among the MRSA isolates in this studywas low The presence of pvl among multi-drug resistantbacteria like MRSA may be fatal and challenging conditionfor clinicians The studied population has a little healtheducation and they commonly use 120573-lactam antibiotics thismay be the underlying reason for high expression of pvlThusdetection of pvl in community isolated S aureus indicates theemergence of pathogenic S aureus in community setup in thestudied region
Data Availability
The data used to support the findings of this study areavailable from the corresponding author upon request
Conflicts of Interest
The authors declare that there are no conflicts of interestregarding the publication of this paper
Acknowledgments
The authors are grateful to the individuals who participatedin the study
Journal of Pathogens 7
References
[1] B Shrestha W Singh V S Raj B M Pokhrel and T MMohapatra ldquoHigh Prevalence of Panton-Valentine leukocidin(PVL) genes in nosocomial-acquired staphylococcus aureusisolated from tertiary care hospitals in Nepalrdquo BioMed ResearchInternational vol 2014 Article ID 790350 7 pages 2014
[2] J Kaneko and Y Kamio ldquoBacterial two-component and hetero-heptameric pore-forming cytolytic toxins structures pore-forming mechanism and organization of the genesrdquo BioscienceBiotechnology and Biochemistry vol 68 no 5 pp 981ndash10032004
[3] G Prevost B Cribier P Couppie et al ldquoPanton-valentineleucocidin and gamma-hemolysin from Staphylococcus aureusATCC 49775 are encoded by distinct genetic loci and havedifferent biological activitiesrdquo Infection and Immunity vol 63no 10 pp 4121ndash4129 1995
[4] D C Melles W B Van Leeuwen H A M Boelens J KPeetersH AVerbrugh andAVanBelkum ldquoPanton-Valentineleukocidin genes in Staphylococcus aureusrdquoEmerging InfectiousDiseases vol 12 no 7 pp 1174-1175 2006
[5] B McGrath F Rutledge and E Broadfield ldquo NecrotisingPneumonia rdquo Journal of the Intensive Care Society vol 9 no2 pp 170ndash172 2008
[6] F Yu Z Chen C Liu et al ldquoPrevalence of Staphylococcusaureus carrying Panton-Valentine leukocidin genes among iso-lates from hospitalised patients in Chinardquo Clinical Microbiologyand Infection vol 14 no 4 pp 381ndash384 2008
[7] S Monecke P Slickers M J Ellington A M Kearns andR Ehricht ldquoHigh diversity of Panton-Valentine leukocidin-positive methicillin-susceptible isolates of Staphylococcusaureus and implications for the evolution of community-associatedmethicillin-resistant S aureusrdquoClinicalMicrobiologyand Infection vol 13 no 12 pp 1157ndash1164 2007
[8] D J Skiest K Brown T W Cooper H Hoffman-RobertsH R Mussa and A C Elliott ldquoProspective comparison ofmethicillin-susceptible and methicillin-resistant community-associated Staphylococcus aureus infections in hospitalizedpatientsrdquo Infection vol 54 no 5 pp 427ndash434 2007
[9] S R Monday and G A Bohach ldquoUse of multiplex PCR todetect classical and newly described pyrogenic toxin genes inStaphylococcal isolatesrdquo Journal of Clinical Microbiology vol 37no 10 pp 3411ndash3414 1999
[10] O G Brakstad K Aasbakk and J A Maeland ldquoDetection ofStaphylococcus aureus by polymerase chain reaction amplifica-tion of the nuc generdquo Journal of Clinical Microbiology vol 30no 7 pp 1654ndash1660 1992
[11] K Zhang J-A McClure S Elsayed T Louie and J MConly ldquoNovel multiplex PCR assay for characterization andconcomitant subtyping of staphylococcal cassette chromosomemec types I to V in methicillin-resistant Staphylococcus aureusrdquoJournal of Clinical Microbiology vol 43 no 10 pp 5026ndash50332005
[12] J-A McClure J M Conly V Lau et al ldquoNovel multiplexPCR assay for detection of the staphylococcal virulence markerPanton-Valentine leukocidin genes and simultaneous discrimi-nation of methicillin-susceptible from -resistant staphylococcirdquoJournal of Clinical Microbiology vol 44 no 3 pp 1141ndash11442006
[13] T Nuryastuti H C van der Mei H J Busscher R Kuijer A TAman and B P Krom ldquorecA mediated spontaneous deletionsof the icaADBC operon of clinical Staphylococcus epidermidis
isolates a new mechanism of phenotypic variationsrdquo Antonievan Leeuwenhoek-Journal ofMicrobiology vol 94 no 2 pp 317ndash328 2008
[14] A Karmakar P Dua and C Ghosh ldquoBiochemical and molec-ular analysis of Staphylococcus aureus clinical isolates fromhospitalized patientsrdquo Canadian Journal of Infectious Diseasesamp Medical Microbiology vol 2016 Article ID 9041636 7 pages2016
[15] G A Lancette and S R Tatini ldquoStaphylococcus aureusrdquo inCompendium of Methods for the Microbiological Examination ofFoods C Vanderzant and D F Splittstoesser Eds pp 533ndash550American Public Health Association Washington DC USA3rd edition 1992
[16] E B Blair J S Emerson and A H Tull ldquoA new mediumsalt mannitol plasma agar for the isolation of Staphylococcusaureusrdquo American Journal of Clinical Pathology vol 47 no 1pp 30ndash39 1967
[17] G H Chapman ldquoThe significance of sodium chloride in studiesof staphylococcirdquo Journal of Bacteriology vol 50 pp 201ndash2031945
[18] R Cruickshank J P Duguid B PMarmion andRHA SwainMed Microbiol vol II Chruchill Livingstone Edinburgh UK12nd edition 1975
[19] A Swan ldquoThe use of a bile-aesculin medium and of Maxtedrsquostechnique of Lancefield grouping in the identification of ente-rococci (Group D Streptococci)rdquo Journal of Clinical Pathologyvol 7 no 2 pp 160ndash163 1954
[20] C R Arciola L Baldassarri and L Montanaro ldquoPresenceof icaA and icaD genes and slime production in a collectionof Staphylococcal strains from catheter-associated infectionsrdquoJournal of Clinical Microbiology vol 39 no 6 pp 2151ndash21562001
[21] P IWatnickCM LaurianoK EKlose LCroal andRKolterldquoThe absence of a flagellum leads to altered colony morphologybiofilm development and virulence in Vibrio cholerae O139rdquoMolecularMicrobiology vol 39 no 2 pp 223ndash235 2001
[22] J-O Cha J I Yoo J S Yoo et al ldquoinvestigation of biofilmformation and its association with the molecular and clinicalcharacteristics of methicillin-resistant staphylococcus aureusrdquoOsong Public Health and Research Perspectives vol 4 no 5 pp225ndash232 2013
[23] AW BauerWMKirby J C Sherris andMTurck ldquoAntibioticsusceptibility testing by a standardized single disk methodrdquoAmerican Journal of Clinical Pathology vol 45 no 4 ts pp 493ndash496 1966
[24] CLSI ldquoPerformance standards for antimicrobial susceptibilitytesting twenty-fifth informational supplementrdquo CLSI Docu-ment M100-S25 Clinical and Laboratory Standards InstituteWayne Pa USA 2015
[25] F Yu Y Liu Y Xu et al ldquoExpression of panton-valentine leuko-cidin mrna among staphylococcus aureus isolates associateswith specific clinical presentationsrdquo PLoS ONE vol 8 no 12p e83368 2013
[26] S V Kazakova J C Hageman M Matava et al ldquoA cloneof methicillin-resistant Staphylococcus aureus among profes-sional football playersrdquo The New England Journal of Medicinevol 352 no 5 pp 468ndash475 2005
[27] D R Bhatta LMCavacoG Nath et al ldquoAssociation of PantonValentine Leukocidin (PVL) genes with methicillin resistantStaphylococcus aureus (MRSA) in Western Nepal A matter ofconcern for community infections (a hospital based prospectivestudy)rdquo BMC Infectious Diseases vol 16 no 1 2016
8 Journal of Pathogens
[28] A Jain and A Agarwal ldquoBiofilm production a marker ofpathogenic potential of colonizing and commensal staphylo-coccirdquo Journal of Microbiological Methods vol 76 no 1 pp 88ndash92 2009
[29] M Zalipour H S Ebrahim-Saraie J Sarvari and R KhasheildquoDetection of biofilm production capability and icaAD genesamong staphylococci isolates from Shiraz Iranrdquo JundishapurJournal of Microbiology vol 9 no 12 pp 1ndash7 2016
[30] T Mathur S Singhal S Khan D J Upadhyay T Fatmaand A Rattan ldquoDetection of biofilm formation among theclinical isolates of staphylococci an evaluation of three differentscreeningmethodsrdquo Indian Journal ofMedicalMicrobiology vol24 no 1 pp 25ndash29 2006
[31] A Gravet M Rondeau C Harf-Monteil et al ldquoPredomi-nant Staphylococcus aureus isolated from antibiotic-associateddiarrhea is clinically relevant and produces enterotoxin Aand the bicomponent toxin LukE-LukDrdquo Journal of ClinicalMicrobiology vol 37 no 12 pp 4012ndash4019 1999
[32] G Lina Y Piemont F Godail-Gamot et al ldquoInvolve-ment of Panton-Valentine leukocidinmdashproducing Staphylococ-cus aureus in primary skin infections and pneumoniardquo ClinicalInfectious Diseases vol 29 no 5 pp 1128ndash1132 1999
[33] S A Havaei S O Moghadam M R Pourmand and JFaghri ldquoPrevalence of genes encoding bi-component leuko-cidins among clinical isolates of methicillin-resistant Staphylo-coccus aureusrdquo Iranian Journal of Public Health vol 39 no 1pp 8ndash14 2010
[34] L G Miller F Perdreau-Remington G Rieg et al ldquoNecrotizingfasciitis caused by community-associated methicillin-resistantStaphylococcus aureus in Los AngelesrdquoTheNewEngland Journalof Medicine vol 352 no 14 pp 1445ndash1453 2005
[35] A Holmes M Ganner S McGuane T L Pitt B D Cooksonand A M Kearns ldquoStaphylococcus aureus isolates carryingPanton-Valentine leucocidin genes in England and Wales fre-quency characterization and association with clinical diseaserdquoJournal of Clinical Microbiology vol 43 no 5 pp 2384ndash23902005
[36] I M Moussa and A M Shibl ldquoMolecular characterizationof methicillin-resistant Staphylococcus aureus recovered fromoutpatient clinics in Riyadh Saudi Arabiardquo Saudi MedicalJournal vol 30 no 5 pp 611ndash617 2009
[37] S Afroz N Kobayashi S Nagashima M M Alam A BM B Hossain and M A Rahman ldquoGenetic characterizationof Staphylococcus aureus isolates carrying Panton ValentineLeukocidin genes in Bangladeshrdquo Japanese Journal of InfectiousDiseases vol 61 pp 393ndash396 2008
[38] H Kaur S Purwar A Saini et al ldquoStatus of methicillinresistant Staphylococcus aureus infections and evaluation ofPVL producing strains in Belgaum South Indiardquo Journal ofKrishna Institute of Medical Sciences University vol 1 no 2 pp43ndash51 2012
[39] D Johnsson P Molling K Stralin and B Soderquist ldquoDetec-tion of Panton-Valentine leukocidin gene in Straphylococ-cus aureus by LightCycler PCR clinical and epidemiologicalaspectsrdquo Clinical Microbiology and Infection vol 10 no 10 pp884ndash889 2004
[40] A Rostamzad and N Rostamneia ldquoPrevalence of the panton-valentine leukocidin gene in clinical isolates of Staphylococcusaureus isolated from hospitals the ilam province of IranrdquoAvicenna Journal of Clinical Microbiology and Infection vol 3no 1 2016
[41] K O Bhutia and T S Singh ldquoThe prevalence and the riskfactors which are associated with Staphylococcus aureus andmethicillin-resistant S aureus which harboured the Panton-Valentine- Leukocidin gene in Sikkimrdquo Journal of Clinical andDiagnostic Research vol 6 no 3 pp 393ndash399 2012
[42] M Li G Y C Cheung J Hu et al ldquoComparative analysis ofvirulence and toxin expression of global community-associatedmethicillin-resistant Staphylococcus aureus strainsrdquo The Jour-nal of Infectious Diseases vol 202 no 12 pp 1866ndash1876 2010
[43] CWitzWWitte CWolz andCGoerke ldquoTranscription of thephage-encoded Panton-Valentine leukocidin of Staphylococcusaureus is dependent on the phage life-cycle and on the hostbackgroundrdquoMicrobiology vol 155 no 11 pp 3491ndash3499 2009
[44] C E Turner and S Sriskandan ldquoPantone-Valentine leucocidinexpression by Staphylococcus aureus exposed to commonantibioticsrdquo Infection vol 71 no 3 pp 338ndash346 2015
[45] J K Rudkin M Laabei A M Edwards et al ldquoOxacillinalters the toxin expression profile of community-associatedmethicillin-resistant Staphylococcus aureusrdquo AntimicrobialAgents and Chemotherapy vol 58 no 2 pp 1100ndash1107 2014
Computational and Mathematical Methods in Medicine
Hindawiwwwhindawicom Volume 2018
Behavioural Neurology
OphthalmologyJournal of
Hindawiwwwhindawicom Volume 2018
Diabetes ResearchJournal of
Hindawiwwwhindawicom Volume 2018
Hindawiwwwhindawicom Volume 2018
Research and TreatmentAIDS
Hindawiwwwhindawicom Volume 2018
Gastroenterology Research and Practice
Hindawiwwwhindawicom Volume 2018
Parkinsonrsquos Disease
Evidence-Based Complementary andAlternative Medicine
Volume 2018Hindawiwwwhindawicom
Submit your manuscripts atwwwhindawicom
2 Journal of Pathogens
Table 1 Source data of collected samples according to collection site sex and age groups of infected individual
Superficial infection site (n=5) Nasal swab (n=20)Male (n=3) Female (n=2) Male (n=7) Female (n=13)
le 30 years ge 31 years le 30 years ge 31 years le 30 years ge 31 year119904 le 30 year119904 ge 31 year1199042 1 1 1 4 3 8 5
∘
E
∘
E
∘
E
∘
E
∘
N∘
N
∘
N∘
N
N
W E
S
KM
Midnapore TownSlum Area
Paschim Medinipur
Midnapore Town
West Bengal
Paschim Medinipur
India
West BengalNepali para
Tantigeria
KabardangaRangamati
Arobindo nagar
Keranitola Golkua chak
Battala chak
Bus stand
Figure 1 Study region including different slum areas in Midnapore town (Lat 22∘ 2310158404157 N to 22∘ 261015840220210158401015840 N Long 87∘171015840236510158401015840 E to 87∘201015840 153510158401015840 E)
To date PVL has become most important and signifi-cant virulence factor of community acquired S aureus Theprevalence of pvl genes among MRSA isolates has not beenadequately reported from IndiaThis studywas undertaken toinvestigate the PVL prevalence and pvl expression level alongwith certain other virulentmarkers from state ofWest BengalIndia The study area is demonstrated in Figure 1
2 Materials and Methods
21 Sample Collection A total of 25 non-repeat communitystrains of S aureus were collected from human subjects ofdifferent slum areas at Midnapore town (Lat 22∘231015840415710158401015840 Nto 22∘261015840220210158401015840 N Long 87∘171015840236510158401015840 E to 87∘201015840153510158401015840 E) inthe state ofWest Bengal India fromNovember 2015 toMarch2016 The protocols in present study with human subjects
got ethical clearance from Institutional Review Board (IRV)who follows the Indian Council of Medical Research (ICMR)ethical guidelines for biomedical research on human subjectsBacterial isolates were categorised into three groups accord-ing to the site of collection age group and sex (Table 1) Forsample collection sterile swabs with rayon tips were usedfor moist wound it was used directly but for dry wound itwas moistened with sterile saline so that it could increase thechance of recovery of the organism from the infected sites
22 Isolation and Identification of S aureus All the sampleswere transferred into 2 gm Luria broth (one for each speci-men) and incubated at 37∘C in shaking incubator After 16-18hours all the samples were inoculated onto mannitol salt agarplate and incubated for 24-25 hours at 37∘C [14] All yellowpigmented colonies were inoculated into LB agar for culture
Journal of Pathogens 3
preservation at 4∘C The resulting growth from respectiveplates of media was examined for colony characteristic andmorphology Each culture underwent Gram staining andwas tested for production of catalase free coagulase yellowpigment and thermonuclease (TNase) according to methoddescribed by Lancette and Tatini [15]
23 DNA Extraction from Bacterial Culture Isolates Forextraction of DNA from bacterial culture the rapid boilingmethod was followed [14] In brief 4 to 5 single coloniesfrom overnight grown culture were inoculated in 200120583l ofsterile Milli-Q water and boiled at 99∘C for 10 minutesAfter centrifugation at 14000 rpm for 10min the supernatantwas collected in a sterile microcentrifuge tube gently Theresultant template DNA was stored at minus20∘C and 2 120583l of eachsample was used for PCR analysis
24 Molecular Identification through PCR Amplification of16S rRNA Gene Phenotypically isolated and confirmed Saureus isolates were further tested by PCR amplificationassay using primer pairs (Table 3) for species-specific 16SrRNA gene These primers amplified 228 bp fragment from16S rRNA gene of these isolated S aureus strains The PCRprocedure was carried out in a 25120583l PCR tube and each ofthe reaction mixtures contained 25120583l PCR buffer (10X) 1 120583l(1U120583l) of Taq DNA polymerase 2 120583l dNTPs (200mM each)1120583l of each primer (10 pmol120583l) 25120583l (10 ng120583l) of templateDNA and 1575120583l PCR grade water The amplifications tookplace by the process of PCR thermocycling in a thermalcycler (Eppendorf Germany) commencing with an initialdenaturation at 94∘C for 2minutes followed by 33 cycles eachconsisting of denaturation at 94∘C for 30 sec annealing at50∘C for 30 sec and polymerization at 72∘C for 45 sec witha final extension at 72∘C for 4minutes S aureusATCC 25923was used as positive control in this experiment
25 Biochemical Characterization of S aureus Strains Thebacterial strains which were confirmed as S aureus byspecies-specific 16S rRNA were further evaluated by differ-ent biochemical tests including mannitol fermentation andgrowth on high salt concentration gelatin hydrolysis ureahydrolysis protease activity on milk agar medium lipaseproduction on egg yolk agar medium (HiMedia Mumbai)and hydrolysis of esculin by standard method [16ndash19] andcongo red agar (CRA) test [20]
26 Biofilm Assay Biofilm assay was done in borosilicateglass tubes according to the method described by Watnicket al [21] Briefly the isolates were grown in 3ml of Luriabroth overnight Then 5120583l of the overnight culture was addedto 500120583l of sterile LB in test tubes to make a dilution of1100 Test tubes were kept standing statically for 24 hrs andafter that the test tubes were rinsed vigorously with distilledwater to remove all nonadherent cells Further 600120583l of 01(wv) crystal violet was added and incubated for 30 minutesTest tubes were then rinsed vigorously with distilled water1ml DMSO was added to it followed by vortexing Afterincubation for 10 minutes optical density was measured at
570nm Each assay was performed in triplicate The meanvalues of optical densities obtained were categorised intothree classes as suggested by Cha et al [22] such that thestrains with OD
570lt 02 02ge OD
570le10 and OD
570gt 10
were defined as biofilm nonformers and biofilm formers ofweak level and strong level respectively
27 Antibiotic Susceptibility Profile of Isolated S aureus Theantibiotic susceptibility pattern of S aureus isolates wasdone by disk diffusion method using commercial antibioticdisks procured from HiMedia Mumbai [23] The antibioticdisks used were as follows ampicillin (30120583g) nalidixicacid (30120583g) chloramphenicol (30120583g) streptomycin (10120583g)kanamycin (30120583g) cefoxitin (30120583g) novobiocin (30120583g) anderythromycin (10120583g) Antibiotic susceptibility of those iso-lates was evaluated according to the Clinical and LaboratoryStandard Institute (CLSI) [23]Minimum InhibitoryConcen-tration (MIC) of vancomycin was done by the broth dilutionmethod to distinguish VSSA from VRSA according to theClinical and Laboratory Standard Institute (CLSI 2015) [24]
28 Detection ofmecA nuc and pvl Gene Detection ofmecAnuc and pvl genes of S aureuswas done by PCR amplificationassay using gene-specific primer pairs (Table 3) The PCRprocedure was carried out in a 25120583l PCR tube and each ofthe reaction mixtures contained 25120583l PCR buffer (10X) 1 120583l(1U120583l) of Taq DNA polymerase 2 120583l dNTPs (200mM each)1 120583l of each primer (10 pmol120583l) 25120583l (10 ng120583l) of templateDNA and 1575120583l PCR grade water The amplifications tookplace by the process of PCR thermocycling in a thermalcycler (Eppendorf Germany) commencing with an initialdenaturation at 94∘C for 2 minutes followed by 33 cycleseach comprising 94∘C for 1 minute and annealing at 55∘C for45 sec 50∘C for 50 sec and 50∘C for 45 sec formecA nuc andpvl gene respectively and a common extension of 72∘C for45 sec with a final extension at 72∘C for 4 minutes A positivecontrol of ATCC 33591 ATCC 25923 and ATCC 49775 Saureus was used respectively in each of these experimentsThe PCR amplicons were visualized in agarose gel containingethidiumbromide (1 120583gml) under Gel DocXR system (Bio-Rad USA) and photographs were taken for image analysisFragments of DNA 147 bp 270 bp and 433 bp correspondedtomecA nuc and pvl gene respectively
29 Real-Time Polymerase Chain Reaction of pvl GeneTotal RNA was extracted using Pure Link RNA MiniKit (Invitrogen Carlsbad CA) and quantified using UV-Visspectrophotometer (Nano Drop 2000 Thermo Fisher) TotalRNA was then converted to cDNA by using Verso cDNAsynthesis kit (Thermo Scientific USA)The standard reactioncontained 1x Power SYBR Green PCR master mix (AppliedBiosystems) primers pairs (Table 3) and cDNA as templatestrand The temperature program for 40 cycles was set todenaturation at 94∘C for 1 minute and annealing at 55∘C for30 sec The reaction was conducted on Step One Plus 96-WellReal-Time PCR System (Applied Biosystems) The sampleswere analysed in triplicate and recA was used as endogenouscontrol for normalization [25]
4 Journal of Pathogens
Table 2 Biochemical identification and characterization of S aureus
TestsSource of the isolates Total
Superficial infection site Nasal swabNolowast () Nolowast () Nolowast ()
210 Statistical Analysis Data analysis and plotting of datawere done using Microsoft Excel Mean and standard error ofmean was also calculated for each quantitative variable usingMicrosoft Excel
3 Results
A total of 25 nonduplicate isolates of S aureus were obtainedfrom different slum areas in Midnapore town West BengalIndia and included in this study Samples were collectedfromnasal swab and superficial infection sites of the subjectsAmong 25 isolates 22 strains (88) were isolated usingselective MSA media and then these isolates were identifiedas S aureus by several phenotypic expressions by means ofdifferent biochemical tests (Table 2) All S aureus isolates inthis study were confirmed by PCR using the species-specificprimer pair (Table 3) In this study we found that 100 91and 23 isolates were positive for catalase coagulase andheat-stable nuclease respectivelyOf the isolated strains 6855 and 68 produced protease lipase and nonwhite pig-mented colonies respectively Rates of resistance of S aureus
ranged from 27 to 91 to different antibiotics Resistance of Saureus isolates to ampicillin (91) nalidixic acid (91) chlo-ramphenicol (36) streptomycin (27) kanamycin (55)cefoxitin (68) novobiocin (64) and erythromycin (82)was found S aureus isolates showing resistance to at leastone agent from three or more antimicrobial categories islabeled as multi-drug resistant Accordingly all S aureusstrains isolated in this study were found to be multi-drugresistant In the present study the prevalence rate of MRSAwas found to be 68 It was also found that 5 of theisolated S aureus were vancomycin intermediate and 95of the isolated S aureus were vancomycin sensitive None ofthe strains were found to be VRSA In general observationsslime-producing strains appear as black colonies whereasnon-slime-producing strains appear as red colonies in CRAplates Among the community acquired isolates 15 out of22 (68) S aureus strains appeared to be slime-producingafter 48 hrs of incubation at 37∘C Moreover in the presentstudy 7 (32) were found as strong biofilm forming and15 (68) isolates were moderate biofilm forming None ofthe isolates were biofilm-negative (Figure 2) Twenty-seven
Journal of Pathogens 5
02468
10121416
lt02 02-1 gt1Low Biofilm Former Moderate Biofilm Former High Biofilm Former
No of strains
Figure 2 Biofilm formation ability of community strains from slum area
228 bp
M 1 2 3 4 5 6 7 8 9 10 11 12 1413
(a)
270 bp
M 1 2 3 4 5 6 7
(b)
M 1 2 3 4 5 6 7
147 bp
1000 bp
500 bp
200 bp
(c)
M 1 2
433 bp
(d)
Figure 3 (a) Agarose gel electrophoresis pattern for identification of Staphylococcus aureus specific 16S rRNA gene M molecular weightmarker Lane 1-13 different clinical strains of Staphylococcus aureus Lane 14 negative control Escherichia coli (SM10 120582pir) (b) nuc geneM DNA molecular weight marker (100 bp DNA ladder) Lane 1 positive control Lanes 2 3 4 and 5 270 bp band obtained with DNAfrom clinical strains Lane 6 negative control (Staphylococcus epidermidis) (c) Agarose gel electrophoresis patterns showing PCR-amplifiedproducts for the mecA gene M DNA molecular weight marker (100 bp DNA ladder) Lane 1 positive control Lanes 2 3 4 5 and 6 147 bpband obtained with DNA from MRSA clinical strains Lane 7 negative control (Staphylococcus epidermidis) (d) Agarose gel electrophoresispatterns showing PCR-amplified products for the pvl gene Lanes 1 and 2 433 bp band obtained with DNA fromMRSA clinical strains
percent (27) strains were found to harbour nuc gene(Figure 3) mecA and pvl genes were detected in 17 out of22 (68) and 2 out of 22 isolates (9) respectively All thepvl harbouring isolates possessed mecA gene (Figure 3) Thelow 998779Ct value (0493) in qRT-PCR was indicative of highexpression of pvl in one S aureus strain (Figure 4)
4 Discussion
In the present study very high rates of resistance particu-larly towards penicillin and also for kanamycin cefoxitinnovobiocin and erythromycin were observed in the isolatedcommunity acquired S aureus strains In 2005 Kazakova
6 Journal of Pathogens
recA pvlGenes
23
235
24
245
25
255
26
265
27
CT M
ean
Figure 4 Comparative analysis of the cycle threshold by qRT-PCRassay Black bar represents the housekeeping gene recA and graybar represents pvl gene
et al [26] reported a community-associated MRSA (CA-MRSA) clone isolated from US football players with skinabscesses The strain was susceptible to most antimicrobialagents except 120573-lactams and macrolides Bhatta et al [27]reported the detection of mecA gene in 568 of the isolatesfrom clinical and community settings In this study outof twenty-two clinical isolates fifteen (68) isolates turnedout to be slime producer Jain and Agarwal [28] reporteda higher rate where 64 of staphylococcal intravascularisolates were biofilm producers by CRA method Zalipouret al [29] reported that forty-three (544) biofilm formingstrains were found in clinical specimens in Iran by CRAassay In the current study seven (32) isolates were strongbiofilm formers and fifteen (68) isolates were moderatebiofilm formers Mathur et al [30] reported that 578 ofstaphylococcal clinical isolates established biofilm-positivecharacteristics and 1447 and 394 exhibited high andmoderate biofilm production respectively in India Globalemergence of MRSA is serious public health problem andchallenge to clinicians Various factors devote to drug resis-tance and the pathogenicity of S aureus The first PVLpositive MRSA was noticed in the late 1990s and these strainsgot scattered worldwide in recent years [31] The role of PVLin boosting virulence of S aureus and their pathogenicityis being deliberated Panton-Valentine leukocidin rises thepathogenicity of S aureus by necrosis quickening apoptosisand damage of polymorphonuclear and mononuclear cellsthereby contributing to mortality and morbidity [32] PVL isgenerally used as a marker for community acquired MRSAliable for deep dermal infections including soft tissue [3334] However the worldwide scheme of PVL among MRSAisolates varies A lower prevalence of PVL has been reportedfrom other parts of world (5 in France 49 in UK81 in Saudi Arabia and 143 in Bangladesh) [32 35ndash37] reflecting the significant variation in prevalence of PVLamong geographical areas and communities Kaur et al [38]from India have reported overall 6285 prevalence of PVL
among MRSA and MSSA (MRSA 851 and MSSA 488)which delineates higher prevalence of PVL among MRSA incomparison to present findings Johnsson et al [39] detectedpvl gene in one isolate (1) from 65 patients with S aureusbacteraemia in two (219) isolates from 91 patients withcutaneous infections and in four (727) isolates from 55patients with respiratory tract infections Rostamzad et al[40] reported that out of thirty-two MRSA isolates thirteenisolates (4062)were positive for presence of the luk-pv genefrom hospitals isolates Higher prevalence of PVL amongchildren (lt14 years of age) was observed as compared toadults and old age group patients Similar observations weremade in another study from India [41] Epidemiological datasuggest that high virulence of community acquired MRSA isassociated with pvl gene but direct evidence of association ofPVL to pathogenesis remains limited [42]The low998779Ct value(0493) in qRT-PCR indicated high expression of pvl gene inS aureus strain in the present study However in previousstudy low expression of luk-pvwas reported [43] In S aureusexpression of toxins and others factors can be measured byusing Panton-Valentine leukocidin (PVL) expression in invivo condition [44] In addition expression of pvl gene isenhanced by sub-MIC values of 120573-lactam antibiotics [45]The studied population has a little health education andthey commonly use 120573-lactam antibiotics to treat any kindof infections this may be the reason of high expression ofpvl gene in in vitro condition Thus detection of pvl genein community isolated S aureus is indication of emergenceof pathogenic S aureus in community setup in the studiedregion
5 Conclusion
The current study reflects the elevated level of multi-drugresistant S aureus infections in community The prevalenceof the pvl gene among the MRSA isolates in this studywas low The presence of pvl among multi-drug resistantbacteria like MRSA may be fatal and challenging conditionfor clinicians The studied population has a little healtheducation and they commonly use 120573-lactam antibiotics thismay be the underlying reason for high expression of pvlThusdetection of pvl in community isolated S aureus indicates theemergence of pathogenic S aureus in community setup in thestudied region
Data Availability
The data used to support the findings of this study areavailable from the corresponding author upon request
Conflicts of Interest
The authors declare that there are no conflicts of interestregarding the publication of this paper
Acknowledgments
The authors are grateful to the individuals who participatedin the study
Journal of Pathogens 7
References
[1] B Shrestha W Singh V S Raj B M Pokhrel and T MMohapatra ldquoHigh Prevalence of Panton-Valentine leukocidin(PVL) genes in nosocomial-acquired staphylococcus aureusisolated from tertiary care hospitals in Nepalrdquo BioMed ResearchInternational vol 2014 Article ID 790350 7 pages 2014
[2] J Kaneko and Y Kamio ldquoBacterial two-component and hetero-heptameric pore-forming cytolytic toxins structures pore-forming mechanism and organization of the genesrdquo BioscienceBiotechnology and Biochemistry vol 68 no 5 pp 981ndash10032004
[3] G Prevost B Cribier P Couppie et al ldquoPanton-valentineleucocidin and gamma-hemolysin from Staphylococcus aureusATCC 49775 are encoded by distinct genetic loci and havedifferent biological activitiesrdquo Infection and Immunity vol 63no 10 pp 4121ndash4129 1995
[4] D C Melles W B Van Leeuwen H A M Boelens J KPeetersH AVerbrugh andAVanBelkum ldquoPanton-Valentineleukocidin genes in Staphylococcus aureusrdquoEmerging InfectiousDiseases vol 12 no 7 pp 1174-1175 2006
[5] B McGrath F Rutledge and E Broadfield ldquo NecrotisingPneumonia rdquo Journal of the Intensive Care Society vol 9 no2 pp 170ndash172 2008
[6] F Yu Z Chen C Liu et al ldquoPrevalence of Staphylococcusaureus carrying Panton-Valentine leukocidin genes among iso-lates from hospitalised patients in Chinardquo Clinical Microbiologyand Infection vol 14 no 4 pp 381ndash384 2008
[7] S Monecke P Slickers M J Ellington A M Kearns andR Ehricht ldquoHigh diversity of Panton-Valentine leukocidin-positive methicillin-susceptible isolates of Staphylococcusaureus and implications for the evolution of community-associatedmethicillin-resistant S aureusrdquoClinicalMicrobiologyand Infection vol 13 no 12 pp 1157ndash1164 2007
[8] D J Skiest K Brown T W Cooper H Hoffman-RobertsH R Mussa and A C Elliott ldquoProspective comparison ofmethicillin-susceptible and methicillin-resistant community-associated Staphylococcus aureus infections in hospitalizedpatientsrdquo Infection vol 54 no 5 pp 427ndash434 2007
[9] S R Monday and G A Bohach ldquoUse of multiplex PCR todetect classical and newly described pyrogenic toxin genes inStaphylococcal isolatesrdquo Journal of Clinical Microbiology vol 37no 10 pp 3411ndash3414 1999
[10] O G Brakstad K Aasbakk and J A Maeland ldquoDetection ofStaphylococcus aureus by polymerase chain reaction amplifica-tion of the nuc generdquo Journal of Clinical Microbiology vol 30no 7 pp 1654ndash1660 1992
[11] K Zhang J-A McClure S Elsayed T Louie and J MConly ldquoNovel multiplex PCR assay for characterization andconcomitant subtyping of staphylococcal cassette chromosomemec types I to V in methicillin-resistant Staphylococcus aureusrdquoJournal of Clinical Microbiology vol 43 no 10 pp 5026ndash50332005
[12] J-A McClure J M Conly V Lau et al ldquoNovel multiplexPCR assay for detection of the staphylococcal virulence markerPanton-Valentine leukocidin genes and simultaneous discrimi-nation of methicillin-susceptible from -resistant staphylococcirdquoJournal of Clinical Microbiology vol 44 no 3 pp 1141ndash11442006
[13] T Nuryastuti H C van der Mei H J Busscher R Kuijer A TAman and B P Krom ldquorecA mediated spontaneous deletionsof the icaADBC operon of clinical Staphylococcus epidermidis
isolates a new mechanism of phenotypic variationsrdquo Antonievan Leeuwenhoek-Journal ofMicrobiology vol 94 no 2 pp 317ndash328 2008
[14] A Karmakar P Dua and C Ghosh ldquoBiochemical and molec-ular analysis of Staphylococcus aureus clinical isolates fromhospitalized patientsrdquo Canadian Journal of Infectious Diseasesamp Medical Microbiology vol 2016 Article ID 9041636 7 pages2016
[15] G A Lancette and S R Tatini ldquoStaphylococcus aureusrdquo inCompendium of Methods for the Microbiological Examination ofFoods C Vanderzant and D F Splittstoesser Eds pp 533ndash550American Public Health Association Washington DC USA3rd edition 1992
[16] E B Blair J S Emerson and A H Tull ldquoA new mediumsalt mannitol plasma agar for the isolation of Staphylococcusaureusrdquo American Journal of Clinical Pathology vol 47 no 1pp 30ndash39 1967
[17] G H Chapman ldquoThe significance of sodium chloride in studiesof staphylococcirdquo Journal of Bacteriology vol 50 pp 201ndash2031945
[18] R Cruickshank J P Duguid B PMarmion andRHA SwainMed Microbiol vol II Chruchill Livingstone Edinburgh UK12nd edition 1975
[19] A Swan ldquoThe use of a bile-aesculin medium and of Maxtedrsquostechnique of Lancefield grouping in the identification of ente-rococci (Group D Streptococci)rdquo Journal of Clinical Pathologyvol 7 no 2 pp 160ndash163 1954
[20] C R Arciola L Baldassarri and L Montanaro ldquoPresenceof icaA and icaD genes and slime production in a collectionof Staphylococcal strains from catheter-associated infectionsrdquoJournal of Clinical Microbiology vol 39 no 6 pp 2151ndash21562001
[21] P IWatnickCM LaurianoK EKlose LCroal andRKolterldquoThe absence of a flagellum leads to altered colony morphologybiofilm development and virulence in Vibrio cholerae O139rdquoMolecularMicrobiology vol 39 no 2 pp 223ndash235 2001
[22] J-O Cha J I Yoo J S Yoo et al ldquoinvestigation of biofilmformation and its association with the molecular and clinicalcharacteristics of methicillin-resistant staphylococcus aureusrdquoOsong Public Health and Research Perspectives vol 4 no 5 pp225ndash232 2013
[23] AW BauerWMKirby J C Sherris andMTurck ldquoAntibioticsusceptibility testing by a standardized single disk methodrdquoAmerican Journal of Clinical Pathology vol 45 no 4 ts pp 493ndash496 1966
[24] CLSI ldquoPerformance standards for antimicrobial susceptibilitytesting twenty-fifth informational supplementrdquo CLSI Docu-ment M100-S25 Clinical and Laboratory Standards InstituteWayne Pa USA 2015
[25] F Yu Y Liu Y Xu et al ldquoExpression of panton-valentine leuko-cidin mrna among staphylococcus aureus isolates associateswith specific clinical presentationsrdquo PLoS ONE vol 8 no 12p e83368 2013
[26] S V Kazakova J C Hageman M Matava et al ldquoA cloneof methicillin-resistant Staphylococcus aureus among profes-sional football playersrdquo The New England Journal of Medicinevol 352 no 5 pp 468ndash475 2005
[27] D R Bhatta LMCavacoG Nath et al ldquoAssociation of PantonValentine Leukocidin (PVL) genes with methicillin resistantStaphylococcus aureus (MRSA) in Western Nepal A matter ofconcern for community infections (a hospital based prospectivestudy)rdquo BMC Infectious Diseases vol 16 no 1 2016
8 Journal of Pathogens
[28] A Jain and A Agarwal ldquoBiofilm production a marker ofpathogenic potential of colonizing and commensal staphylo-coccirdquo Journal of Microbiological Methods vol 76 no 1 pp 88ndash92 2009
[29] M Zalipour H S Ebrahim-Saraie J Sarvari and R KhasheildquoDetection of biofilm production capability and icaAD genesamong staphylococci isolates from Shiraz Iranrdquo JundishapurJournal of Microbiology vol 9 no 12 pp 1ndash7 2016
[30] T Mathur S Singhal S Khan D J Upadhyay T Fatmaand A Rattan ldquoDetection of biofilm formation among theclinical isolates of staphylococci an evaluation of three differentscreeningmethodsrdquo Indian Journal ofMedicalMicrobiology vol24 no 1 pp 25ndash29 2006
[31] A Gravet M Rondeau C Harf-Monteil et al ldquoPredomi-nant Staphylococcus aureus isolated from antibiotic-associateddiarrhea is clinically relevant and produces enterotoxin Aand the bicomponent toxin LukE-LukDrdquo Journal of ClinicalMicrobiology vol 37 no 12 pp 4012ndash4019 1999
[32] G Lina Y Piemont F Godail-Gamot et al ldquoInvolve-ment of Panton-Valentine leukocidinmdashproducing Staphylococ-cus aureus in primary skin infections and pneumoniardquo ClinicalInfectious Diseases vol 29 no 5 pp 1128ndash1132 1999
[33] S A Havaei S O Moghadam M R Pourmand and JFaghri ldquoPrevalence of genes encoding bi-component leuko-cidins among clinical isolates of methicillin-resistant Staphylo-coccus aureusrdquo Iranian Journal of Public Health vol 39 no 1pp 8ndash14 2010
[34] L G Miller F Perdreau-Remington G Rieg et al ldquoNecrotizingfasciitis caused by community-associated methicillin-resistantStaphylococcus aureus in Los AngelesrdquoTheNewEngland Journalof Medicine vol 352 no 14 pp 1445ndash1453 2005
[35] A Holmes M Ganner S McGuane T L Pitt B D Cooksonand A M Kearns ldquoStaphylococcus aureus isolates carryingPanton-Valentine leucocidin genes in England and Wales fre-quency characterization and association with clinical diseaserdquoJournal of Clinical Microbiology vol 43 no 5 pp 2384ndash23902005
[36] I M Moussa and A M Shibl ldquoMolecular characterizationof methicillin-resistant Staphylococcus aureus recovered fromoutpatient clinics in Riyadh Saudi Arabiardquo Saudi MedicalJournal vol 30 no 5 pp 611ndash617 2009
[37] S Afroz N Kobayashi S Nagashima M M Alam A BM B Hossain and M A Rahman ldquoGenetic characterizationof Staphylococcus aureus isolates carrying Panton ValentineLeukocidin genes in Bangladeshrdquo Japanese Journal of InfectiousDiseases vol 61 pp 393ndash396 2008
[38] H Kaur S Purwar A Saini et al ldquoStatus of methicillinresistant Staphylococcus aureus infections and evaluation ofPVL producing strains in Belgaum South Indiardquo Journal ofKrishna Institute of Medical Sciences University vol 1 no 2 pp43ndash51 2012
[39] D Johnsson P Molling K Stralin and B Soderquist ldquoDetec-tion of Panton-Valentine leukocidin gene in Straphylococ-cus aureus by LightCycler PCR clinical and epidemiologicalaspectsrdquo Clinical Microbiology and Infection vol 10 no 10 pp884ndash889 2004
[40] A Rostamzad and N Rostamneia ldquoPrevalence of the panton-valentine leukocidin gene in clinical isolates of Staphylococcusaureus isolated from hospitals the ilam province of IranrdquoAvicenna Journal of Clinical Microbiology and Infection vol 3no 1 2016
[41] K O Bhutia and T S Singh ldquoThe prevalence and the riskfactors which are associated with Staphylococcus aureus andmethicillin-resistant S aureus which harboured the Panton-Valentine- Leukocidin gene in Sikkimrdquo Journal of Clinical andDiagnostic Research vol 6 no 3 pp 393ndash399 2012
[42] M Li G Y C Cheung J Hu et al ldquoComparative analysis ofvirulence and toxin expression of global community-associatedmethicillin-resistant Staphylococcus aureus strainsrdquo The Jour-nal of Infectious Diseases vol 202 no 12 pp 1866ndash1876 2010
[43] CWitzWWitte CWolz andCGoerke ldquoTranscription of thephage-encoded Panton-Valentine leukocidin of Staphylococcusaureus is dependent on the phage life-cycle and on the hostbackgroundrdquoMicrobiology vol 155 no 11 pp 3491ndash3499 2009
[44] C E Turner and S Sriskandan ldquoPantone-Valentine leucocidinexpression by Staphylococcus aureus exposed to commonantibioticsrdquo Infection vol 71 no 3 pp 338ndash346 2015
[45] J K Rudkin M Laabei A M Edwards et al ldquoOxacillinalters the toxin expression profile of community-associatedmethicillin-resistant Staphylococcus aureusrdquo AntimicrobialAgents and Chemotherapy vol 58 no 2 pp 1100ndash1107 2014
Computational and Mathematical Methods in Medicine
Hindawiwwwhindawicom Volume 2018
Behavioural Neurology
OphthalmologyJournal of
Hindawiwwwhindawicom Volume 2018
Diabetes ResearchJournal of
Hindawiwwwhindawicom Volume 2018
Hindawiwwwhindawicom Volume 2018
Research and TreatmentAIDS
Hindawiwwwhindawicom Volume 2018
Gastroenterology Research and Practice
Hindawiwwwhindawicom Volume 2018
Parkinsonrsquos Disease
Evidence-Based Complementary andAlternative Medicine
Volume 2018Hindawiwwwhindawicom
Submit your manuscripts atwwwhindawicom
Journal of Pathogens 3
preservation at 4∘C The resulting growth from respectiveplates of media was examined for colony characteristic andmorphology Each culture underwent Gram staining andwas tested for production of catalase free coagulase yellowpigment and thermonuclease (TNase) according to methoddescribed by Lancette and Tatini [15]
23 DNA Extraction from Bacterial Culture Isolates Forextraction of DNA from bacterial culture the rapid boilingmethod was followed [14] In brief 4 to 5 single coloniesfrom overnight grown culture were inoculated in 200120583l ofsterile Milli-Q water and boiled at 99∘C for 10 minutesAfter centrifugation at 14000 rpm for 10min the supernatantwas collected in a sterile microcentrifuge tube gently Theresultant template DNA was stored at minus20∘C and 2 120583l of eachsample was used for PCR analysis
24 Molecular Identification through PCR Amplification of16S rRNA Gene Phenotypically isolated and confirmed Saureus isolates were further tested by PCR amplificationassay using primer pairs (Table 3) for species-specific 16SrRNA gene These primers amplified 228 bp fragment from16S rRNA gene of these isolated S aureus strains The PCRprocedure was carried out in a 25120583l PCR tube and each ofthe reaction mixtures contained 25120583l PCR buffer (10X) 1 120583l(1U120583l) of Taq DNA polymerase 2 120583l dNTPs (200mM each)1120583l of each primer (10 pmol120583l) 25120583l (10 ng120583l) of templateDNA and 1575120583l PCR grade water The amplifications tookplace by the process of PCR thermocycling in a thermalcycler (Eppendorf Germany) commencing with an initialdenaturation at 94∘C for 2minutes followed by 33 cycles eachconsisting of denaturation at 94∘C for 30 sec annealing at50∘C for 30 sec and polymerization at 72∘C for 45 sec witha final extension at 72∘C for 4minutes S aureusATCC 25923was used as positive control in this experiment
25 Biochemical Characterization of S aureus Strains Thebacterial strains which were confirmed as S aureus byspecies-specific 16S rRNA were further evaluated by differ-ent biochemical tests including mannitol fermentation andgrowth on high salt concentration gelatin hydrolysis ureahydrolysis protease activity on milk agar medium lipaseproduction on egg yolk agar medium (HiMedia Mumbai)and hydrolysis of esculin by standard method [16ndash19] andcongo red agar (CRA) test [20]
26 Biofilm Assay Biofilm assay was done in borosilicateglass tubes according to the method described by Watnicket al [21] Briefly the isolates were grown in 3ml of Luriabroth overnight Then 5120583l of the overnight culture was addedto 500120583l of sterile LB in test tubes to make a dilution of1100 Test tubes were kept standing statically for 24 hrs andafter that the test tubes were rinsed vigorously with distilledwater to remove all nonadherent cells Further 600120583l of 01(wv) crystal violet was added and incubated for 30 minutesTest tubes were then rinsed vigorously with distilled water1ml DMSO was added to it followed by vortexing Afterincubation for 10 minutes optical density was measured at
570nm Each assay was performed in triplicate The meanvalues of optical densities obtained were categorised intothree classes as suggested by Cha et al [22] such that thestrains with OD
570lt 02 02ge OD
570le10 and OD
570gt 10
were defined as biofilm nonformers and biofilm formers ofweak level and strong level respectively
27 Antibiotic Susceptibility Profile of Isolated S aureus Theantibiotic susceptibility pattern of S aureus isolates wasdone by disk diffusion method using commercial antibioticdisks procured from HiMedia Mumbai [23] The antibioticdisks used were as follows ampicillin (30120583g) nalidixicacid (30120583g) chloramphenicol (30120583g) streptomycin (10120583g)kanamycin (30120583g) cefoxitin (30120583g) novobiocin (30120583g) anderythromycin (10120583g) Antibiotic susceptibility of those iso-lates was evaluated according to the Clinical and LaboratoryStandard Institute (CLSI) [23]Minimum InhibitoryConcen-tration (MIC) of vancomycin was done by the broth dilutionmethod to distinguish VSSA from VRSA according to theClinical and Laboratory Standard Institute (CLSI 2015) [24]
28 Detection ofmecA nuc and pvl Gene Detection ofmecAnuc and pvl genes of S aureuswas done by PCR amplificationassay using gene-specific primer pairs (Table 3) The PCRprocedure was carried out in a 25120583l PCR tube and each ofthe reaction mixtures contained 25120583l PCR buffer (10X) 1 120583l(1U120583l) of Taq DNA polymerase 2 120583l dNTPs (200mM each)1 120583l of each primer (10 pmol120583l) 25120583l (10 ng120583l) of templateDNA and 1575120583l PCR grade water The amplifications tookplace by the process of PCR thermocycling in a thermalcycler (Eppendorf Germany) commencing with an initialdenaturation at 94∘C for 2 minutes followed by 33 cycleseach comprising 94∘C for 1 minute and annealing at 55∘C for45 sec 50∘C for 50 sec and 50∘C for 45 sec formecA nuc andpvl gene respectively and a common extension of 72∘C for45 sec with a final extension at 72∘C for 4 minutes A positivecontrol of ATCC 33591 ATCC 25923 and ATCC 49775 Saureus was used respectively in each of these experimentsThe PCR amplicons were visualized in agarose gel containingethidiumbromide (1 120583gml) under Gel DocXR system (Bio-Rad USA) and photographs were taken for image analysisFragments of DNA 147 bp 270 bp and 433 bp correspondedtomecA nuc and pvl gene respectively
29 Real-Time Polymerase Chain Reaction of pvl GeneTotal RNA was extracted using Pure Link RNA MiniKit (Invitrogen Carlsbad CA) and quantified using UV-Visspectrophotometer (Nano Drop 2000 Thermo Fisher) TotalRNA was then converted to cDNA by using Verso cDNAsynthesis kit (Thermo Scientific USA)The standard reactioncontained 1x Power SYBR Green PCR master mix (AppliedBiosystems) primers pairs (Table 3) and cDNA as templatestrand The temperature program for 40 cycles was set todenaturation at 94∘C for 1 minute and annealing at 55∘C for30 sec The reaction was conducted on Step One Plus 96-WellReal-Time PCR System (Applied Biosystems) The sampleswere analysed in triplicate and recA was used as endogenouscontrol for normalization [25]
4 Journal of Pathogens
Table 2 Biochemical identification and characterization of S aureus
TestsSource of the isolates Total
Superficial infection site Nasal swabNolowast () Nolowast () Nolowast ()
210 Statistical Analysis Data analysis and plotting of datawere done using Microsoft Excel Mean and standard error ofmean was also calculated for each quantitative variable usingMicrosoft Excel
3 Results
A total of 25 nonduplicate isolates of S aureus were obtainedfrom different slum areas in Midnapore town West BengalIndia and included in this study Samples were collectedfromnasal swab and superficial infection sites of the subjectsAmong 25 isolates 22 strains (88) were isolated usingselective MSA media and then these isolates were identifiedas S aureus by several phenotypic expressions by means ofdifferent biochemical tests (Table 2) All S aureus isolates inthis study were confirmed by PCR using the species-specificprimer pair (Table 3) In this study we found that 100 91and 23 isolates were positive for catalase coagulase andheat-stable nuclease respectivelyOf the isolated strains 6855 and 68 produced protease lipase and nonwhite pig-mented colonies respectively Rates of resistance of S aureus
ranged from 27 to 91 to different antibiotics Resistance of Saureus isolates to ampicillin (91) nalidixic acid (91) chlo-ramphenicol (36) streptomycin (27) kanamycin (55)cefoxitin (68) novobiocin (64) and erythromycin (82)was found S aureus isolates showing resistance to at leastone agent from three or more antimicrobial categories islabeled as multi-drug resistant Accordingly all S aureusstrains isolated in this study were found to be multi-drugresistant In the present study the prevalence rate of MRSAwas found to be 68 It was also found that 5 of theisolated S aureus were vancomycin intermediate and 95of the isolated S aureus were vancomycin sensitive None ofthe strains were found to be VRSA In general observationsslime-producing strains appear as black colonies whereasnon-slime-producing strains appear as red colonies in CRAplates Among the community acquired isolates 15 out of22 (68) S aureus strains appeared to be slime-producingafter 48 hrs of incubation at 37∘C Moreover in the presentstudy 7 (32) were found as strong biofilm forming and15 (68) isolates were moderate biofilm forming None ofthe isolates were biofilm-negative (Figure 2) Twenty-seven
Journal of Pathogens 5
02468
10121416
lt02 02-1 gt1Low Biofilm Former Moderate Biofilm Former High Biofilm Former
No of strains
Figure 2 Biofilm formation ability of community strains from slum area
228 bp
M 1 2 3 4 5 6 7 8 9 10 11 12 1413
(a)
270 bp
M 1 2 3 4 5 6 7
(b)
M 1 2 3 4 5 6 7
147 bp
1000 bp
500 bp
200 bp
(c)
M 1 2
433 bp
(d)
Figure 3 (a) Agarose gel electrophoresis pattern for identification of Staphylococcus aureus specific 16S rRNA gene M molecular weightmarker Lane 1-13 different clinical strains of Staphylococcus aureus Lane 14 negative control Escherichia coli (SM10 120582pir) (b) nuc geneM DNA molecular weight marker (100 bp DNA ladder) Lane 1 positive control Lanes 2 3 4 and 5 270 bp band obtained with DNAfrom clinical strains Lane 6 negative control (Staphylococcus epidermidis) (c) Agarose gel electrophoresis patterns showing PCR-amplifiedproducts for the mecA gene M DNA molecular weight marker (100 bp DNA ladder) Lane 1 positive control Lanes 2 3 4 5 and 6 147 bpband obtained with DNA from MRSA clinical strains Lane 7 negative control (Staphylococcus epidermidis) (d) Agarose gel electrophoresispatterns showing PCR-amplified products for the pvl gene Lanes 1 and 2 433 bp band obtained with DNA fromMRSA clinical strains
percent (27) strains were found to harbour nuc gene(Figure 3) mecA and pvl genes were detected in 17 out of22 (68) and 2 out of 22 isolates (9) respectively All thepvl harbouring isolates possessed mecA gene (Figure 3) Thelow 998779Ct value (0493) in qRT-PCR was indicative of highexpression of pvl in one S aureus strain (Figure 4)
4 Discussion
In the present study very high rates of resistance particu-larly towards penicillin and also for kanamycin cefoxitinnovobiocin and erythromycin were observed in the isolatedcommunity acquired S aureus strains In 2005 Kazakova
6 Journal of Pathogens
recA pvlGenes
23
235
24
245
25
255
26
265
27
CT M
ean
Figure 4 Comparative analysis of the cycle threshold by qRT-PCRassay Black bar represents the housekeeping gene recA and graybar represents pvl gene
et al [26] reported a community-associated MRSA (CA-MRSA) clone isolated from US football players with skinabscesses The strain was susceptible to most antimicrobialagents except 120573-lactams and macrolides Bhatta et al [27]reported the detection of mecA gene in 568 of the isolatesfrom clinical and community settings In this study outof twenty-two clinical isolates fifteen (68) isolates turnedout to be slime producer Jain and Agarwal [28] reporteda higher rate where 64 of staphylococcal intravascularisolates were biofilm producers by CRA method Zalipouret al [29] reported that forty-three (544) biofilm formingstrains were found in clinical specimens in Iran by CRAassay In the current study seven (32) isolates were strongbiofilm formers and fifteen (68) isolates were moderatebiofilm formers Mathur et al [30] reported that 578 ofstaphylococcal clinical isolates established biofilm-positivecharacteristics and 1447 and 394 exhibited high andmoderate biofilm production respectively in India Globalemergence of MRSA is serious public health problem andchallenge to clinicians Various factors devote to drug resis-tance and the pathogenicity of S aureus The first PVLpositive MRSA was noticed in the late 1990s and these strainsgot scattered worldwide in recent years [31] The role of PVLin boosting virulence of S aureus and their pathogenicityis being deliberated Panton-Valentine leukocidin rises thepathogenicity of S aureus by necrosis quickening apoptosisand damage of polymorphonuclear and mononuclear cellsthereby contributing to mortality and morbidity [32] PVL isgenerally used as a marker for community acquired MRSAliable for deep dermal infections including soft tissue [3334] However the worldwide scheme of PVL among MRSAisolates varies A lower prevalence of PVL has been reportedfrom other parts of world (5 in France 49 in UK81 in Saudi Arabia and 143 in Bangladesh) [32 35ndash37] reflecting the significant variation in prevalence of PVLamong geographical areas and communities Kaur et al [38]from India have reported overall 6285 prevalence of PVL
among MRSA and MSSA (MRSA 851 and MSSA 488)which delineates higher prevalence of PVL among MRSA incomparison to present findings Johnsson et al [39] detectedpvl gene in one isolate (1) from 65 patients with S aureusbacteraemia in two (219) isolates from 91 patients withcutaneous infections and in four (727) isolates from 55patients with respiratory tract infections Rostamzad et al[40] reported that out of thirty-two MRSA isolates thirteenisolates (4062)were positive for presence of the luk-pv genefrom hospitals isolates Higher prevalence of PVL amongchildren (lt14 years of age) was observed as compared toadults and old age group patients Similar observations weremade in another study from India [41] Epidemiological datasuggest that high virulence of community acquired MRSA isassociated with pvl gene but direct evidence of association ofPVL to pathogenesis remains limited [42]The low998779Ct value(0493) in qRT-PCR indicated high expression of pvl gene inS aureus strain in the present study However in previousstudy low expression of luk-pvwas reported [43] In S aureusexpression of toxins and others factors can be measured byusing Panton-Valentine leukocidin (PVL) expression in invivo condition [44] In addition expression of pvl gene isenhanced by sub-MIC values of 120573-lactam antibiotics [45]The studied population has a little health education andthey commonly use 120573-lactam antibiotics to treat any kindof infections this may be the reason of high expression ofpvl gene in in vitro condition Thus detection of pvl genein community isolated S aureus is indication of emergenceof pathogenic S aureus in community setup in the studiedregion
5 Conclusion
The current study reflects the elevated level of multi-drugresistant S aureus infections in community The prevalenceof the pvl gene among the MRSA isolates in this studywas low The presence of pvl among multi-drug resistantbacteria like MRSA may be fatal and challenging conditionfor clinicians The studied population has a little healtheducation and they commonly use 120573-lactam antibiotics thismay be the underlying reason for high expression of pvlThusdetection of pvl in community isolated S aureus indicates theemergence of pathogenic S aureus in community setup in thestudied region
Data Availability
The data used to support the findings of this study areavailable from the corresponding author upon request
Conflicts of Interest
The authors declare that there are no conflicts of interestregarding the publication of this paper
Acknowledgments
The authors are grateful to the individuals who participatedin the study
Journal of Pathogens 7
References
[1] B Shrestha W Singh V S Raj B M Pokhrel and T MMohapatra ldquoHigh Prevalence of Panton-Valentine leukocidin(PVL) genes in nosocomial-acquired staphylococcus aureusisolated from tertiary care hospitals in Nepalrdquo BioMed ResearchInternational vol 2014 Article ID 790350 7 pages 2014
[2] J Kaneko and Y Kamio ldquoBacterial two-component and hetero-heptameric pore-forming cytolytic toxins structures pore-forming mechanism and organization of the genesrdquo BioscienceBiotechnology and Biochemistry vol 68 no 5 pp 981ndash10032004
[3] G Prevost B Cribier P Couppie et al ldquoPanton-valentineleucocidin and gamma-hemolysin from Staphylococcus aureusATCC 49775 are encoded by distinct genetic loci and havedifferent biological activitiesrdquo Infection and Immunity vol 63no 10 pp 4121ndash4129 1995
[4] D C Melles W B Van Leeuwen H A M Boelens J KPeetersH AVerbrugh andAVanBelkum ldquoPanton-Valentineleukocidin genes in Staphylococcus aureusrdquoEmerging InfectiousDiseases vol 12 no 7 pp 1174-1175 2006
[5] B McGrath F Rutledge and E Broadfield ldquo NecrotisingPneumonia rdquo Journal of the Intensive Care Society vol 9 no2 pp 170ndash172 2008
[6] F Yu Z Chen C Liu et al ldquoPrevalence of Staphylococcusaureus carrying Panton-Valentine leukocidin genes among iso-lates from hospitalised patients in Chinardquo Clinical Microbiologyand Infection vol 14 no 4 pp 381ndash384 2008
[7] S Monecke P Slickers M J Ellington A M Kearns andR Ehricht ldquoHigh diversity of Panton-Valentine leukocidin-positive methicillin-susceptible isolates of Staphylococcusaureus and implications for the evolution of community-associatedmethicillin-resistant S aureusrdquoClinicalMicrobiologyand Infection vol 13 no 12 pp 1157ndash1164 2007
[8] D J Skiest K Brown T W Cooper H Hoffman-RobertsH R Mussa and A C Elliott ldquoProspective comparison ofmethicillin-susceptible and methicillin-resistant community-associated Staphylococcus aureus infections in hospitalizedpatientsrdquo Infection vol 54 no 5 pp 427ndash434 2007
[9] S R Monday and G A Bohach ldquoUse of multiplex PCR todetect classical and newly described pyrogenic toxin genes inStaphylococcal isolatesrdquo Journal of Clinical Microbiology vol 37no 10 pp 3411ndash3414 1999
[10] O G Brakstad K Aasbakk and J A Maeland ldquoDetection ofStaphylococcus aureus by polymerase chain reaction amplifica-tion of the nuc generdquo Journal of Clinical Microbiology vol 30no 7 pp 1654ndash1660 1992
[11] K Zhang J-A McClure S Elsayed T Louie and J MConly ldquoNovel multiplex PCR assay for characterization andconcomitant subtyping of staphylococcal cassette chromosomemec types I to V in methicillin-resistant Staphylococcus aureusrdquoJournal of Clinical Microbiology vol 43 no 10 pp 5026ndash50332005
[12] J-A McClure J M Conly V Lau et al ldquoNovel multiplexPCR assay for detection of the staphylococcal virulence markerPanton-Valentine leukocidin genes and simultaneous discrimi-nation of methicillin-susceptible from -resistant staphylococcirdquoJournal of Clinical Microbiology vol 44 no 3 pp 1141ndash11442006
[13] T Nuryastuti H C van der Mei H J Busscher R Kuijer A TAman and B P Krom ldquorecA mediated spontaneous deletionsof the icaADBC operon of clinical Staphylococcus epidermidis
isolates a new mechanism of phenotypic variationsrdquo Antonievan Leeuwenhoek-Journal ofMicrobiology vol 94 no 2 pp 317ndash328 2008
[14] A Karmakar P Dua and C Ghosh ldquoBiochemical and molec-ular analysis of Staphylococcus aureus clinical isolates fromhospitalized patientsrdquo Canadian Journal of Infectious Diseasesamp Medical Microbiology vol 2016 Article ID 9041636 7 pages2016
[15] G A Lancette and S R Tatini ldquoStaphylococcus aureusrdquo inCompendium of Methods for the Microbiological Examination ofFoods C Vanderzant and D F Splittstoesser Eds pp 533ndash550American Public Health Association Washington DC USA3rd edition 1992
[16] E B Blair J S Emerson and A H Tull ldquoA new mediumsalt mannitol plasma agar for the isolation of Staphylococcusaureusrdquo American Journal of Clinical Pathology vol 47 no 1pp 30ndash39 1967
[17] G H Chapman ldquoThe significance of sodium chloride in studiesof staphylococcirdquo Journal of Bacteriology vol 50 pp 201ndash2031945
[18] R Cruickshank J P Duguid B PMarmion andRHA SwainMed Microbiol vol II Chruchill Livingstone Edinburgh UK12nd edition 1975
[19] A Swan ldquoThe use of a bile-aesculin medium and of Maxtedrsquostechnique of Lancefield grouping in the identification of ente-rococci (Group D Streptococci)rdquo Journal of Clinical Pathologyvol 7 no 2 pp 160ndash163 1954
[20] C R Arciola L Baldassarri and L Montanaro ldquoPresenceof icaA and icaD genes and slime production in a collectionof Staphylococcal strains from catheter-associated infectionsrdquoJournal of Clinical Microbiology vol 39 no 6 pp 2151ndash21562001
[21] P IWatnickCM LaurianoK EKlose LCroal andRKolterldquoThe absence of a flagellum leads to altered colony morphologybiofilm development and virulence in Vibrio cholerae O139rdquoMolecularMicrobiology vol 39 no 2 pp 223ndash235 2001
[22] J-O Cha J I Yoo J S Yoo et al ldquoinvestigation of biofilmformation and its association with the molecular and clinicalcharacteristics of methicillin-resistant staphylococcus aureusrdquoOsong Public Health and Research Perspectives vol 4 no 5 pp225ndash232 2013
[23] AW BauerWMKirby J C Sherris andMTurck ldquoAntibioticsusceptibility testing by a standardized single disk methodrdquoAmerican Journal of Clinical Pathology vol 45 no 4 ts pp 493ndash496 1966
[24] CLSI ldquoPerformance standards for antimicrobial susceptibilitytesting twenty-fifth informational supplementrdquo CLSI Docu-ment M100-S25 Clinical and Laboratory Standards InstituteWayne Pa USA 2015
[25] F Yu Y Liu Y Xu et al ldquoExpression of panton-valentine leuko-cidin mrna among staphylococcus aureus isolates associateswith specific clinical presentationsrdquo PLoS ONE vol 8 no 12p e83368 2013
[26] S V Kazakova J C Hageman M Matava et al ldquoA cloneof methicillin-resistant Staphylococcus aureus among profes-sional football playersrdquo The New England Journal of Medicinevol 352 no 5 pp 468ndash475 2005
[27] D R Bhatta LMCavacoG Nath et al ldquoAssociation of PantonValentine Leukocidin (PVL) genes with methicillin resistantStaphylococcus aureus (MRSA) in Western Nepal A matter ofconcern for community infections (a hospital based prospectivestudy)rdquo BMC Infectious Diseases vol 16 no 1 2016
8 Journal of Pathogens
[28] A Jain and A Agarwal ldquoBiofilm production a marker ofpathogenic potential of colonizing and commensal staphylo-coccirdquo Journal of Microbiological Methods vol 76 no 1 pp 88ndash92 2009
[29] M Zalipour H S Ebrahim-Saraie J Sarvari and R KhasheildquoDetection of biofilm production capability and icaAD genesamong staphylococci isolates from Shiraz Iranrdquo JundishapurJournal of Microbiology vol 9 no 12 pp 1ndash7 2016
[30] T Mathur S Singhal S Khan D J Upadhyay T Fatmaand A Rattan ldquoDetection of biofilm formation among theclinical isolates of staphylococci an evaluation of three differentscreeningmethodsrdquo Indian Journal ofMedicalMicrobiology vol24 no 1 pp 25ndash29 2006
[31] A Gravet M Rondeau C Harf-Monteil et al ldquoPredomi-nant Staphylococcus aureus isolated from antibiotic-associateddiarrhea is clinically relevant and produces enterotoxin Aand the bicomponent toxin LukE-LukDrdquo Journal of ClinicalMicrobiology vol 37 no 12 pp 4012ndash4019 1999
[32] G Lina Y Piemont F Godail-Gamot et al ldquoInvolve-ment of Panton-Valentine leukocidinmdashproducing Staphylococ-cus aureus in primary skin infections and pneumoniardquo ClinicalInfectious Diseases vol 29 no 5 pp 1128ndash1132 1999
[33] S A Havaei S O Moghadam M R Pourmand and JFaghri ldquoPrevalence of genes encoding bi-component leuko-cidins among clinical isolates of methicillin-resistant Staphylo-coccus aureusrdquo Iranian Journal of Public Health vol 39 no 1pp 8ndash14 2010
[34] L G Miller F Perdreau-Remington G Rieg et al ldquoNecrotizingfasciitis caused by community-associated methicillin-resistantStaphylococcus aureus in Los AngelesrdquoTheNewEngland Journalof Medicine vol 352 no 14 pp 1445ndash1453 2005
[35] A Holmes M Ganner S McGuane T L Pitt B D Cooksonand A M Kearns ldquoStaphylococcus aureus isolates carryingPanton-Valentine leucocidin genes in England and Wales fre-quency characterization and association with clinical diseaserdquoJournal of Clinical Microbiology vol 43 no 5 pp 2384ndash23902005
[36] I M Moussa and A M Shibl ldquoMolecular characterizationof methicillin-resistant Staphylococcus aureus recovered fromoutpatient clinics in Riyadh Saudi Arabiardquo Saudi MedicalJournal vol 30 no 5 pp 611ndash617 2009
[37] S Afroz N Kobayashi S Nagashima M M Alam A BM B Hossain and M A Rahman ldquoGenetic characterizationof Staphylococcus aureus isolates carrying Panton ValentineLeukocidin genes in Bangladeshrdquo Japanese Journal of InfectiousDiseases vol 61 pp 393ndash396 2008
[38] H Kaur S Purwar A Saini et al ldquoStatus of methicillinresistant Staphylococcus aureus infections and evaluation ofPVL producing strains in Belgaum South Indiardquo Journal ofKrishna Institute of Medical Sciences University vol 1 no 2 pp43ndash51 2012
[39] D Johnsson P Molling K Stralin and B Soderquist ldquoDetec-tion of Panton-Valentine leukocidin gene in Straphylococ-cus aureus by LightCycler PCR clinical and epidemiologicalaspectsrdquo Clinical Microbiology and Infection vol 10 no 10 pp884ndash889 2004
[40] A Rostamzad and N Rostamneia ldquoPrevalence of the panton-valentine leukocidin gene in clinical isolates of Staphylococcusaureus isolated from hospitals the ilam province of IranrdquoAvicenna Journal of Clinical Microbiology and Infection vol 3no 1 2016
[41] K O Bhutia and T S Singh ldquoThe prevalence and the riskfactors which are associated with Staphylococcus aureus andmethicillin-resistant S aureus which harboured the Panton-Valentine- Leukocidin gene in Sikkimrdquo Journal of Clinical andDiagnostic Research vol 6 no 3 pp 393ndash399 2012
[42] M Li G Y C Cheung J Hu et al ldquoComparative analysis ofvirulence and toxin expression of global community-associatedmethicillin-resistant Staphylococcus aureus strainsrdquo The Jour-nal of Infectious Diseases vol 202 no 12 pp 1866ndash1876 2010
[43] CWitzWWitte CWolz andCGoerke ldquoTranscription of thephage-encoded Panton-Valentine leukocidin of Staphylococcusaureus is dependent on the phage life-cycle and on the hostbackgroundrdquoMicrobiology vol 155 no 11 pp 3491ndash3499 2009
[44] C E Turner and S Sriskandan ldquoPantone-Valentine leucocidinexpression by Staphylococcus aureus exposed to commonantibioticsrdquo Infection vol 71 no 3 pp 338ndash346 2015
[45] J K Rudkin M Laabei A M Edwards et al ldquoOxacillinalters the toxin expression profile of community-associatedmethicillin-resistant Staphylococcus aureusrdquo AntimicrobialAgents and Chemotherapy vol 58 no 2 pp 1100ndash1107 2014
210 Statistical Analysis Data analysis and plotting of datawere done using Microsoft Excel Mean and standard error ofmean was also calculated for each quantitative variable usingMicrosoft Excel
3 Results
A total of 25 nonduplicate isolates of S aureus were obtainedfrom different slum areas in Midnapore town West BengalIndia and included in this study Samples were collectedfromnasal swab and superficial infection sites of the subjectsAmong 25 isolates 22 strains (88) were isolated usingselective MSA media and then these isolates were identifiedas S aureus by several phenotypic expressions by means ofdifferent biochemical tests (Table 2) All S aureus isolates inthis study were confirmed by PCR using the species-specificprimer pair (Table 3) In this study we found that 100 91and 23 isolates were positive for catalase coagulase andheat-stable nuclease respectivelyOf the isolated strains 6855 and 68 produced protease lipase and nonwhite pig-mented colonies respectively Rates of resistance of S aureus
ranged from 27 to 91 to different antibiotics Resistance of Saureus isolates to ampicillin (91) nalidixic acid (91) chlo-ramphenicol (36) streptomycin (27) kanamycin (55)cefoxitin (68) novobiocin (64) and erythromycin (82)was found S aureus isolates showing resistance to at leastone agent from three or more antimicrobial categories islabeled as multi-drug resistant Accordingly all S aureusstrains isolated in this study were found to be multi-drugresistant In the present study the prevalence rate of MRSAwas found to be 68 It was also found that 5 of theisolated S aureus were vancomycin intermediate and 95of the isolated S aureus were vancomycin sensitive None ofthe strains were found to be VRSA In general observationsslime-producing strains appear as black colonies whereasnon-slime-producing strains appear as red colonies in CRAplates Among the community acquired isolates 15 out of22 (68) S aureus strains appeared to be slime-producingafter 48 hrs of incubation at 37∘C Moreover in the presentstudy 7 (32) were found as strong biofilm forming and15 (68) isolates were moderate biofilm forming None ofthe isolates were biofilm-negative (Figure 2) Twenty-seven
Journal of Pathogens 5
02468
10121416
lt02 02-1 gt1Low Biofilm Former Moderate Biofilm Former High Biofilm Former
No of strains
Figure 2 Biofilm formation ability of community strains from slum area
228 bp
M 1 2 3 4 5 6 7 8 9 10 11 12 1413
(a)
270 bp
M 1 2 3 4 5 6 7
(b)
M 1 2 3 4 5 6 7
147 bp
1000 bp
500 bp
200 bp
(c)
M 1 2
433 bp
(d)
Figure 3 (a) Agarose gel electrophoresis pattern for identification of Staphylococcus aureus specific 16S rRNA gene M molecular weightmarker Lane 1-13 different clinical strains of Staphylococcus aureus Lane 14 negative control Escherichia coli (SM10 120582pir) (b) nuc geneM DNA molecular weight marker (100 bp DNA ladder) Lane 1 positive control Lanes 2 3 4 and 5 270 bp band obtained with DNAfrom clinical strains Lane 6 negative control (Staphylococcus epidermidis) (c) Agarose gel electrophoresis patterns showing PCR-amplifiedproducts for the mecA gene M DNA molecular weight marker (100 bp DNA ladder) Lane 1 positive control Lanes 2 3 4 5 and 6 147 bpband obtained with DNA from MRSA clinical strains Lane 7 negative control (Staphylococcus epidermidis) (d) Agarose gel electrophoresispatterns showing PCR-amplified products for the pvl gene Lanes 1 and 2 433 bp band obtained with DNA fromMRSA clinical strains
percent (27) strains were found to harbour nuc gene(Figure 3) mecA and pvl genes were detected in 17 out of22 (68) and 2 out of 22 isolates (9) respectively All thepvl harbouring isolates possessed mecA gene (Figure 3) Thelow 998779Ct value (0493) in qRT-PCR was indicative of highexpression of pvl in one S aureus strain (Figure 4)
4 Discussion
In the present study very high rates of resistance particu-larly towards penicillin and also for kanamycin cefoxitinnovobiocin and erythromycin were observed in the isolatedcommunity acquired S aureus strains In 2005 Kazakova
6 Journal of Pathogens
recA pvlGenes
23
235
24
245
25
255
26
265
27
CT M
ean
Figure 4 Comparative analysis of the cycle threshold by qRT-PCRassay Black bar represents the housekeeping gene recA and graybar represents pvl gene
et al [26] reported a community-associated MRSA (CA-MRSA) clone isolated from US football players with skinabscesses The strain was susceptible to most antimicrobialagents except 120573-lactams and macrolides Bhatta et al [27]reported the detection of mecA gene in 568 of the isolatesfrom clinical and community settings In this study outof twenty-two clinical isolates fifteen (68) isolates turnedout to be slime producer Jain and Agarwal [28] reporteda higher rate where 64 of staphylococcal intravascularisolates were biofilm producers by CRA method Zalipouret al [29] reported that forty-three (544) biofilm formingstrains were found in clinical specimens in Iran by CRAassay In the current study seven (32) isolates were strongbiofilm formers and fifteen (68) isolates were moderatebiofilm formers Mathur et al [30] reported that 578 ofstaphylococcal clinical isolates established biofilm-positivecharacteristics and 1447 and 394 exhibited high andmoderate biofilm production respectively in India Globalemergence of MRSA is serious public health problem andchallenge to clinicians Various factors devote to drug resis-tance and the pathogenicity of S aureus The first PVLpositive MRSA was noticed in the late 1990s and these strainsgot scattered worldwide in recent years [31] The role of PVLin boosting virulence of S aureus and their pathogenicityis being deliberated Panton-Valentine leukocidin rises thepathogenicity of S aureus by necrosis quickening apoptosisand damage of polymorphonuclear and mononuclear cellsthereby contributing to mortality and morbidity [32] PVL isgenerally used as a marker for community acquired MRSAliable for deep dermal infections including soft tissue [3334] However the worldwide scheme of PVL among MRSAisolates varies A lower prevalence of PVL has been reportedfrom other parts of world (5 in France 49 in UK81 in Saudi Arabia and 143 in Bangladesh) [32 35ndash37] reflecting the significant variation in prevalence of PVLamong geographical areas and communities Kaur et al [38]from India have reported overall 6285 prevalence of PVL
among MRSA and MSSA (MRSA 851 and MSSA 488)which delineates higher prevalence of PVL among MRSA incomparison to present findings Johnsson et al [39] detectedpvl gene in one isolate (1) from 65 patients with S aureusbacteraemia in two (219) isolates from 91 patients withcutaneous infections and in four (727) isolates from 55patients with respiratory tract infections Rostamzad et al[40] reported that out of thirty-two MRSA isolates thirteenisolates (4062)were positive for presence of the luk-pv genefrom hospitals isolates Higher prevalence of PVL amongchildren (lt14 years of age) was observed as compared toadults and old age group patients Similar observations weremade in another study from India [41] Epidemiological datasuggest that high virulence of community acquired MRSA isassociated with pvl gene but direct evidence of association ofPVL to pathogenesis remains limited [42]The low998779Ct value(0493) in qRT-PCR indicated high expression of pvl gene inS aureus strain in the present study However in previousstudy low expression of luk-pvwas reported [43] In S aureusexpression of toxins and others factors can be measured byusing Panton-Valentine leukocidin (PVL) expression in invivo condition [44] In addition expression of pvl gene isenhanced by sub-MIC values of 120573-lactam antibiotics [45]The studied population has a little health education andthey commonly use 120573-lactam antibiotics to treat any kindof infections this may be the reason of high expression ofpvl gene in in vitro condition Thus detection of pvl genein community isolated S aureus is indication of emergenceof pathogenic S aureus in community setup in the studiedregion
5 Conclusion
The current study reflects the elevated level of multi-drugresistant S aureus infections in community The prevalenceof the pvl gene among the MRSA isolates in this studywas low The presence of pvl among multi-drug resistantbacteria like MRSA may be fatal and challenging conditionfor clinicians The studied population has a little healtheducation and they commonly use 120573-lactam antibiotics thismay be the underlying reason for high expression of pvlThusdetection of pvl in community isolated S aureus indicates theemergence of pathogenic S aureus in community setup in thestudied region
Data Availability
The data used to support the findings of this study areavailable from the corresponding author upon request
Conflicts of Interest
The authors declare that there are no conflicts of interestregarding the publication of this paper
Acknowledgments
The authors are grateful to the individuals who participatedin the study
Journal of Pathogens 7
References
[1] B Shrestha W Singh V S Raj B M Pokhrel and T MMohapatra ldquoHigh Prevalence of Panton-Valentine leukocidin(PVL) genes in nosocomial-acquired staphylococcus aureusisolated from tertiary care hospitals in Nepalrdquo BioMed ResearchInternational vol 2014 Article ID 790350 7 pages 2014
[2] J Kaneko and Y Kamio ldquoBacterial two-component and hetero-heptameric pore-forming cytolytic toxins structures pore-forming mechanism and organization of the genesrdquo BioscienceBiotechnology and Biochemistry vol 68 no 5 pp 981ndash10032004
[3] G Prevost B Cribier P Couppie et al ldquoPanton-valentineleucocidin and gamma-hemolysin from Staphylococcus aureusATCC 49775 are encoded by distinct genetic loci and havedifferent biological activitiesrdquo Infection and Immunity vol 63no 10 pp 4121ndash4129 1995
[4] D C Melles W B Van Leeuwen H A M Boelens J KPeetersH AVerbrugh andAVanBelkum ldquoPanton-Valentineleukocidin genes in Staphylococcus aureusrdquoEmerging InfectiousDiseases vol 12 no 7 pp 1174-1175 2006
[5] B McGrath F Rutledge and E Broadfield ldquo NecrotisingPneumonia rdquo Journal of the Intensive Care Society vol 9 no2 pp 170ndash172 2008
[6] F Yu Z Chen C Liu et al ldquoPrevalence of Staphylococcusaureus carrying Panton-Valentine leukocidin genes among iso-lates from hospitalised patients in Chinardquo Clinical Microbiologyand Infection vol 14 no 4 pp 381ndash384 2008
[7] S Monecke P Slickers M J Ellington A M Kearns andR Ehricht ldquoHigh diversity of Panton-Valentine leukocidin-positive methicillin-susceptible isolates of Staphylococcusaureus and implications for the evolution of community-associatedmethicillin-resistant S aureusrdquoClinicalMicrobiologyand Infection vol 13 no 12 pp 1157ndash1164 2007
[8] D J Skiest K Brown T W Cooper H Hoffman-RobertsH R Mussa and A C Elliott ldquoProspective comparison ofmethicillin-susceptible and methicillin-resistant community-associated Staphylococcus aureus infections in hospitalizedpatientsrdquo Infection vol 54 no 5 pp 427ndash434 2007
[9] S R Monday and G A Bohach ldquoUse of multiplex PCR todetect classical and newly described pyrogenic toxin genes inStaphylococcal isolatesrdquo Journal of Clinical Microbiology vol 37no 10 pp 3411ndash3414 1999
[10] O G Brakstad K Aasbakk and J A Maeland ldquoDetection ofStaphylococcus aureus by polymerase chain reaction amplifica-tion of the nuc generdquo Journal of Clinical Microbiology vol 30no 7 pp 1654ndash1660 1992
[11] K Zhang J-A McClure S Elsayed T Louie and J MConly ldquoNovel multiplex PCR assay for characterization andconcomitant subtyping of staphylococcal cassette chromosomemec types I to V in methicillin-resistant Staphylococcus aureusrdquoJournal of Clinical Microbiology vol 43 no 10 pp 5026ndash50332005
[12] J-A McClure J M Conly V Lau et al ldquoNovel multiplexPCR assay for detection of the staphylococcal virulence markerPanton-Valentine leukocidin genes and simultaneous discrimi-nation of methicillin-susceptible from -resistant staphylococcirdquoJournal of Clinical Microbiology vol 44 no 3 pp 1141ndash11442006
[13] T Nuryastuti H C van der Mei H J Busscher R Kuijer A TAman and B P Krom ldquorecA mediated spontaneous deletionsof the icaADBC operon of clinical Staphylococcus epidermidis
isolates a new mechanism of phenotypic variationsrdquo Antonievan Leeuwenhoek-Journal ofMicrobiology vol 94 no 2 pp 317ndash328 2008
[14] A Karmakar P Dua and C Ghosh ldquoBiochemical and molec-ular analysis of Staphylococcus aureus clinical isolates fromhospitalized patientsrdquo Canadian Journal of Infectious Diseasesamp Medical Microbiology vol 2016 Article ID 9041636 7 pages2016
[15] G A Lancette and S R Tatini ldquoStaphylococcus aureusrdquo inCompendium of Methods for the Microbiological Examination ofFoods C Vanderzant and D F Splittstoesser Eds pp 533ndash550American Public Health Association Washington DC USA3rd edition 1992
[16] E B Blair J S Emerson and A H Tull ldquoA new mediumsalt mannitol plasma agar for the isolation of Staphylococcusaureusrdquo American Journal of Clinical Pathology vol 47 no 1pp 30ndash39 1967
[17] G H Chapman ldquoThe significance of sodium chloride in studiesof staphylococcirdquo Journal of Bacteriology vol 50 pp 201ndash2031945
[18] R Cruickshank J P Duguid B PMarmion andRHA SwainMed Microbiol vol II Chruchill Livingstone Edinburgh UK12nd edition 1975
[19] A Swan ldquoThe use of a bile-aesculin medium and of Maxtedrsquostechnique of Lancefield grouping in the identification of ente-rococci (Group D Streptococci)rdquo Journal of Clinical Pathologyvol 7 no 2 pp 160ndash163 1954
[20] C R Arciola L Baldassarri and L Montanaro ldquoPresenceof icaA and icaD genes and slime production in a collectionof Staphylococcal strains from catheter-associated infectionsrdquoJournal of Clinical Microbiology vol 39 no 6 pp 2151ndash21562001
[21] P IWatnickCM LaurianoK EKlose LCroal andRKolterldquoThe absence of a flagellum leads to altered colony morphologybiofilm development and virulence in Vibrio cholerae O139rdquoMolecularMicrobiology vol 39 no 2 pp 223ndash235 2001
[22] J-O Cha J I Yoo J S Yoo et al ldquoinvestigation of biofilmformation and its association with the molecular and clinicalcharacteristics of methicillin-resistant staphylococcus aureusrdquoOsong Public Health and Research Perspectives vol 4 no 5 pp225ndash232 2013
[23] AW BauerWMKirby J C Sherris andMTurck ldquoAntibioticsusceptibility testing by a standardized single disk methodrdquoAmerican Journal of Clinical Pathology vol 45 no 4 ts pp 493ndash496 1966
[24] CLSI ldquoPerformance standards for antimicrobial susceptibilitytesting twenty-fifth informational supplementrdquo CLSI Docu-ment M100-S25 Clinical and Laboratory Standards InstituteWayne Pa USA 2015
[25] F Yu Y Liu Y Xu et al ldquoExpression of panton-valentine leuko-cidin mrna among staphylococcus aureus isolates associateswith specific clinical presentationsrdquo PLoS ONE vol 8 no 12p e83368 2013
[26] S V Kazakova J C Hageman M Matava et al ldquoA cloneof methicillin-resistant Staphylococcus aureus among profes-sional football playersrdquo The New England Journal of Medicinevol 352 no 5 pp 468ndash475 2005
[27] D R Bhatta LMCavacoG Nath et al ldquoAssociation of PantonValentine Leukocidin (PVL) genes with methicillin resistantStaphylococcus aureus (MRSA) in Western Nepal A matter ofconcern for community infections (a hospital based prospectivestudy)rdquo BMC Infectious Diseases vol 16 no 1 2016
8 Journal of Pathogens
[28] A Jain and A Agarwal ldquoBiofilm production a marker ofpathogenic potential of colonizing and commensal staphylo-coccirdquo Journal of Microbiological Methods vol 76 no 1 pp 88ndash92 2009
[29] M Zalipour H S Ebrahim-Saraie J Sarvari and R KhasheildquoDetection of biofilm production capability and icaAD genesamong staphylococci isolates from Shiraz Iranrdquo JundishapurJournal of Microbiology vol 9 no 12 pp 1ndash7 2016
[30] T Mathur S Singhal S Khan D J Upadhyay T Fatmaand A Rattan ldquoDetection of biofilm formation among theclinical isolates of staphylococci an evaluation of three differentscreeningmethodsrdquo Indian Journal ofMedicalMicrobiology vol24 no 1 pp 25ndash29 2006
[31] A Gravet M Rondeau C Harf-Monteil et al ldquoPredomi-nant Staphylococcus aureus isolated from antibiotic-associateddiarrhea is clinically relevant and produces enterotoxin Aand the bicomponent toxin LukE-LukDrdquo Journal of ClinicalMicrobiology vol 37 no 12 pp 4012ndash4019 1999
[32] G Lina Y Piemont F Godail-Gamot et al ldquoInvolve-ment of Panton-Valentine leukocidinmdashproducing Staphylococ-cus aureus in primary skin infections and pneumoniardquo ClinicalInfectious Diseases vol 29 no 5 pp 1128ndash1132 1999
[33] S A Havaei S O Moghadam M R Pourmand and JFaghri ldquoPrevalence of genes encoding bi-component leuko-cidins among clinical isolates of methicillin-resistant Staphylo-coccus aureusrdquo Iranian Journal of Public Health vol 39 no 1pp 8ndash14 2010
[34] L G Miller F Perdreau-Remington G Rieg et al ldquoNecrotizingfasciitis caused by community-associated methicillin-resistantStaphylococcus aureus in Los AngelesrdquoTheNewEngland Journalof Medicine vol 352 no 14 pp 1445ndash1453 2005
[35] A Holmes M Ganner S McGuane T L Pitt B D Cooksonand A M Kearns ldquoStaphylococcus aureus isolates carryingPanton-Valentine leucocidin genes in England and Wales fre-quency characterization and association with clinical diseaserdquoJournal of Clinical Microbiology vol 43 no 5 pp 2384ndash23902005
[36] I M Moussa and A M Shibl ldquoMolecular characterizationof methicillin-resistant Staphylococcus aureus recovered fromoutpatient clinics in Riyadh Saudi Arabiardquo Saudi MedicalJournal vol 30 no 5 pp 611ndash617 2009
[37] S Afroz N Kobayashi S Nagashima M M Alam A BM B Hossain and M A Rahman ldquoGenetic characterizationof Staphylococcus aureus isolates carrying Panton ValentineLeukocidin genes in Bangladeshrdquo Japanese Journal of InfectiousDiseases vol 61 pp 393ndash396 2008
[38] H Kaur S Purwar A Saini et al ldquoStatus of methicillinresistant Staphylococcus aureus infections and evaluation ofPVL producing strains in Belgaum South Indiardquo Journal ofKrishna Institute of Medical Sciences University vol 1 no 2 pp43ndash51 2012
[39] D Johnsson P Molling K Stralin and B Soderquist ldquoDetec-tion of Panton-Valentine leukocidin gene in Straphylococ-cus aureus by LightCycler PCR clinical and epidemiologicalaspectsrdquo Clinical Microbiology and Infection vol 10 no 10 pp884ndash889 2004
[40] A Rostamzad and N Rostamneia ldquoPrevalence of the panton-valentine leukocidin gene in clinical isolates of Staphylococcusaureus isolated from hospitals the ilam province of IranrdquoAvicenna Journal of Clinical Microbiology and Infection vol 3no 1 2016
[41] K O Bhutia and T S Singh ldquoThe prevalence and the riskfactors which are associated with Staphylococcus aureus andmethicillin-resistant S aureus which harboured the Panton-Valentine- Leukocidin gene in Sikkimrdquo Journal of Clinical andDiagnostic Research vol 6 no 3 pp 393ndash399 2012
[42] M Li G Y C Cheung J Hu et al ldquoComparative analysis ofvirulence and toxin expression of global community-associatedmethicillin-resistant Staphylococcus aureus strainsrdquo The Jour-nal of Infectious Diseases vol 202 no 12 pp 1866ndash1876 2010
[43] CWitzWWitte CWolz andCGoerke ldquoTranscription of thephage-encoded Panton-Valentine leukocidin of Staphylococcusaureus is dependent on the phage life-cycle and on the hostbackgroundrdquoMicrobiology vol 155 no 11 pp 3491ndash3499 2009
[44] C E Turner and S Sriskandan ldquoPantone-Valentine leucocidinexpression by Staphylococcus aureus exposed to commonantibioticsrdquo Infection vol 71 no 3 pp 338ndash346 2015
[45] J K Rudkin M Laabei A M Edwards et al ldquoOxacillinalters the toxin expression profile of community-associatedmethicillin-resistant Staphylococcus aureusrdquo AntimicrobialAgents and Chemotherapy vol 58 no 2 pp 1100ndash1107 2014
Computational and Mathematical Methods in Medicine
Hindawiwwwhindawicom Volume 2018
Behavioural Neurology
OphthalmologyJournal of
Hindawiwwwhindawicom Volume 2018
Diabetes ResearchJournal of
Hindawiwwwhindawicom Volume 2018
Hindawiwwwhindawicom Volume 2018
Research and TreatmentAIDS
Hindawiwwwhindawicom Volume 2018
Gastroenterology Research and Practice
Hindawiwwwhindawicom Volume 2018
Parkinsonrsquos Disease
Evidence-Based Complementary andAlternative Medicine
Volume 2018Hindawiwwwhindawicom
Submit your manuscripts atwwwhindawicom
Journal of Pathogens 5
02468
10121416
lt02 02-1 gt1Low Biofilm Former Moderate Biofilm Former High Biofilm Former
No of strains
Figure 2 Biofilm formation ability of community strains from slum area
228 bp
M 1 2 3 4 5 6 7 8 9 10 11 12 1413
(a)
270 bp
M 1 2 3 4 5 6 7
(b)
M 1 2 3 4 5 6 7
147 bp
1000 bp
500 bp
200 bp
(c)
M 1 2
433 bp
(d)
Figure 3 (a) Agarose gel electrophoresis pattern for identification of Staphylococcus aureus specific 16S rRNA gene M molecular weightmarker Lane 1-13 different clinical strains of Staphylococcus aureus Lane 14 negative control Escherichia coli (SM10 120582pir) (b) nuc geneM DNA molecular weight marker (100 bp DNA ladder) Lane 1 positive control Lanes 2 3 4 and 5 270 bp band obtained with DNAfrom clinical strains Lane 6 negative control (Staphylococcus epidermidis) (c) Agarose gel electrophoresis patterns showing PCR-amplifiedproducts for the mecA gene M DNA molecular weight marker (100 bp DNA ladder) Lane 1 positive control Lanes 2 3 4 5 and 6 147 bpband obtained with DNA from MRSA clinical strains Lane 7 negative control (Staphylococcus epidermidis) (d) Agarose gel electrophoresispatterns showing PCR-amplified products for the pvl gene Lanes 1 and 2 433 bp band obtained with DNA fromMRSA clinical strains
percent (27) strains were found to harbour nuc gene(Figure 3) mecA and pvl genes were detected in 17 out of22 (68) and 2 out of 22 isolates (9) respectively All thepvl harbouring isolates possessed mecA gene (Figure 3) Thelow 998779Ct value (0493) in qRT-PCR was indicative of highexpression of pvl in one S aureus strain (Figure 4)
4 Discussion
In the present study very high rates of resistance particu-larly towards penicillin and also for kanamycin cefoxitinnovobiocin and erythromycin were observed in the isolatedcommunity acquired S aureus strains In 2005 Kazakova
6 Journal of Pathogens
recA pvlGenes
23
235
24
245
25
255
26
265
27
CT M
ean
Figure 4 Comparative analysis of the cycle threshold by qRT-PCRassay Black bar represents the housekeeping gene recA and graybar represents pvl gene
et al [26] reported a community-associated MRSA (CA-MRSA) clone isolated from US football players with skinabscesses The strain was susceptible to most antimicrobialagents except 120573-lactams and macrolides Bhatta et al [27]reported the detection of mecA gene in 568 of the isolatesfrom clinical and community settings In this study outof twenty-two clinical isolates fifteen (68) isolates turnedout to be slime producer Jain and Agarwal [28] reporteda higher rate where 64 of staphylococcal intravascularisolates were biofilm producers by CRA method Zalipouret al [29] reported that forty-three (544) biofilm formingstrains were found in clinical specimens in Iran by CRAassay In the current study seven (32) isolates were strongbiofilm formers and fifteen (68) isolates were moderatebiofilm formers Mathur et al [30] reported that 578 ofstaphylococcal clinical isolates established biofilm-positivecharacteristics and 1447 and 394 exhibited high andmoderate biofilm production respectively in India Globalemergence of MRSA is serious public health problem andchallenge to clinicians Various factors devote to drug resis-tance and the pathogenicity of S aureus The first PVLpositive MRSA was noticed in the late 1990s and these strainsgot scattered worldwide in recent years [31] The role of PVLin boosting virulence of S aureus and their pathogenicityis being deliberated Panton-Valentine leukocidin rises thepathogenicity of S aureus by necrosis quickening apoptosisand damage of polymorphonuclear and mononuclear cellsthereby contributing to mortality and morbidity [32] PVL isgenerally used as a marker for community acquired MRSAliable for deep dermal infections including soft tissue [3334] However the worldwide scheme of PVL among MRSAisolates varies A lower prevalence of PVL has been reportedfrom other parts of world (5 in France 49 in UK81 in Saudi Arabia and 143 in Bangladesh) [32 35ndash37] reflecting the significant variation in prevalence of PVLamong geographical areas and communities Kaur et al [38]from India have reported overall 6285 prevalence of PVL
among MRSA and MSSA (MRSA 851 and MSSA 488)which delineates higher prevalence of PVL among MRSA incomparison to present findings Johnsson et al [39] detectedpvl gene in one isolate (1) from 65 patients with S aureusbacteraemia in two (219) isolates from 91 patients withcutaneous infections and in four (727) isolates from 55patients with respiratory tract infections Rostamzad et al[40] reported that out of thirty-two MRSA isolates thirteenisolates (4062)were positive for presence of the luk-pv genefrom hospitals isolates Higher prevalence of PVL amongchildren (lt14 years of age) was observed as compared toadults and old age group patients Similar observations weremade in another study from India [41] Epidemiological datasuggest that high virulence of community acquired MRSA isassociated with pvl gene but direct evidence of association ofPVL to pathogenesis remains limited [42]The low998779Ct value(0493) in qRT-PCR indicated high expression of pvl gene inS aureus strain in the present study However in previousstudy low expression of luk-pvwas reported [43] In S aureusexpression of toxins and others factors can be measured byusing Panton-Valentine leukocidin (PVL) expression in invivo condition [44] In addition expression of pvl gene isenhanced by sub-MIC values of 120573-lactam antibiotics [45]The studied population has a little health education andthey commonly use 120573-lactam antibiotics to treat any kindof infections this may be the reason of high expression ofpvl gene in in vitro condition Thus detection of pvl genein community isolated S aureus is indication of emergenceof pathogenic S aureus in community setup in the studiedregion
5 Conclusion
The current study reflects the elevated level of multi-drugresistant S aureus infections in community The prevalenceof the pvl gene among the MRSA isolates in this studywas low The presence of pvl among multi-drug resistantbacteria like MRSA may be fatal and challenging conditionfor clinicians The studied population has a little healtheducation and they commonly use 120573-lactam antibiotics thismay be the underlying reason for high expression of pvlThusdetection of pvl in community isolated S aureus indicates theemergence of pathogenic S aureus in community setup in thestudied region
Data Availability
The data used to support the findings of this study areavailable from the corresponding author upon request
Conflicts of Interest
The authors declare that there are no conflicts of interestregarding the publication of this paper
Acknowledgments
The authors are grateful to the individuals who participatedin the study
Journal of Pathogens 7
References
[1] B Shrestha W Singh V S Raj B M Pokhrel and T MMohapatra ldquoHigh Prevalence of Panton-Valentine leukocidin(PVL) genes in nosocomial-acquired staphylococcus aureusisolated from tertiary care hospitals in Nepalrdquo BioMed ResearchInternational vol 2014 Article ID 790350 7 pages 2014
[2] J Kaneko and Y Kamio ldquoBacterial two-component and hetero-heptameric pore-forming cytolytic toxins structures pore-forming mechanism and organization of the genesrdquo BioscienceBiotechnology and Biochemistry vol 68 no 5 pp 981ndash10032004
[3] G Prevost B Cribier P Couppie et al ldquoPanton-valentineleucocidin and gamma-hemolysin from Staphylococcus aureusATCC 49775 are encoded by distinct genetic loci and havedifferent biological activitiesrdquo Infection and Immunity vol 63no 10 pp 4121ndash4129 1995
[4] D C Melles W B Van Leeuwen H A M Boelens J KPeetersH AVerbrugh andAVanBelkum ldquoPanton-Valentineleukocidin genes in Staphylococcus aureusrdquoEmerging InfectiousDiseases vol 12 no 7 pp 1174-1175 2006
[5] B McGrath F Rutledge and E Broadfield ldquo NecrotisingPneumonia rdquo Journal of the Intensive Care Society vol 9 no2 pp 170ndash172 2008
[6] F Yu Z Chen C Liu et al ldquoPrevalence of Staphylococcusaureus carrying Panton-Valentine leukocidin genes among iso-lates from hospitalised patients in Chinardquo Clinical Microbiologyand Infection vol 14 no 4 pp 381ndash384 2008
[7] S Monecke P Slickers M J Ellington A M Kearns andR Ehricht ldquoHigh diversity of Panton-Valentine leukocidin-positive methicillin-susceptible isolates of Staphylococcusaureus and implications for the evolution of community-associatedmethicillin-resistant S aureusrdquoClinicalMicrobiologyand Infection vol 13 no 12 pp 1157ndash1164 2007
[8] D J Skiest K Brown T W Cooper H Hoffman-RobertsH R Mussa and A C Elliott ldquoProspective comparison ofmethicillin-susceptible and methicillin-resistant community-associated Staphylococcus aureus infections in hospitalizedpatientsrdquo Infection vol 54 no 5 pp 427ndash434 2007
[9] S R Monday and G A Bohach ldquoUse of multiplex PCR todetect classical and newly described pyrogenic toxin genes inStaphylococcal isolatesrdquo Journal of Clinical Microbiology vol 37no 10 pp 3411ndash3414 1999
[10] O G Brakstad K Aasbakk and J A Maeland ldquoDetection ofStaphylococcus aureus by polymerase chain reaction amplifica-tion of the nuc generdquo Journal of Clinical Microbiology vol 30no 7 pp 1654ndash1660 1992
[11] K Zhang J-A McClure S Elsayed T Louie and J MConly ldquoNovel multiplex PCR assay for characterization andconcomitant subtyping of staphylococcal cassette chromosomemec types I to V in methicillin-resistant Staphylococcus aureusrdquoJournal of Clinical Microbiology vol 43 no 10 pp 5026ndash50332005
[12] J-A McClure J M Conly V Lau et al ldquoNovel multiplexPCR assay for detection of the staphylococcal virulence markerPanton-Valentine leukocidin genes and simultaneous discrimi-nation of methicillin-susceptible from -resistant staphylococcirdquoJournal of Clinical Microbiology vol 44 no 3 pp 1141ndash11442006
[13] T Nuryastuti H C van der Mei H J Busscher R Kuijer A TAman and B P Krom ldquorecA mediated spontaneous deletionsof the icaADBC operon of clinical Staphylococcus epidermidis
isolates a new mechanism of phenotypic variationsrdquo Antonievan Leeuwenhoek-Journal ofMicrobiology vol 94 no 2 pp 317ndash328 2008
[14] A Karmakar P Dua and C Ghosh ldquoBiochemical and molec-ular analysis of Staphylococcus aureus clinical isolates fromhospitalized patientsrdquo Canadian Journal of Infectious Diseasesamp Medical Microbiology vol 2016 Article ID 9041636 7 pages2016
[15] G A Lancette and S R Tatini ldquoStaphylococcus aureusrdquo inCompendium of Methods for the Microbiological Examination ofFoods C Vanderzant and D F Splittstoesser Eds pp 533ndash550American Public Health Association Washington DC USA3rd edition 1992
[16] E B Blair J S Emerson and A H Tull ldquoA new mediumsalt mannitol plasma agar for the isolation of Staphylococcusaureusrdquo American Journal of Clinical Pathology vol 47 no 1pp 30ndash39 1967
[17] G H Chapman ldquoThe significance of sodium chloride in studiesof staphylococcirdquo Journal of Bacteriology vol 50 pp 201ndash2031945
[18] R Cruickshank J P Duguid B PMarmion andRHA SwainMed Microbiol vol II Chruchill Livingstone Edinburgh UK12nd edition 1975
[19] A Swan ldquoThe use of a bile-aesculin medium and of Maxtedrsquostechnique of Lancefield grouping in the identification of ente-rococci (Group D Streptococci)rdquo Journal of Clinical Pathologyvol 7 no 2 pp 160ndash163 1954
[20] C R Arciola L Baldassarri and L Montanaro ldquoPresenceof icaA and icaD genes and slime production in a collectionof Staphylococcal strains from catheter-associated infectionsrdquoJournal of Clinical Microbiology vol 39 no 6 pp 2151ndash21562001
[21] P IWatnickCM LaurianoK EKlose LCroal andRKolterldquoThe absence of a flagellum leads to altered colony morphologybiofilm development and virulence in Vibrio cholerae O139rdquoMolecularMicrobiology vol 39 no 2 pp 223ndash235 2001
[22] J-O Cha J I Yoo J S Yoo et al ldquoinvestigation of biofilmformation and its association with the molecular and clinicalcharacteristics of methicillin-resistant staphylococcus aureusrdquoOsong Public Health and Research Perspectives vol 4 no 5 pp225ndash232 2013
[23] AW BauerWMKirby J C Sherris andMTurck ldquoAntibioticsusceptibility testing by a standardized single disk methodrdquoAmerican Journal of Clinical Pathology vol 45 no 4 ts pp 493ndash496 1966
[24] CLSI ldquoPerformance standards for antimicrobial susceptibilitytesting twenty-fifth informational supplementrdquo CLSI Docu-ment M100-S25 Clinical and Laboratory Standards InstituteWayne Pa USA 2015
[25] F Yu Y Liu Y Xu et al ldquoExpression of panton-valentine leuko-cidin mrna among staphylococcus aureus isolates associateswith specific clinical presentationsrdquo PLoS ONE vol 8 no 12p e83368 2013
[26] S V Kazakova J C Hageman M Matava et al ldquoA cloneof methicillin-resistant Staphylococcus aureus among profes-sional football playersrdquo The New England Journal of Medicinevol 352 no 5 pp 468ndash475 2005
[27] D R Bhatta LMCavacoG Nath et al ldquoAssociation of PantonValentine Leukocidin (PVL) genes with methicillin resistantStaphylococcus aureus (MRSA) in Western Nepal A matter ofconcern for community infections (a hospital based prospectivestudy)rdquo BMC Infectious Diseases vol 16 no 1 2016
8 Journal of Pathogens
[28] A Jain and A Agarwal ldquoBiofilm production a marker ofpathogenic potential of colonizing and commensal staphylo-coccirdquo Journal of Microbiological Methods vol 76 no 1 pp 88ndash92 2009
[29] M Zalipour H S Ebrahim-Saraie J Sarvari and R KhasheildquoDetection of biofilm production capability and icaAD genesamong staphylococci isolates from Shiraz Iranrdquo JundishapurJournal of Microbiology vol 9 no 12 pp 1ndash7 2016
[30] T Mathur S Singhal S Khan D J Upadhyay T Fatmaand A Rattan ldquoDetection of biofilm formation among theclinical isolates of staphylococci an evaluation of three differentscreeningmethodsrdquo Indian Journal ofMedicalMicrobiology vol24 no 1 pp 25ndash29 2006
[31] A Gravet M Rondeau C Harf-Monteil et al ldquoPredomi-nant Staphylococcus aureus isolated from antibiotic-associateddiarrhea is clinically relevant and produces enterotoxin Aand the bicomponent toxin LukE-LukDrdquo Journal of ClinicalMicrobiology vol 37 no 12 pp 4012ndash4019 1999
[32] G Lina Y Piemont F Godail-Gamot et al ldquoInvolve-ment of Panton-Valentine leukocidinmdashproducing Staphylococ-cus aureus in primary skin infections and pneumoniardquo ClinicalInfectious Diseases vol 29 no 5 pp 1128ndash1132 1999
[33] S A Havaei S O Moghadam M R Pourmand and JFaghri ldquoPrevalence of genes encoding bi-component leuko-cidins among clinical isolates of methicillin-resistant Staphylo-coccus aureusrdquo Iranian Journal of Public Health vol 39 no 1pp 8ndash14 2010
[34] L G Miller F Perdreau-Remington G Rieg et al ldquoNecrotizingfasciitis caused by community-associated methicillin-resistantStaphylococcus aureus in Los AngelesrdquoTheNewEngland Journalof Medicine vol 352 no 14 pp 1445ndash1453 2005
[35] A Holmes M Ganner S McGuane T L Pitt B D Cooksonand A M Kearns ldquoStaphylococcus aureus isolates carryingPanton-Valentine leucocidin genes in England and Wales fre-quency characterization and association with clinical diseaserdquoJournal of Clinical Microbiology vol 43 no 5 pp 2384ndash23902005
[36] I M Moussa and A M Shibl ldquoMolecular characterizationof methicillin-resistant Staphylococcus aureus recovered fromoutpatient clinics in Riyadh Saudi Arabiardquo Saudi MedicalJournal vol 30 no 5 pp 611ndash617 2009
[37] S Afroz N Kobayashi S Nagashima M M Alam A BM B Hossain and M A Rahman ldquoGenetic characterizationof Staphylococcus aureus isolates carrying Panton ValentineLeukocidin genes in Bangladeshrdquo Japanese Journal of InfectiousDiseases vol 61 pp 393ndash396 2008
[38] H Kaur S Purwar A Saini et al ldquoStatus of methicillinresistant Staphylococcus aureus infections and evaluation ofPVL producing strains in Belgaum South Indiardquo Journal ofKrishna Institute of Medical Sciences University vol 1 no 2 pp43ndash51 2012
[39] D Johnsson P Molling K Stralin and B Soderquist ldquoDetec-tion of Panton-Valentine leukocidin gene in Straphylococ-cus aureus by LightCycler PCR clinical and epidemiologicalaspectsrdquo Clinical Microbiology and Infection vol 10 no 10 pp884ndash889 2004
[40] A Rostamzad and N Rostamneia ldquoPrevalence of the panton-valentine leukocidin gene in clinical isolates of Staphylococcusaureus isolated from hospitals the ilam province of IranrdquoAvicenna Journal of Clinical Microbiology and Infection vol 3no 1 2016
[41] K O Bhutia and T S Singh ldquoThe prevalence and the riskfactors which are associated with Staphylococcus aureus andmethicillin-resistant S aureus which harboured the Panton-Valentine- Leukocidin gene in Sikkimrdquo Journal of Clinical andDiagnostic Research vol 6 no 3 pp 393ndash399 2012
[42] M Li G Y C Cheung J Hu et al ldquoComparative analysis ofvirulence and toxin expression of global community-associatedmethicillin-resistant Staphylococcus aureus strainsrdquo The Jour-nal of Infectious Diseases vol 202 no 12 pp 1866ndash1876 2010
[43] CWitzWWitte CWolz andCGoerke ldquoTranscription of thephage-encoded Panton-Valentine leukocidin of Staphylococcusaureus is dependent on the phage life-cycle and on the hostbackgroundrdquoMicrobiology vol 155 no 11 pp 3491ndash3499 2009
[44] C E Turner and S Sriskandan ldquoPantone-Valentine leucocidinexpression by Staphylococcus aureus exposed to commonantibioticsrdquo Infection vol 71 no 3 pp 338ndash346 2015
[45] J K Rudkin M Laabei A M Edwards et al ldquoOxacillinalters the toxin expression profile of community-associatedmethicillin-resistant Staphylococcus aureusrdquo AntimicrobialAgents and Chemotherapy vol 58 no 2 pp 1100ndash1107 2014
Computational and Mathematical Methods in Medicine
Hindawiwwwhindawicom Volume 2018
Behavioural Neurology
OphthalmologyJournal of
Hindawiwwwhindawicom Volume 2018
Diabetes ResearchJournal of
Hindawiwwwhindawicom Volume 2018
Hindawiwwwhindawicom Volume 2018
Research and TreatmentAIDS
Hindawiwwwhindawicom Volume 2018
Gastroenterology Research and Practice
Hindawiwwwhindawicom Volume 2018
Parkinsonrsquos Disease
Evidence-Based Complementary andAlternative Medicine
Volume 2018Hindawiwwwhindawicom
Submit your manuscripts atwwwhindawicom
6 Journal of Pathogens
recA pvlGenes
23
235
24
245
25
255
26
265
27
CT M
ean
Figure 4 Comparative analysis of the cycle threshold by qRT-PCRassay Black bar represents the housekeeping gene recA and graybar represents pvl gene
et al [26] reported a community-associated MRSA (CA-MRSA) clone isolated from US football players with skinabscesses The strain was susceptible to most antimicrobialagents except 120573-lactams and macrolides Bhatta et al [27]reported the detection of mecA gene in 568 of the isolatesfrom clinical and community settings In this study outof twenty-two clinical isolates fifteen (68) isolates turnedout to be slime producer Jain and Agarwal [28] reporteda higher rate where 64 of staphylococcal intravascularisolates were biofilm producers by CRA method Zalipouret al [29] reported that forty-three (544) biofilm formingstrains were found in clinical specimens in Iran by CRAassay In the current study seven (32) isolates were strongbiofilm formers and fifteen (68) isolates were moderatebiofilm formers Mathur et al [30] reported that 578 ofstaphylococcal clinical isolates established biofilm-positivecharacteristics and 1447 and 394 exhibited high andmoderate biofilm production respectively in India Globalemergence of MRSA is serious public health problem andchallenge to clinicians Various factors devote to drug resis-tance and the pathogenicity of S aureus The first PVLpositive MRSA was noticed in the late 1990s and these strainsgot scattered worldwide in recent years [31] The role of PVLin boosting virulence of S aureus and their pathogenicityis being deliberated Panton-Valentine leukocidin rises thepathogenicity of S aureus by necrosis quickening apoptosisand damage of polymorphonuclear and mononuclear cellsthereby contributing to mortality and morbidity [32] PVL isgenerally used as a marker for community acquired MRSAliable for deep dermal infections including soft tissue [3334] However the worldwide scheme of PVL among MRSAisolates varies A lower prevalence of PVL has been reportedfrom other parts of world (5 in France 49 in UK81 in Saudi Arabia and 143 in Bangladesh) [32 35ndash37] reflecting the significant variation in prevalence of PVLamong geographical areas and communities Kaur et al [38]from India have reported overall 6285 prevalence of PVL
among MRSA and MSSA (MRSA 851 and MSSA 488)which delineates higher prevalence of PVL among MRSA incomparison to present findings Johnsson et al [39] detectedpvl gene in one isolate (1) from 65 patients with S aureusbacteraemia in two (219) isolates from 91 patients withcutaneous infections and in four (727) isolates from 55patients with respiratory tract infections Rostamzad et al[40] reported that out of thirty-two MRSA isolates thirteenisolates (4062)were positive for presence of the luk-pv genefrom hospitals isolates Higher prevalence of PVL amongchildren (lt14 years of age) was observed as compared toadults and old age group patients Similar observations weremade in another study from India [41] Epidemiological datasuggest that high virulence of community acquired MRSA isassociated with pvl gene but direct evidence of association ofPVL to pathogenesis remains limited [42]The low998779Ct value(0493) in qRT-PCR indicated high expression of pvl gene inS aureus strain in the present study However in previousstudy low expression of luk-pvwas reported [43] In S aureusexpression of toxins and others factors can be measured byusing Panton-Valentine leukocidin (PVL) expression in invivo condition [44] In addition expression of pvl gene isenhanced by sub-MIC values of 120573-lactam antibiotics [45]The studied population has a little health education andthey commonly use 120573-lactam antibiotics to treat any kindof infections this may be the reason of high expression ofpvl gene in in vitro condition Thus detection of pvl genein community isolated S aureus is indication of emergenceof pathogenic S aureus in community setup in the studiedregion
5 Conclusion
The current study reflects the elevated level of multi-drugresistant S aureus infections in community The prevalenceof the pvl gene among the MRSA isolates in this studywas low The presence of pvl among multi-drug resistantbacteria like MRSA may be fatal and challenging conditionfor clinicians The studied population has a little healtheducation and they commonly use 120573-lactam antibiotics thismay be the underlying reason for high expression of pvlThusdetection of pvl in community isolated S aureus indicates theemergence of pathogenic S aureus in community setup in thestudied region
Data Availability
The data used to support the findings of this study areavailable from the corresponding author upon request
Conflicts of Interest
The authors declare that there are no conflicts of interestregarding the publication of this paper
Acknowledgments
The authors are grateful to the individuals who participatedin the study
Journal of Pathogens 7
References
[1] B Shrestha W Singh V S Raj B M Pokhrel and T MMohapatra ldquoHigh Prevalence of Panton-Valentine leukocidin(PVL) genes in nosocomial-acquired staphylococcus aureusisolated from tertiary care hospitals in Nepalrdquo BioMed ResearchInternational vol 2014 Article ID 790350 7 pages 2014
[2] J Kaneko and Y Kamio ldquoBacterial two-component and hetero-heptameric pore-forming cytolytic toxins structures pore-forming mechanism and organization of the genesrdquo BioscienceBiotechnology and Biochemistry vol 68 no 5 pp 981ndash10032004
[3] G Prevost B Cribier P Couppie et al ldquoPanton-valentineleucocidin and gamma-hemolysin from Staphylococcus aureusATCC 49775 are encoded by distinct genetic loci and havedifferent biological activitiesrdquo Infection and Immunity vol 63no 10 pp 4121ndash4129 1995
[4] D C Melles W B Van Leeuwen H A M Boelens J KPeetersH AVerbrugh andAVanBelkum ldquoPanton-Valentineleukocidin genes in Staphylococcus aureusrdquoEmerging InfectiousDiseases vol 12 no 7 pp 1174-1175 2006
[5] B McGrath F Rutledge and E Broadfield ldquo NecrotisingPneumonia rdquo Journal of the Intensive Care Society vol 9 no2 pp 170ndash172 2008
[6] F Yu Z Chen C Liu et al ldquoPrevalence of Staphylococcusaureus carrying Panton-Valentine leukocidin genes among iso-lates from hospitalised patients in Chinardquo Clinical Microbiologyand Infection vol 14 no 4 pp 381ndash384 2008
[7] S Monecke P Slickers M J Ellington A M Kearns andR Ehricht ldquoHigh diversity of Panton-Valentine leukocidin-positive methicillin-susceptible isolates of Staphylococcusaureus and implications for the evolution of community-associatedmethicillin-resistant S aureusrdquoClinicalMicrobiologyand Infection vol 13 no 12 pp 1157ndash1164 2007
[8] D J Skiest K Brown T W Cooper H Hoffman-RobertsH R Mussa and A C Elliott ldquoProspective comparison ofmethicillin-susceptible and methicillin-resistant community-associated Staphylococcus aureus infections in hospitalizedpatientsrdquo Infection vol 54 no 5 pp 427ndash434 2007
[9] S R Monday and G A Bohach ldquoUse of multiplex PCR todetect classical and newly described pyrogenic toxin genes inStaphylococcal isolatesrdquo Journal of Clinical Microbiology vol 37no 10 pp 3411ndash3414 1999
[10] O G Brakstad K Aasbakk and J A Maeland ldquoDetection ofStaphylococcus aureus by polymerase chain reaction amplifica-tion of the nuc generdquo Journal of Clinical Microbiology vol 30no 7 pp 1654ndash1660 1992
[11] K Zhang J-A McClure S Elsayed T Louie and J MConly ldquoNovel multiplex PCR assay for characterization andconcomitant subtyping of staphylococcal cassette chromosomemec types I to V in methicillin-resistant Staphylococcus aureusrdquoJournal of Clinical Microbiology vol 43 no 10 pp 5026ndash50332005
[12] J-A McClure J M Conly V Lau et al ldquoNovel multiplexPCR assay for detection of the staphylococcal virulence markerPanton-Valentine leukocidin genes and simultaneous discrimi-nation of methicillin-susceptible from -resistant staphylococcirdquoJournal of Clinical Microbiology vol 44 no 3 pp 1141ndash11442006
[13] T Nuryastuti H C van der Mei H J Busscher R Kuijer A TAman and B P Krom ldquorecA mediated spontaneous deletionsof the icaADBC operon of clinical Staphylococcus epidermidis
isolates a new mechanism of phenotypic variationsrdquo Antonievan Leeuwenhoek-Journal ofMicrobiology vol 94 no 2 pp 317ndash328 2008
[14] A Karmakar P Dua and C Ghosh ldquoBiochemical and molec-ular analysis of Staphylococcus aureus clinical isolates fromhospitalized patientsrdquo Canadian Journal of Infectious Diseasesamp Medical Microbiology vol 2016 Article ID 9041636 7 pages2016
[15] G A Lancette and S R Tatini ldquoStaphylococcus aureusrdquo inCompendium of Methods for the Microbiological Examination ofFoods C Vanderzant and D F Splittstoesser Eds pp 533ndash550American Public Health Association Washington DC USA3rd edition 1992
[16] E B Blair J S Emerson and A H Tull ldquoA new mediumsalt mannitol plasma agar for the isolation of Staphylococcusaureusrdquo American Journal of Clinical Pathology vol 47 no 1pp 30ndash39 1967
[17] G H Chapman ldquoThe significance of sodium chloride in studiesof staphylococcirdquo Journal of Bacteriology vol 50 pp 201ndash2031945
[18] R Cruickshank J P Duguid B PMarmion andRHA SwainMed Microbiol vol II Chruchill Livingstone Edinburgh UK12nd edition 1975
[19] A Swan ldquoThe use of a bile-aesculin medium and of Maxtedrsquostechnique of Lancefield grouping in the identification of ente-rococci (Group D Streptococci)rdquo Journal of Clinical Pathologyvol 7 no 2 pp 160ndash163 1954
[20] C R Arciola L Baldassarri and L Montanaro ldquoPresenceof icaA and icaD genes and slime production in a collectionof Staphylococcal strains from catheter-associated infectionsrdquoJournal of Clinical Microbiology vol 39 no 6 pp 2151ndash21562001
[21] P IWatnickCM LaurianoK EKlose LCroal andRKolterldquoThe absence of a flagellum leads to altered colony morphologybiofilm development and virulence in Vibrio cholerae O139rdquoMolecularMicrobiology vol 39 no 2 pp 223ndash235 2001
[22] J-O Cha J I Yoo J S Yoo et al ldquoinvestigation of biofilmformation and its association with the molecular and clinicalcharacteristics of methicillin-resistant staphylococcus aureusrdquoOsong Public Health and Research Perspectives vol 4 no 5 pp225ndash232 2013
[23] AW BauerWMKirby J C Sherris andMTurck ldquoAntibioticsusceptibility testing by a standardized single disk methodrdquoAmerican Journal of Clinical Pathology vol 45 no 4 ts pp 493ndash496 1966
[24] CLSI ldquoPerformance standards for antimicrobial susceptibilitytesting twenty-fifth informational supplementrdquo CLSI Docu-ment M100-S25 Clinical and Laboratory Standards InstituteWayne Pa USA 2015
[25] F Yu Y Liu Y Xu et al ldquoExpression of panton-valentine leuko-cidin mrna among staphylococcus aureus isolates associateswith specific clinical presentationsrdquo PLoS ONE vol 8 no 12p e83368 2013
[26] S V Kazakova J C Hageman M Matava et al ldquoA cloneof methicillin-resistant Staphylococcus aureus among profes-sional football playersrdquo The New England Journal of Medicinevol 352 no 5 pp 468ndash475 2005
[27] D R Bhatta LMCavacoG Nath et al ldquoAssociation of PantonValentine Leukocidin (PVL) genes with methicillin resistantStaphylococcus aureus (MRSA) in Western Nepal A matter ofconcern for community infections (a hospital based prospectivestudy)rdquo BMC Infectious Diseases vol 16 no 1 2016
8 Journal of Pathogens
[28] A Jain and A Agarwal ldquoBiofilm production a marker ofpathogenic potential of colonizing and commensal staphylo-coccirdquo Journal of Microbiological Methods vol 76 no 1 pp 88ndash92 2009
[29] M Zalipour H S Ebrahim-Saraie J Sarvari and R KhasheildquoDetection of biofilm production capability and icaAD genesamong staphylococci isolates from Shiraz Iranrdquo JundishapurJournal of Microbiology vol 9 no 12 pp 1ndash7 2016
[30] T Mathur S Singhal S Khan D J Upadhyay T Fatmaand A Rattan ldquoDetection of biofilm formation among theclinical isolates of staphylococci an evaluation of three differentscreeningmethodsrdquo Indian Journal ofMedicalMicrobiology vol24 no 1 pp 25ndash29 2006
[31] A Gravet M Rondeau C Harf-Monteil et al ldquoPredomi-nant Staphylococcus aureus isolated from antibiotic-associateddiarrhea is clinically relevant and produces enterotoxin Aand the bicomponent toxin LukE-LukDrdquo Journal of ClinicalMicrobiology vol 37 no 12 pp 4012ndash4019 1999
[32] G Lina Y Piemont F Godail-Gamot et al ldquoInvolve-ment of Panton-Valentine leukocidinmdashproducing Staphylococ-cus aureus in primary skin infections and pneumoniardquo ClinicalInfectious Diseases vol 29 no 5 pp 1128ndash1132 1999
[33] S A Havaei S O Moghadam M R Pourmand and JFaghri ldquoPrevalence of genes encoding bi-component leuko-cidins among clinical isolates of methicillin-resistant Staphylo-coccus aureusrdquo Iranian Journal of Public Health vol 39 no 1pp 8ndash14 2010
[34] L G Miller F Perdreau-Remington G Rieg et al ldquoNecrotizingfasciitis caused by community-associated methicillin-resistantStaphylococcus aureus in Los AngelesrdquoTheNewEngland Journalof Medicine vol 352 no 14 pp 1445ndash1453 2005
[35] A Holmes M Ganner S McGuane T L Pitt B D Cooksonand A M Kearns ldquoStaphylococcus aureus isolates carryingPanton-Valentine leucocidin genes in England and Wales fre-quency characterization and association with clinical diseaserdquoJournal of Clinical Microbiology vol 43 no 5 pp 2384ndash23902005
[36] I M Moussa and A M Shibl ldquoMolecular characterizationof methicillin-resistant Staphylococcus aureus recovered fromoutpatient clinics in Riyadh Saudi Arabiardquo Saudi MedicalJournal vol 30 no 5 pp 611ndash617 2009
[37] S Afroz N Kobayashi S Nagashima M M Alam A BM B Hossain and M A Rahman ldquoGenetic characterizationof Staphylococcus aureus isolates carrying Panton ValentineLeukocidin genes in Bangladeshrdquo Japanese Journal of InfectiousDiseases vol 61 pp 393ndash396 2008
[38] H Kaur S Purwar A Saini et al ldquoStatus of methicillinresistant Staphylococcus aureus infections and evaluation ofPVL producing strains in Belgaum South Indiardquo Journal ofKrishna Institute of Medical Sciences University vol 1 no 2 pp43ndash51 2012
[39] D Johnsson P Molling K Stralin and B Soderquist ldquoDetec-tion of Panton-Valentine leukocidin gene in Straphylococ-cus aureus by LightCycler PCR clinical and epidemiologicalaspectsrdquo Clinical Microbiology and Infection vol 10 no 10 pp884ndash889 2004
[40] A Rostamzad and N Rostamneia ldquoPrevalence of the panton-valentine leukocidin gene in clinical isolates of Staphylococcusaureus isolated from hospitals the ilam province of IranrdquoAvicenna Journal of Clinical Microbiology and Infection vol 3no 1 2016
[41] K O Bhutia and T S Singh ldquoThe prevalence and the riskfactors which are associated with Staphylococcus aureus andmethicillin-resistant S aureus which harboured the Panton-Valentine- Leukocidin gene in Sikkimrdquo Journal of Clinical andDiagnostic Research vol 6 no 3 pp 393ndash399 2012
[42] M Li G Y C Cheung J Hu et al ldquoComparative analysis ofvirulence and toxin expression of global community-associatedmethicillin-resistant Staphylococcus aureus strainsrdquo The Jour-nal of Infectious Diseases vol 202 no 12 pp 1866ndash1876 2010
[43] CWitzWWitte CWolz andCGoerke ldquoTranscription of thephage-encoded Panton-Valentine leukocidin of Staphylococcusaureus is dependent on the phage life-cycle and on the hostbackgroundrdquoMicrobiology vol 155 no 11 pp 3491ndash3499 2009
[44] C E Turner and S Sriskandan ldquoPantone-Valentine leucocidinexpression by Staphylococcus aureus exposed to commonantibioticsrdquo Infection vol 71 no 3 pp 338ndash346 2015
[45] J K Rudkin M Laabei A M Edwards et al ldquoOxacillinalters the toxin expression profile of community-associatedmethicillin-resistant Staphylococcus aureusrdquo AntimicrobialAgents and Chemotherapy vol 58 no 2 pp 1100ndash1107 2014
Computational and Mathematical Methods in Medicine
Hindawiwwwhindawicom Volume 2018
Behavioural Neurology
OphthalmologyJournal of
Hindawiwwwhindawicom Volume 2018
Diabetes ResearchJournal of
Hindawiwwwhindawicom Volume 2018
Hindawiwwwhindawicom Volume 2018
Research and TreatmentAIDS
Hindawiwwwhindawicom Volume 2018
Gastroenterology Research and Practice
Hindawiwwwhindawicom Volume 2018
Parkinsonrsquos Disease
Evidence-Based Complementary andAlternative Medicine
Volume 2018Hindawiwwwhindawicom
Submit your manuscripts atwwwhindawicom
Journal of Pathogens 7
References
[1] B Shrestha W Singh V S Raj B M Pokhrel and T MMohapatra ldquoHigh Prevalence of Panton-Valentine leukocidin(PVL) genes in nosocomial-acquired staphylococcus aureusisolated from tertiary care hospitals in Nepalrdquo BioMed ResearchInternational vol 2014 Article ID 790350 7 pages 2014
[2] J Kaneko and Y Kamio ldquoBacterial two-component and hetero-heptameric pore-forming cytolytic toxins structures pore-forming mechanism and organization of the genesrdquo BioscienceBiotechnology and Biochemistry vol 68 no 5 pp 981ndash10032004
[3] G Prevost B Cribier P Couppie et al ldquoPanton-valentineleucocidin and gamma-hemolysin from Staphylococcus aureusATCC 49775 are encoded by distinct genetic loci and havedifferent biological activitiesrdquo Infection and Immunity vol 63no 10 pp 4121ndash4129 1995
[4] D C Melles W B Van Leeuwen H A M Boelens J KPeetersH AVerbrugh andAVanBelkum ldquoPanton-Valentineleukocidin genes in Staphylococcus aureusrdquoEmerging InfectiousDiseases vol 12 no 7 pp 1174-1175 2006
[5] B McGrath F Rutledge and E Broadfield ldquo NecrotisingPneumonia rdquo Journal of the Intensive Care Society vol 9 no2 pp 170ndash172 2008
[6] F Yu Z Chen C Liu et al ldquoPrevalence of Staphylococcusaureus carrying Panton-Valentine leukocidin genes among iso-lates from hospitalised patients in Chinardquo Clinical Microbiologyand Infection vol 14 no 4 pp 381ndash384 2008
[7] S Monecke P Slickers M J Ellington A M Kearns andR Ehricht ldquoHigh diversity of Panton-Valentine leukocidin-positive methicillin-susceptible isolates of Staphylococcusaureus and implications for the evolution of community-associatedmethicillin-resistant S aureusrdquoClinicalMicrobiologyand Infection vol 13 no 12 pp 1157ndash1164 2007
[8] D J Skiest K Brown T W Cooper H Hoffman-RobertsH R Mussa and A C Elliott ldquoProspective comparison ofmethicillin-susceptible and methicillin-resistant community-associated Staphylococcus aureus infections in hospitalizedpatientsrdquo Infection vol 54 no 5 pp 427ndash434 2007
[9] S R Monday and G A Bohach ldquoUse of multiplex PCR todetect classical and newly described pyrogenic toxin genes inStaphylococcal isolatesrdquo Journal of Clinical Microbiology vol 37no 10 pp 3411ndash3414 1999
[10] O G Brakstad K Aasbakk and J A Maeland ldquoDetection ofStaphylococcus aureus by polymerase chain reaction amplifica-tion of the nuc generdquo Journal of Clinical Microbiology vol 30no 7 pp 1654ndash1660 1992
[11] K Zhang J-A McClure S Elsayed T Louie and J MConly ldquoNovel multiplex PCR assay for characterization andconcomitant subtyping of staphylococcal cassette chromosomemec types I to V in methicillin-resistant Staphylococcus aureusrdquoJournal of Clinical Microbiology vol 43 no 10 pp 5026ndash50332005
[12] J-A McClure J M Conly V Lau et al ldquoNovel multiplexPCR assay for detection of the staphylococcal virulence markerPanton-Valentine leukocidin genes and simultaneous discrimi-nation of methicillin-susceptible from -resistant staphylococcirdquoJournal of Clinical Microbiology vol 44 no 3 pp 1141ndash11442006
[13] T Nuryastuti H C van der Mei H J Busscher R Kuijer A TAman and B P Krom ldquorecA mediated spontaneous deletionsof the icaADBC operon of clinical Staphylococcus epidermidis
isolates a new mechanism of phenotypic variationsrdquo Antonievan Leeuwenhoek-Journal ofMicrobiology vol 94 no 2 pp 317ndash328 2008
[14] A Karmakar P Dua and C Ghosh ldquoBiochemical and molec-ular analysis of Staphylococcus aureus clinical isolates fromhospitalized patientsrdquo Canadian Journal of Infectious Diseasesamp Medical Microbiology vol 2016 Article ID 9041636 7 pages2016
[15] G A Lancette and S R Tatini ldquoStaphylococcus aureusrdquo inCompendium of Methods for the Microbiological Examination ofFoods C Vanderzant and D F Splittstoesser Eds pp 533ndash550American Public Health Association Washington DC USA3rd edition 1992
[16] E B Blair J S Emerson and A H Tull ldquoA new mediumsalt mannitol plasma agar for the isolation of Staphylococcusaureusrdquo American Journal of Clinical Pathology vol 47 no 1pp 30ndash39 1967
[17] G H Chapman ldquoThe significance of sodium chloride in studiesof staphylococcirdquo Journal of Bacteriology vol 50 pp 201ndash2031945
[18] R Cruickshank J P Duguid B PMarmion andRHA SwainMed Microbiol vol II Chruchill Livingstone Edinburgh UK12nd edition 1975
[19] A Swan ldquoThe use of a bile-aesculin medium and of Maxtedrsquostechnique of Lancefield grouping in the identification of ente-rococci (Group D Streptococci)rdquo Journal of Clinical Pathologyvol 7 no 2 pp 160ndash163 1954
[20] C R Arciola L Baldassarri and L Montanaro ldquoPresenceof icaA and icaD genes and slime production in a collectionof Staphylococcal strains from catheter-associated infectionsrdquoJournal of Clinical Microbiology vol 39 no 6 pp 2151ndash21562001
[21] P IWatnickCM LaurianoK EKlose LCroal andRKolterldquoThe absence of a flagellum leads to altered colony morphologybiofilm development and virulence in Vibrio cholerae O139rdquoMolecularMicrobiology vol 39 no 2 pp 223ndash235 2001
[22] J-O Cha J I Yoo J S Yoo et al ldquoinvestigation of biofilmformation and its association with the molecular and clinicalcharacteristics of methicillin-resistant staphylococcus aureusrdquoOsong Public Health and Research Perspectives vol 4 no 5 pp225ndash232 2013
[23] AW BauerWMKirby J C Sherris andMTurck ldquoAntibioticsusceptibility testing by a standardized single disk methodrdquoAmerican Journal of Clinical Pathology vol 45 no 4 ts pp 493ndash496 1966
[24] CLSI ldquoPerformance standards for antimicrobial susceptibilitytesting twenty-fifth informational supplementrdquo CLSI Docu-ment M100-S25 Clinical and Laboratory Standards InstituteWayne Pa USA 2015
[25] F Yu Y Liu Y Xu et al ldquoExpression of panton-valentine leuko-cidin mrna among staphylococcus aureus isolates associateswith specific clinical presentationsrdquo PLoS ONE vol 8 no 12p e83368 2013
[26] S V Kazakova J C Hageman M Matava et al ldquoA cloneof methicillin-resistant Staphylococcus aureus among profes-sional football playersrdquo The New England Journal of Medicinevol 352 no 5 pp 468ndash475 2005
[27] D R Bhatta LMCavacoG Nath et al ldquoAssociation of PantonValentine Leukocidin (PVL) genes with methicillin resistantStaphylococcus aureus (MRSA) in Western Nepal A matter ofconcern for community infections (a hospital based prospectivestudy)rdquo BMC Infectious Diseases vol 16 no 1 2016
8 Journal of Pathogens
[28] A Jain and A Agarwal ldquoBiofilm production a marker ofpathogenic potential of colonizing and commensal staphylo-coccirdquo Journal of Microbiological Methods vol 76 no 1 pp 88ndash92 2009
[29] M Zalipour H S Ebrahim-Saraie J Sarvari and R KhasheildquoDetection of biofilm production capability and icaAD genesamong staphylococci isolates from Shiraz Iranrdquo JundishapurJournal of Microbiology vol 9 no 12 pp 1ndash7 2016
[30] T Mathur S Singhal S Khan D J Upadhyay T Fatmaand A Rattan ldquoDetection of biofilm formation among theclinical isolates of staphylococci an evaluation of three differentscreeningmethodsrdquo Indian Journal ofMedicalMicrobiology vol24 no 1 pp 25ndash29 2006
[31] A Gravet M Rondeau C Harf-Monteil et al ldquoPredomi-nant Staphylococcus aureus isolated from antibiotic-associateddiarrhea is clinically relevant and produces enterotoxin Aand the bicomponent toxin LukE-LukDrdquo Journal of ClinicalMicrobiology vol 37 no 12 pp 4012ndash4019 1999
[32] G Lina Y Piemont F Godail-Gamot et al ldquoInvolve-ment of Panton-Valentine leukocidinmdashproducing Staphylococ-cus aureus in primary skin infections and pneumoniardquo ClinicalInfectious Diseases vol 29 no 5 pp 1128ndash1132 1999
[33] S A Havaei S O Moghadam M R Pourmand and JFaghri ldquoPrevalence of genes encoding bi-component leuko-cidins among clinical isolates of methicillin-resistant Staphylo-coccus aureusrdquo Iranian Journal of Public Health vol 39 no 1pp 8ndash14 2010
[34] L G Miller F Perdreau-Remington G Rieg et al ldquoNecrotizingfasciitis caused by community-associated methicillin-resistantStaphylococcus aureus in Los AngelesrdquoTheNewEngland Journalof Medicine vol 352 no 14 pp 1445ndash1453 2005
[35] A Holmes M Ganner S McGuane T L Pitt B D Cooksonand A M Kearns ldquoStaphylococcus aureus isolates carryingPanton-Valentine leucocidin genes in England and Wales fre-quency characterization and association with clinical diseaserdquoJournal of Clinical Microbiology vol 43 no 5 pp 2384ndash23902005
[36] I M Moussa and A M Shibl ldquoMolecular characterizationof methicillin-resistant Staphylococcus aureus recovered fromoutpatient clinics in Riyadh Saudi Arabiardquo Saudi MedicalJournal vol 30 no 5 pp 611ndash617 2009
[37] S Afroz N Kobayashi S Nagashima M M Alam A BM B Hossain and M A Rahman ldquoGenetic characterizationof Staphylococcus aureus isolates carrying Panton ValentineLeukocidin genes in Bangladeshrdquo Japanese Journal of InfectiousDiseases vol 61 pp 393ndash396 2008
[38] H Kaur S Purwar A Saini et al ldquoStatus of methicillinresistant Staphylococcus aureus infections and evaluation ofPVL producing strains in Belgaum South Indiardquo Journal ofKrishna Institute of Medical Sciences University vol 1 no 2 pp43ndash51 2012
[39] D Johnsson P Molling K Stralin and B Soderquist ldquoDetec-tion of Panton-Valentine leukocidin gene in Straphylococ-cus aureus by LightCycler PCR clinical and epidemiologicalaspectsrdquo Clinical Microbiology and Infection vol 10 no 10 pp884ndash889 2004
[40] A Rostamzad and N Rostamneia ldquoPrevalence of the panton-valentine leukocidin gene in clinical isolates of Staphylococcusaureus isolated from hospitals the ilam province of IranrdquoAvicenna Journal of Clinical Microbiology and Infection vol 3no 1 2016
[41] K O Bhutia and T S Singh ldquoThe prevalence and the riskfactors which are associated with Staphylococcus aureus andmethicillin-resistant S aureus which harboured the Panton-Valentine- Leukocidin gene in Sikkimrdquo Journal of Clinical andDiagnostic Research vol 6 no 3 pp 393ndash399 2012
[42] M Li G Y C Cheung J Hu et al ldquoComparative analysis ofvirulence and toxin expression of global community-associatedmethicillin-resistant Staphylococcus aureus strainsrdquo The Jour-nal of Infectious Diseases vol 202 no 12 pp 1866ndash1876 2010
[43] CWitzWWitte CWolz andCGoerke ldquoTranscription of thephage-encoded Panton-Valentine leukocidin of Staphylococcusaureus is dependent on the phage life-cycle and on the hostbackgroundrdquoMicrobiology vol 155 no 11 pp 3491ndash3499 2009
[44] C E Turner and S Sriskandan ldquoPantone-Valentine leucocidinexpression by Staphylococcus aureus exposed to commonantibioticsrdquo Infection vol 71 no 3 pp 338ndash346 2015
[45] J K Rudkin M Laabei A M Edwards et al ldquoOxacillinalters the toxin expression profile of community-associatedmethicillin-resistant Staphylococcus aureusrdquo AntimicrobialAgents and Chemotherapy vol 58 no 2 pp 1100ndash1107 2014
Computational and Mathematical Methods in Medicine
Hindawiwwwhindawicom Volume 2018
Behavioural Neurology
OphthalmologyJournal of
Hindawiwwwhindawicom Volume 2018
Diabetes ResearchJournal of
Hindawiwwwhindawicom Volume 2018
Hindawiwwwhindawicom Volume 2018
Research and TreatmentAIDS
Hindawiwwwhindawicom Volume 2018
Gastroenterology Research and Practice
Hindawiwwwhindawicom Volume 2018
Parkinsonrsquos Disease
Evidence-Based Complementary andAlternative Medicine
Volume 2018Hindawiwwwhindawicom
Submit your manuscripts atwwwhindawicom
8 Journal of Pathogens
[28] A Jain and A Agarwal ldquoBiofilm production a marker ofpathogenic potential of colonizing and commensal staphylo-coccirdquo Journal of Microbiological Methods vol 76 no 1 pp 88ndash92 2009
[29] M Zalipour H S Ebrahim-Saraie J Sarvari and R KhasheildquoDetection of biofilm production capability and icaAD genesamong staphylococci isolates from Shiraz Iranrdquo JundishapurJournal of Microbiology vol 9 no 12 pp 1ndash7 2016
[30] T Mathur S Singhal S Khan D J Upadhyay T Fatmaand A Rattan ldquoDetection of biofilm formation among theclinical isolates of staphylococci an evaluation of three differentscreeningmethodsrdquo Indian Journal ofMedicalMicrobiology vol24 no 1 pp 25ndash29 2006
[31] A Gravet M Rondeau C Harf-Monteil et al ldquoPredomi-nant Staphylococcus aureus isolated from antibiotic-associateddiarrhea is clinically relevant and produces enterotoxin Aand the bicomponent toxin LukE-LukDrdquo Journal of ClinicalMicrobiology vol 37 no 12 pp 4012ndash4019 1999
[32] G Lina Y Piemont F Godail-Gamot et al ldquoInvolve-ment of Panton-Valentine leukocidinmdashproducing Staphylococ-cus aureus in primary skin infections and pneumoniardquo ClinicalInfectious Diseases vol 29 no 5 pp 1128ndash1132 1999
[33] S A Havaei S O Moghadam M R Pourmand and JFaghri ldquoPrevalence of genes encoding bi-component leuko-cidins among clinical isolates of methicillin-resistant Staphylo-coccus aureusrdquo Iranian Journal of Public Health vol 39 no 1pp 8ndash14 2010
[34] L G Miller F Perdreau-Remington G Rieg et al ldquoNecrotizingfasciitis caused by community-associated methicillin-resistantStaphylococcus aureus in Los AngelesrdquoTheNewEngland Journalof Medicine vol 352 no 14 pp 1445ndash1453 2005
[35] A Holmes M Ganner S McGuane T L Pitt B D Cooksonand A M Kearns ldquoStaphylococcus aureus isolates carryingPanton-Valentine leucocidin genes in England and Wales fre-quency characterization and association with clinical diseaserdquoJournal of Clinical Microbiology vol 43 no 5 pp 2384ndash23902005
[36] I M Moussa and A M Shibl ldquoMolecular characterizationof methicillin-resistant Staphylococcus aureus recovered fromoutpatient clinics in Riyadh Saudi Arabiardquo Saudi MedicalJournal vol 30 no 5 pp 611ndash617 2009
[37] S Afroz N Kobayashi S Nagashima M M Alam A BM B Hossain and M A Rahman ldquoGenetic characterizationof Staphylococcus aureus isolates carrying Panton ValentineLeukocidin genes in Bangladeshrdquo Japanese Journal of InfectiousDiseases vol 61 pp 393ndash396 2008
[38] H Kaur S Purwar A Saini et al ldquoStatus of methicillinresistant Staphylococcus aureus infections and evaluation ofPVL producing strains in Belgaum South Indiardquo Journal ofKrishna Institute of Medical Sciences University vol 1 no 2 pp43ndash51 2012
[39] D Johnsson P Molling K Stralin and B Soderquist ldquoDetec-tion of Panton-Valentine leukocidin gene in Straphylococ-cus aureus by LightCycler PCR clinical and epidemiologicalaspectsrdquo Clinical Microbiology and Infection vol 10 no 10 pp884ndash889 2004
[40] A Rostamzad and N Rostamneia ldquoPrevalence of the panton-valentine leukocidin gene in clinical isolates of Staphylococcusaureus isolated from hospitals the ilam province of IranrdquoAvicenna Journal of Clinical Microbiology and Infection vol 3no 1 2016
[41] K O Bhutia and T S Singh ldquoThe prevalence and the riskfactors which are associated with Staphylococcus aureus andmethicillin-resistant S aureus which harboured the Panton-Valentine- Leukocidin gene in Sikkimrdquo Journal of Clinical andDiagnostic Research vol 6 no 3 pp 393ndash399 2012
[42] M Li G Y C Cheung J Hu et al ldquoComparative analysis ofvirulence and toxin expression of global community-associatedmethicillin-resistant Staphylococcus aureus strainsrdquo The Jour-nal of Infectious Diseases vol 202 no 12 pp 1866ndash1876 2010
[43] CWitzWWitte CWolz andCGoerke ldquoTranscription of thephage-encoded Panton-Valentine leukocidin of Staphylococcusaureus is dependent on the phage life-cycle and on the hostbackgroundrdquoMicrobiology vol 155 no 11 pp 3491ndash3499 2009
[44] C E Turner and S Sriskandan ldquoPantone-Valentine leucocidinexpression by Staphylococcus aureus exposed to commonantibioticsrdquo Infection vol 71 no 3 pp 338ndash346 2015
[45] J K Rudkin M Laabei A M Edwards et al ldquoOxacillinalters the toxin expression profile of community-associatedmethicillin-resistant Staphylococcus aureusrdquo AntimicrobialAgents and Chemotherapy vol 58 no 2 pp 1100ndash1107 2014
