Salmonella spp., and Yersinia Strains from · 2019. 7. 31. · 2. Materials and Methods.. Bacterial...

Research ArticleDetection and Characterization of Shiga Toxin ProducingEscherichia coli Salmonella spp and Yersinia Strains fromHuman Animal and Food Samples in San Luis Argentina

Gabriela Isabel Favier Cecilia Lucero EstradaTeresa Ineacutes Cortintildeas and Mariacutea Esther Escudero

General Microbiology Faculty of Chemistry Biochemistry and Pharmacy National University of San LuisEjercito de los Andes 950 Bloque 1 Piso 1 5700 San Luis Argentina

Correspondence should be addressed to Marıa Esther Escudero escudemegmailcom

Received 7 February 2014 Revised 24 May 2014 Accepted 14 July 2014 Published 7 August 2014

Academic Editor Marcel H Zwietering

Copyright copy 2014 Gabriela Isabel Favier et al This is an open access article distributed under the Creative Commons AttributionLicense which permits unrestricted use distribution and reproduction in any medium provided the original work is properlycited

Shiga toxin producing Escherichia coli (STEC) Salmonella spp andYersinia species was investigated in humans animals and foodsin San Luis Argentina A total of 453 samples were analyzed by culture and PCR The antimicrobial susceptibility of all the strainswas studied the genomic relationships among isolates of the same species were determined by PFGE and the potencial virulence ofY enterocolitica strains was analyzed Yersinia species showed higher prevalence (9453 20 95 CI 07ndash33) than STEC (445309 95 CI 0ndash18) and Salmonella spp (3453 07 95 CI 0ndash15) Y enterocolitica and Y intermedia were isolated fromchicken carcasses (680 75 95 CI 15ndash135) and porcine skin and bones (310 30 95 CI 0ndash65) One STEC strain wasrecovered from human feces (170 14 95 CI 0ndash42) and STEC stx1stx2 genes were detected in bovine stools (3129 2395 CI 0ndash50) S Typhimurium was isolated from human feces (170 14 95 CI 0ndash42) while one S Newport and two SGaminara strains were recovered from one wild boar (13 33 95 CI 0ndash99) The knowledge of prevalence and characteristicsof these enteropathogens in our region would allow public health services to take adequate preventive measures

1 Introduction

The detection and characterization of Shiga toxin producingEscherichia coli (STEC) Salmonella spp and Yersinia ente-rocolitica strains in human patients animal reservoirs andfoods of animal origin intended for human consumption arerelevant to public health These organisms are transmittedthrough contaminated drinking water and food and cancause intestinal and extraintestinal clinical manifestations inhumans [1 2] STEC is associated with hemorrhagic colitisand hemolytic uremic syndrome (HUS) its pathogenicityis attributed to virulence factors that facilitate effectivecolonization of the human gastrointestinal tract and subse-quent release of Shiga toxins [3] Argentina has the highestincidence of HUS in the world with E coli O157H7 as theprimary etiological agent [4] Previous studies in our country

have been focused on the prevalence of STEC in cattle andbyproducts intended for human consumption [4]

Salmonella spp can enter the food chain thereby trigger-ing either sporadic cases or outbreaks of human salmonellosis[5] Foodborne outbreaks triggered by Salmonella serotypesare of rare occurrence in our province however S PanamaS Newport S Sandiego S Enteritidis S Montevideo SAnatum and S Typhimurium strains have been isolated fromsources of diverse origin [6]

Pigs are the most important Y enterocolitica carriersPathogenic strains usually carry a 72 kb virulence plasmid(pYV) bearing yadA yops and virF genes and chromosomalvirulence markers such as ail yst myfA and inv genes [7]The assessment of virulence markers contributes to properlyestablish the pathogenic potential of the Y enterocoliticaisolates Since Y enterocolitica is not routinely investigated in

Hindawi Publishing CorporationInternational Journal of MicrobiologyVolume 2014 Article ID 284649 11 pageshttpdxdoiorg1011552014284649

2 International Journal of Microbiology

the clinical laboratories of Argentina a systematic epidemi-ological study of Y enterocolitica in environment reservoirsfoods and humans is still pending

For contributing to the knowledge of prevalence anddistribution of these enteropathogens in patients presumableanimal reservoirs and foods of our region this study wasaimed (i) to detect STEC Salmonella spp and Y enterocol-itica in human and animal feces and foods of animal originintended for human consumption (ii) to assess the patho-genic potential of Y enterocolitica strains through phenotypicand molecular virulence markers (iii) to test the antimi-crobial susceptibility of all strains and (iv) to determinepossible genetic relationships among isolates of each speciesby subtyping using pulsed field gel electrophoresis (PFGE) Inaddition counts of total coliformswere performed in samplesof foods

2 Materials and Methods

21 Bacterial Strains Y enterocolitica O9 W1024 pYV+(Belgium)Y enterocoliticaMCH700 B4O3 pYV+ (Canada)Y enterocolitica 29C-43 B4 O3 pYV+ (Norway) and Yenterocolitica B2 O9 pYV+ (Argentina) were used as pos-itive controls in phenotypic virulence assays assessmentof genetic virulence markers by PCR and comparison ofDNA profiles by PFGE Escherichia coli O157H7 EDL933Sor-120573glu-E-Hly+eae+ biotype C producer of Stx1 andStx2 was employed as positive control in PCR targetingSTEC stx1stx2 genes and Salmonella Braenderup H9812 wasused as molecular size marker in PFGE Y enterocoliticaB1A O5 CLO229 and Y enterocolitica B1A O630 CLO225local strains isolated from sausages were utilized as positivecontrols in ystB gene PCR [8] These strains were maintainedat 4∘C on trypticase soy agar slants (TSA Merck DarmstadtGermany) For determining culture purity isolations onMac Conkey agar (MC Merck) Sorbitol Mac Conkey agar(SMAC Merck) and Salmonella Shigella agar (SS Merck)for Y enterocolitica E coli O157H7 and S Braenderuprespectively were made prior to each experiment Plates wereincubated 48 h at 25∘C forY enterocolitica and 24 h at 37∘C forE coli O157H7 and S Braenderup

22 Samples A total of 453 samples from human and animalsources in San Luis city and upcoming rural areas Argentinawere analyzed during the period June 2008ndashNovember 2011They included 70 samples of human stools from patients withenterocolitis symptoms attending a local clinical laboratory167 stool samples obtained from feedlot bovines (119899 = 61)grazing bovines (119899 = 68) porcine (119899 = 20) ovine (119899 =10) goats (119899 = 6) and equines (119899 = 2) and 216 samplesof animal origin intended for human consumption such aschicken carcasses (119899 = 80) porcine skin and bones (119899 = 10)goat cheeses (119899 = 30) fresh sausages ldquochorizosrdquo (119899 = 90) andsix samples of three wild boars tongues (119899 = 3) and tonsils(119899 = 3) Samples of animal stools were randomly collected inregional cattle markets and farms immediately after defeca-tion Samples of wild boars were hunt products from ourregion Samples of foods of animal origin intended for humanconsumptionwere purchased at five retailmarkets in San Luis

city All samples were packed in individual sterile plastic bagsand stored at 4∘C for up to 6 h before processing

23 Investigation of Total Coliforms Thepresence of total col-iforms was investigated in samples of animal origin intendedfor human consumption except tonsils and tongues of wildboars Decimal dilutions of each sample were prepared in01 peptone water pH 72 (PW Merck) one milliliter ofeach dilution was seeded in violet-lactose-neutral red-bileagar (VLRB Merck) and incubated at 37∘C for 24 h Countsof characteristic colonies were performed and results wereexpressed as log

10CFUg

24 Investigation of Shiga Toxin-Producing Escherichia coli(STEC) Salmonella spp and Y enterocolitica

241 STEC Samples of 25 g were homogenized in stomacher(IULMasticator Germany) for 90 s and incubated in 225mLof EC broth (Merck) for 24 h at 37∘C For avoiding falsenegative PCR results produced by interference of organiccompounds and background microflora present in samplesstx1stx2 PCR was not immediately performed Instead ECenriched samples were streaked on SMAC incubating 24 h at37∘C [9] Each plate showed confluent growth in the initialstreaking zone where bacteria were clustered and individualcolonies in the final streaking zone where bacteria wereisolatedThen DNAwas extracted from confluent growth forscreening stx1stx2 genes by PCR [9] if amplified five sorbitolnonfermenting and five fermenting colonies were randomlyselected from SMAC plates and their DNA were individuallytested by PCR targeted to stx1 and stx2 genes When posi-tive results were observed Gram staining and biotyping ofeach colony was performed Sorbitol nonfermenting E colicolonies were challenged against O157 antiserum (NationalInstitute of Infectious Diseases-INEI ANLIS-ldquoDr Carlos GMalbranrdquo Buenos Aires Argentina) by a slide agglutinationtest

242 Salmonella spp This microorganism was investigatedaccording to US FDA Bacteriological Analytical Manual(httpwwwfdagovFoodFoodScienceResearchLaborato-ryMethodsucm2006949htm accessed in May 2008)Twenty five grams of each homogenized sample wasenriched in 225mL of lactose broth (LB Merck) at 37∘Cfor 24 h then one-mL LB aliquots was transferred intotwo tubes containing 9mL of tetrathionate broth (Merck)and two tubes with 9mL of Rappaport-Vassiliadis broth(Merck) One tube of each selective broth was incubatedat 37∘C for 24 h and the other one was incubated at 42∘Cfor 24 h Isolations were done on SS agar (Merck) for 24 hat 37∘C and suspect lactose nonfermenting colonies wereexamined by Gram staining and biochemical assays Sero-typing was performed in the National Reference Center forEnterobacteria INEI-ANLIS Buenos Aires Argentina

243 Y enterocolitica This species was searched by enrich-ment of 25-g homogenized sample in 225mL of phos-phate buffered saline pH 76 added with 1 sorbitol and 015bile salts and incubated 21 days at 4∘C Isolations were made

International Journal of Microbiology 3

on MC agar for 48 h at 25∘C and presumptive Yersinia col-onies were examined by Gram staining and identified by bio-chemical assays (httpwwwfdagovFoodFoodScienceRe-searchLaboratoryMethodsucm072633htm accessed May2008) The final characterization in biotypes and serotypeswas performed by Dr E Carniel National Reference Centerof Yersinia Institute Pasteur Paris France

25 PCRTargeting STEC stx1 and stx2Genes DNAextractionwas performed by the boiling method [9] Each strain wassuspended in an Eppendorf tube containing 150120583L of TE1X buffer (10mM Tris (Sigma Aldrich St Louis MO USA)1mM EDTA (Sigma) pH 80) added with 1 Triton-X100(Parafarm Buenos Aires Argentina) Suspensions wereboiled 15min and centrifuged at 10000 rpm for 5min(Sigma centrifuge model 3K30 Germany) Each supernatantcontaining DNA was transferred to other tube and stored atminus20∘C before use A 25 120583L volume of a PCR mix containing1X PCR buffer 01mM dNTP 15mM MgCl

2 2 pmol120583L of

stx1 primers (forward 51015840-GAAGAGTCCGTGGGATTACG-31015840 and reverse 51015840-AGCGATGCAGCTATTAATAA-31015840) for anamplicon of 130 bp 04 pmol120583L of stx2 primers (forward51015840-TTAACCACACCCCACCGGGCAGT-31015840 and reverse 51015840-GCTCTGGATGCATCTCTGGT-31015840) for an amplicon of346 bp 002U120583L Taq DNA polymerase (Productos Biologi-cos Quilmes Argentina) 2 120583L DNA template and ultrapurewater was prepared Cycling conditions consisted of an initialdenaturation at 94∘C for 5min followed by 30 cycles of 94∘Cfor 30 s 58∘C for 30 s and 72∘C for 30 s and a final extensionof 72∘C for 3min [9] PCR was performed in a TechneTC-512 thermal cycler (Techne Inc Duxford UK) The PCRproducts were electrophoresed in a 2 agarose gel (ProductosBiologicos) added with GelRed Acid Gel Stain (BiotiumHayward CA USA) 15 120583L stock solution40120583L gel at 80Vfor 40min and visualized in an UV transilluminator (UVPUpland CA USA) For comparison a 100-bp DNA ladder(Productos Biologicos) was used

26 PFGE DNA preparation was according to Ribot et al[10] Each bacterial strain was isolated on Mueller Hintonagar (MH Britania Buenos Aires Argentina) and incubated24 h at 37∘C for Salmonella and E coli or 48 h at 25∘Cfor Yersinia strains Colonies of each strain were directlysuspended in 4mL of the suspension buffer (100mM Tris100mM EDTA pH 80) to OD

610 nm 10 Two hundredmicroliters of bacterial suspension were mixed with equalvolume of 1 SeaKem Gold agarose (Cambrex RocklandME USA) and poured in molds to obtain ldquoplugsrdquo Plugswere treated for 20 h in a lysis solution (50mmol Tris-EDTA (Sigma) 1 sodium lauroyl sarcosine (Sigma) andProteinase K 01mgmL (Fluka Chemie Buchs Switzerland)pH 80) at 37∘C and then washed four times with TE buffer(10mM Tris (Sigma) 1mM EDTA (Sigma) pH 80) for30min at 37∘C Chromosomal DNA contained in agaroseplugs was digested with 10 U XbaI (Fermentas BurlingtonOntario Canada) for two hours according to manufacturerrsquosinstructions Plugs were cut in approximately 1-mm-thickslices placed in the CHEF-DRIII chamber (Bio-Rad Her-cules CA USA) and PFGE was performed using an electric

field of 6Vcm at 14∘C angle of 120∘ and switching timesof 22 (initial time) to 638 s (final time) for Salmonella andE coli O157H7 and 18 s (initial time) to 20 s (final time)for Y enterocolitica over 20 h [6 8] Migration of the DNAfragments was achieved in a 10 pulsed-field agarose gel(Bio-Rad) submerged in 05X TBE buffer (45mMTris-borate(Sigma) and 1mMEDTA (Sigma))The gels were stainedwithGel Red Acid Gel Stain (Biotium Hayward CA USA) underthe conditions suggested by the manufacturer visualized inUV transilluminator and photographed The size standardstrain was S Braenderup H9812 kindly donated by Dr NBinsztein (INEI-ANLIS Buenos Aires Argentina)

27 Virulence Phenotypical Assays of Y enterocolitica IsolatesThese assays were performed according to US FDA Bacterio-logical Analytical Manual (httpwwwfdagovFoodFood-ScienceResearchLaboratoryMethodsucm072633htm ac-cessed September 2011)

271 Autoagglutination Each strain was inoculated into twotubes of Methyl Red-Voges Proskauer broth (Merck) Onetube was incubated at 37∘C and the other was incubated at25∘C After 18 to 24 h the tubes were observed for bacterialautoagglutinationTheMR-VP tube incubated at 25∘C for 24 hshould show turbidity from bacterial growth The 37∘C MR-VP tube should show agglutination (clumping) of bacteriaalong walls andor bottom of tube with clear supernatantfluid Isolates giving this result were presumptive positive forthe virulence plasmid Any other pattern for autoagglutina-tion at these two temperatures was considered negative

272 Calcium Dependence at 37∘C and Congo Red BindingCongo Red-Magnesium Oxalate sodium agar (CR-MOX)was composed by TSA added with 268 gL sodium oxalate(Mallinckrodt) 4067 gL MgCl

2sdotH2O (Sigma) and 5mLL

of 1 Congo Red dye (Baker) which allowed visualization ofcalcium-dependent growth and uptake of Congo Red dye onthe same plate The test organism was inoculated into TSBand incubated overnight at 25∘C Decimal dilutions in phys-iologic saline were made to obtain 1000 cellsmL Volumesof 01mL of the appropriate dilution were spread-plated onCR-MOXplates and incubated at 37∘C Presumptive plasmid-bearing Y enterocolitica showed scarce growth of pinpointround convex red and opaque colonies whereas plasmidlessY enterocolitica exhibited abundant growth of large irregularflat and translucent colonies

273 Esculin Hydrolysis This test was performed on esculinagar (10 g polypeptone (Britania) 1 g esculin (Sigma) 1 gferric ammonium citrate (Sigma) and 5 g agar (Britania) in1000mL of distilled water) Strains were inoculated incu-bated at 25∘C and observed up to 7 days after seeding Thedevelopment of dark pigment on the agar surface was indi-cative of lacking of virulence

274 Pyrazinamidase Production Strains were inoculatedover the entire slant of pyrazinamide agar (30 g TSA (Merck)1 g pyrazinecarboxamide (Sigma) and 02M Tris-maleatebuffer (Sigma) for 1000mL) incubated at 25∘C for 48 h and


flooded with 1mL of 1 freshly prepared ferrous ammoniumsulfate Development of pink color within 15min was positivetest indicating presence of pyrazinoic acid formed by thepyrazinamidase enzyme

28 PCR for Y enterocolitica Molecular Virulence Markers(yadA and ystB Genes)

281 Nested yadA PCR DNA extraction was performed asdescribed [9] A nested-PCR targeted to Y enterocoliticayadA gene [11] was applied using two sets of primers Fivemicroliters of the template was used for the first PCR and 2120583Lof the product obtained in this step was used as template forthe second PCR The reaction mixture (50mL) contained 1XPCR buffer 15mMMgCl

2 200mmolmL of each dNTP 1 U

of Taq DNA-polymerase and 01mmolmL of each primer(Productos Biologicos)Thefirst primer pair integrated by thefollowing oligonucleotides YadA1 51015840-TAA GAT CAG TGTCTC TGC GGC A-31015840 and YadA2 51015840-TAG TTA TTT GCGATC CCT AGC AC-31015840 was used under the following cyclingconditions initial denaturation at 95∘C for 3min then 40cycles of denaturation at 95∘C for 30 s annealing at 58∘Cfor 60 s and extension at 72∘C for 90 s followed by a finalextension at 72∘C for 10min for amplifying a 747 bp fragmentThe second primer pair consisting of YadA3 51015840-GCG TTGTTC TCA TCT CCA TAT GC-31015840 and YadA4 51015840-GGC TTTCAT GAC CAA TGG ATA CAC-31015840 was used under thefollowing cycling conditions initial denaturation at 95∘C for3min followed by 20 cycles of denaturation at 95∘C for 30 sannealing at 62∘C for 60 s and extension at 72∘C for 90 s anda final extension at 72∘C for 10min which amplified a 529 pbfragment Products were electrophoresed at 80V for 40minin a 1 agarose gel stained with GelRed visualized in an UVtransilluminator (UVP) and compared with a 100-bp ladderDNA (Productos Biologicos)

282 Simple ystB PCR DNA extraction was performed bythe boiling method [9] The reaction mixture (25 120583L) con-tained PCR buffer 1X 200120583M of each dNTP 15mMMgCl

2

008U120583L of Taq DNA-polymerase and 1 pmol120583L of eachprimer (Productos Biologicos) It used the primer pair con-stituted by YstB-F 51015840GTACATTAGGCCAAGAGACG 31015840 andYstB-R 51015840 GCAACATACCTCACAACACC 31015840 for amplifyinga 146 bp fragment [12]The PCR product was electrophoresedat 80V for 40min in a 2agarose gel stainedwithGelRed andvisualized in an UV transiluminator (UVP) The molecularmass of amplicons was determined as described above

29 Antimicrobial Susceptibility The antimicrobial suscep-tibility of the isolates was addressed by the disk diffusionmethod on Mueller Hinton agar The following antibio-tic disks (Britania) were used ampicillin 10 120583g cephalotin30 120583g aztreonam 30 120583g erythromycin 15 120583g colistin 10 120583gchloramphenicol 30 120583g gentamicin 10 120583g tetracycline30 120583g trimethoprim-sulfamethoxazole 25 120583g ciprofloxacin5 120583g neomycin 30 120583g furazolidone 5 120583g nalidixic acid30 120583g cefotaxime 30 120583g ceftriaxone 30 120583g and phospho-mycin 50 120583g Zones of growth inhibition were evaluated

Table 1 Counts of total coliforms in samples of animal originpurchased at retail markets and intended for human consumption

Type of samples Number of samples log10CFUgplusmn SDlowast

Chicken carcasses 80 50 plusmn 07A

Porcine skin and bones 10 39 plusmn 06B

Goat cheeses 30 07 plusmn 02C

Fresh sausages ldquochorizosrdquo 90 54 plusmn 07A

Total 210lowastSD standard deviationABCValues of log

10CFUg followed by different capital letters are statistically

different (119875 le 005) Counts were performed on violet red bile (VRB) agar

according to Clinical and Laboratory Standards Institute(CLSI) guidelines [13]

210 Statistical Analysis Statistical analysis of counts ofcoliforms related to type of sample was performed usingChi-square test (Analytical Software Tallahassee FL USA)Statistical calculations were based on confidence level equalor higher than 95 (119875 le 005 was considered statisticallysignificant) Confidence intervals for each percentage werecalculated by using the formula 95 CI = percentageplusmn (196 times SE) with SE standard error calculated as SE= percentage(N∘ of positive cases)12 The discriminationindex (DI) values of PFGE were calculated by Simpsonrsquosdiversity index Clustering of the patterns obtained by PFGEwas performed using Statistica 60 software (StatSoft IncTulsa OKUSA) and the unweighted pair groupmethodwitharithmetic average (UPGMA)

3 Results

31 Investigation of Total Coliforms Shiga Toxin-ProducingEscherichia coli (STEC) Salmonella spp and Y enterocoliticaCounts of total coliforms in fresh sausages and chicken car-casses yielded 54plusmn07 and 50plusmn07 log

10CFUg respectively

being significantly higher (119875 le 005) than those observedfor porcine skin and bones (39 plusmn 06 log

10CFUg) and

goat cheeses (07 plusmn 02 log10CFUg) (Table 1) The detection

frequencies of STEC Salmonella serotypes and Yersiniaspecies in 453 samples are shown in Table 2 The number ofYersinia positive samples (9453 20 95CI 07ndash33) washigher (119875 le 005) than those observed for STEC (4453 0995CI 0ndash18) and Salmonella spp (3453 07 95CI 0ndash15)

A sample was considered ldquoSTEC positiverdquo when E colicolonies carrying stx1stx2 genes could be recovered by cul-turing or ldquopresumptive STEC positiverdquo when only stx1stx2signals were detected by PCR on DNA extracted fromconfluent growth on SMACThus four sampleswere positiveone stool sample from a pediatric patient with diarrhea (17014 95 CI 0ndash42) yielded one E coli O157H7 strainby culture on SMAC which was characterized as stx2+ byPCR and three samples of bovine stools (3129 23 95CI 0ndash50) that amplified stx genes from DNA extracted


Table2Frequencyof

detectionof

STEC

SalmonellaserotypesandYersiniaspeciesinsamples

ofdiverseo

rigin

analyzed

inthisstu

dy

Source

Num

bero

fsamples

STEC

Salm

onellaspp

Yersiniaspecies

Positives

amples

(plusmn19

6SE)lowast

Positives

amples

(plusmn19

6SE)lowast

Serovar

(num

bero

fstrains)

Positives

amples

(plusmn19

6SE)lowast

Species

Bioserovar

(Nostr

ains)

Cultu

rePC

RHum

ansto

ols

701(14plusmn28)

mdash1(14plusmn28)

STy

phim

urium

(1)

mdashmdash

Animalsto

ols

167

feedlotb

ovines

61mdash

2(33plusmn46)

mdashmdash

grazingbo

vines

68mdash

1(15plusmn30)

mdashmdash

porcines

20mdash

mdashmdash

mdashovines

10mdash

mdashmdash

mdashgoats

6mdash

mdashmdash

mdashequines

2mdash

mdashmdash

mdashSamples

ofanim

alorigin

forh

uman

consum

ption

216

chickencarcasses

80mdash

mdashmdash

6(75plusmn60)

Yenterocolitica

Yinterm

edia

B1AO12

25-1226(2)

B1AO78-8-819

(2)

B6O17

(1)

B4O40(1)

porcines

kinandbo

nes

10mdash

mdashmdash

3(30plusmn35)

Yenterocolitica

B1AO78-8-819

(3)

goatcheeses

30mdash

mdashmdash

mdashfre

shsausages

90mdash

mdashmdash

mdashwild

boars

3

tonsils

3mdash

mdash1(33plusmn66)

SNew

port(1)

SGam

inara(

1)mdash

tong

ues

3mdash

mdash1(33plusmn66)

SGam

inara(

1)mdash

Total

453

4(09plusmn09)dagger

3(07plusmn08)dagger

9(20plusmn13

)daggerlowast(

)percentage

correspo

ndingto

positives

amplesto

talsam

ples

ofthes

ametypedagger(

)percentage

correspo

ndingto

totalp

ositive

samples

fore

achpathogento

talofsam

plesInbo

thcases19

6SEisthe119905

value

(120572005)m

ultip

liedby

thes

tand

arderror


Table 3 Phenotypic virulence assays corresponding to Y enterocolitica strains

Total of strains Bioserotype Origin Phenotypic assayslowast

Esc Pyr AA Ca2+ CR2 B1A O1225-1226 Chicken carcasses + + + minus minus

3 B1A O78-8-819 Porcine skin and bones + + + minus minus

2 B1A O78-8-819 Chicken carcasses + + + minus minus

1 W1024 B2 O9 Reference strain minus minus + + +1 MCH 700 B4 O3 Reference strain minus minus + + +1 29C-46 B4 O3 Reference strain minus minus + + +1 B2 O9 Eggshell (local strain) minus minus + + +lowastEsc esculin hydrolysis Pyr pyrazinamide hydrolysis AA autoagglutination at 37∘C Ca2+ calcium dependence and CR congo red binding

from confluent growth (two samples were stx1+stx2+and thethird one was stx2+) Individual STEC colonies could not beisolated from these samples

On the other hand three samples yielded four Salmonellaisolates corresponding to different serotypes one S Typhi-murium strain was obtained from stools of a symptomaticpatient (170 14 95 CI 0ndash42) and one S Newportand two S Gaminara strains were isolated from one tonsiland one tongue of wild boar among 216 samples of animalorigin intended for human consumption (2216 09 95CI 0ndash21) In the small number of wild boar samplesthe Salmonella recovery was high (13 33 95 CI 0ndash99) Interestingly the tonsil positive sample carried both SGaminara and S Newport strains Furthermore nineYersiniaisolates were recovered from nine (9216 42 95 CI 15ndash69) samples of animal origin intended for human consump-tion Two isolates were classified as Y enterocolitica B1AO1225-1226 five as Y enterocolitica B1A O78-8-819 oneas Y intermedia B6 O17 and other one as Y intermedia B4O40 (Table 2) They were isolated from six chicken carcasses(680 75 95 CI 15ndash135) and three porcine skin andbones (310 30 95 CI 0ndash65)

32 PFGE XbaI-restricted DNA polymorphisms of Salmo-nella isolates are observed in Figure 1 Two major clustersA and B with a 65 similarity were obtained Even thoughsix S Newport isolates from tonsil and five S Gaminaraisolates from tonsil and tongue were initially recoveredfrom one wild boar the analysis of their DNA restrictionprofiles by PFGE showed that all S Newport strains groupedin cluster A while all S Gaminara ones grouped in thegenotype GTB1 within cluster B Since identical DNA bandpatterns between isolates of the same serovar were observedonly three Salmonella strains were reported (Table 2) Indendrogram S Typhimurium of human source was includedin GTB2 within cluster B showing 68 similarity with GTB1

Although fifteen Yersinia isolates were originally recov-ered from nine positive samples the analysis of DNA restric-tion profiles observed byPFGE allowed to conclude that someisolates were replicates of the same strain Therefore bac-terial isolates were grouped into two major clusters A andB (63 similarity) according to Yersinia species (Figure 2)Thus cluster A comprised GTA1 with five Y enterocoliticaB1A O1225-1226 strains isolated from chicken carcasses

GTA2 with eight Y enterocolitica B1A O78-8-819 isolatesfrom chicken carcasses and porcine skin and GTA3 with thereference Y enterocolitica W1024 strain Cluster B includedGTB1 consisting of Y intermedia B6 O17 strain and GTB2corresponding to Y intermedia B4 O40 strain (86 similar-ity) both strains recovered from chicken carcasses PFGEwasnot applied on the human STEC strain isolated in this study

33 Phenotypic and Molecular Virulence Assays of Y entero-colitica Isolates Five Y enterocolitica B1AO78-8-819 strainsand two Y enterocolitica B1A O1225-1226 strains isolated inthis study produced negative results for calcium dependentgrowth and Congo red binding at 37∘C showed pyrazinami-dase activity hydrolyzed esculin and autoagglutinated at 37∘C(Table 3) Nested PCR targeting yadA gene yielded negativeresults in all cases When ystB gene was assayed one Yenterocolitica B1A O78-8-810 strain isolated from porcineskin and bones was positive

34 Antimicrobial Susceptibility Thehuman STEC strain wassusceptible to all antimicrobials assayed except ampicillin andrifampicin Similarly Salmonella strains were susceptible toall drugs except cephalotin Meanwhile all Y enterocoliticaisolates shared resistance to ampicillin one Y enterocoliticaB1A O1225-1226 strain was susceptible to cephalotin andtwo Y enterocolitica B1A O78-8-810 strains showed resis-tance to both cephalotin and erythromycin

4 Discussion

Total coliforms are considered indicators of hygienic qualityand their presence in foodsmay correlate with the presence ofpathogenic bacteria The low total coliform counts observedin porcine skin and bones and goat cheeses might be attri-buted to the effects of thermal treatments applied to pig car-casses during slaughtering and pasteurization and preserva-tion of dairy products respectively Nomicrobiological speci-fications for porcine skin and bones are included in theArgentinean Alimentary Code (AAC httpwwwanmatgovaralimentosnormativas alimentos caaasp accessed No-vember 2013) On the other hand values up to 500 totalcoliforms per gram at 45∘C are allowed for cheeses with 36 to46moisture (AAC)Thus low coliform counts for this food


would be consistent with good practices of manufacture Incontrast low microbiological quality of ingredients or poorhygiene could explain coliform counts higher than 103MPNg which is the maximal limit established by AAC for freshsausages Although no microbiological standards for chickencarcasses are addressed by AAC counts of coliforms in thiswork were higher than 27 log

10CFUg observed by Capita et

al [14] in Spain Clearly contamination is possible at any stageof the production process from defeathering eviscerationand washing to storage by cooling or freezing

Regarding the search of enteropathogens the human Ecoli O157H7 strain was isolated by culture and characterizedas stx2+ by PCR On the contrary no STEC strain could beisolated from positive stx1stx2 cattle stools probably becausethey were viable but noncultivable strains ConcurrentlyJure et al [15] identified the stx2 gene in seven samples ofmeat in Argentina however only one E coli O157H7 straincould be isolated The low detection of STEC from cattle inSan Luis contrasts with reports of 4 to 39 STEC isolatesrecovered from calves by Meichtri et al [16] in our countrywho enriched stools and rectal swabs in TSB added withantibiotics and then performed screening of stx genes byconventional PCR inDNA extracted from confluent bacterialgrowth on SMAC If amplified PCRwas repeated on individ-ual colonies Similarly Sanz et al [17] recovered 44 STECfrom bovines for slaughtering in other Argentina regions Awide range of protocols have been described for detection orisolation of STEC since that all serotypes cannot be detectedby one method [18] Trypticase soy broth E coli brothbuffered peptone water and brain heart infusion broth addedwith selective agents have been recommended for STECenrichment In the present study samples were enriched inEC broth without antibiotics which may be advisable whenstressed or injured STEC cells are cultured [19] In addition acomparative study of enrichment protocols by Vimont et al[20] showed that the initial level of E coliO157was not greatlyinfluenced by the enrichment protocol tested whereas theinitial level of background microflora appeared to decreasewhen EC brothwas used Other techniques have been recom-mended for improving the sensitivity of detection methodsThe immunomagnetic separation (IMS) can be used afterenrichment and prior to plating for the selective concentra-tion of STEC cells and it is well established for the detectionof E coliO157 in foods yielding detection limits as low as 1-2CFU25 g [18] While IMS was not used in this study subse-quent STEC researches in our laboratorywill include this pro-cedure Otherwise molecular methods such as conventionalPCR and real-time PCR are very sensitive and provide resultsin shorter times than cultures Thus the ISOTS 131362012standard is based on the sample enrichment followed by areal-time PCR targeted to the detection of the stx and eae vir-ulence genes and the determination ofO157O111 O26O103and O145 STEC serogroups in foods and animal foodstuffsWhen genes are detected the STEC strain should be isolatedfor confirmation [18] Also immunoassay-based methodssuch as an available EIA for testing Shiga toxins 1 and 2 havebeen used in the STEC detection from human stools [21]Differences in STEC carriage have been observed betweengrass-fed and feedlot cattle [22] in the present study two

positive stx1stx2 samples corresponded to feedlot animal andthe other one came from a grazing animal Although STECdetection andor recovery were negative in other samplesstudied here Ojo et al [23] demonstrated STEC in feces ofcattle (152) sheep (107) goats (75) and pigs (56) aswell as in beef (38) goat-meat (17) and pork (40)

The isolation of S Typhimurium from stools of a patientwas consistent with studies reporting this one as the mostfrequently isolated serovar from humans in Argentina since2006 [24] We report the isolation of SNewport and SGam-inara from wild boars for the first time in our region TheseSalmonella serotypes have been previously isolated from clin-ical samples during an outbreak caused by consumption ofunpasteurized orange juice in USA [25] and recovered frompatients with diarrhea in Caribbean zone of Colombia [26]Environmental factors and seasonal variations as well as dif-ferent supply sources of samples might have influenced in thelow recovery of Salmonella from animal samples in our study

The Yersinia prevalence observed in this work was lowerthan 55 from pork and beef sausages and minced meatobtained by Lucero Estrada et al [8] who detected YenterocoliticaB1A (O5 andO630) B2O9 andY intermediain our region Previously Floccari et al [27] isolated 10 YenterocoliticaB1AO5Y intermedia andY frederiksenii from70 chicken carcasses inArgentina AACestablishes noY ente-rocolitica limits in relation to any of the foods here analyzedbut the absence of this pathogen is desirable Although Yenterocolitica was not detected in human and animal stoolsinvestigated in the present study this microorganism hasbeen isolated from human diarrheic feces [28] and animalstools [29] in our country

In this study virulence phenotypic tests for Y ente-rocolitica B1A strains produced negative results exceptingautoagglutination at 37∘C however the yadA gene was notdetected by PCR Lack of correlation betweenY enterocoliticaphenotypic and genotypic virulence markers such as theabove mentioned has been reported by Zheng et al [30]These authors found that some Y enterocolitica strains con-tain other unknown virulencemarkers that interact with eachother and play an important role in the pathogenesis In thisregard the chromosomal gene ystB had been strongly linkedto the production of diarrhea by B1A strains Opportunelyamong 115 Y enterocolitica isolates of pig origin analyzed byBonardi et al [31] 757 corresponded to B1A with ystB asthe most common virulence gene (724) In our study thisgene was demonstrated in one Y enterocolitica B1A O78-8-810 strain (17 14 95 CI 0ndash42) isolated from porcineskin and bones The presence of Y enterocolitica B1A and Yintermedia in chicken carcasses and porcine skin and bonescould be the result of cross-contamination during processingof these products or carriage by slaughtered pigs respectively

Related to STEC antimicrobial susceptibility since anti-microbials can injure the bacterial membrane causing anacute release of preformed Shiga toxin [32] the treatment ofHUS in patients is mostly supportive with adequate corporalfluid and electrolytemanagement control of the haematolog-ical complications antihypertensive and analgesic therapymechanical ventilation and dialysis when necessary [33]avoiding antibiotic administration In our region STEC


GTB2

GTB1

127A

127B

127G

127H

127I

127P

127Q

128B

128I

128O

128P

OSA

100 95 90 85 80 75 70 65

Similarity ()

A

B

Figure 1 Fingerprints and dendrogram obtained by PFGE of twelve Salmonella isolates in this study GT genomic type Six SNewport (127AB G H I and P) and one S Gaminara (127Q) isolates from a tonsil and four S Gaminara (128B I O and P) isolates from tongue of thesame wild boar S Typhimurium strain of human origin (OSA)

strains isolated from patients with diarrhea have demon-strated in vitro susceptibility to antibiotics commonly used inthe treatment of infections triggered by other enterobacteriaContrary to the antibiotic sensitivity demonstrated by ourSalmonella isolates Ibar et al [34] observed multidrug resist-ance in different Salmonella serotypes isolated from porcinein Argentina against antimicrobials commonly used inveterinary medicine Regarding Y enterocolitica antimicro-bial susceptibility our results matched those reported byLucero Estrada et al [8] and Bonardi et al [31] who observedresistance to cephalotin and ampicillin

5 Conclusions

A low prevalence of STEC Salmonella spp and Yersiniaspecies was observed in human animal and food samplesin this region of Argentina The low number of STEC foundin this study one E coli O157H7 from human stool ascompared to otherworksmight be attributed to the detectionmethods used Otherwise the detection of stx1stx2 genes incattle stools highlights the risk of exposure to STEC animalcarriers and reinforces the requirement of the good practicesof hygiene during slaughtering and meat processing On the

other hand the high Salmonella frequency observed in thesmall number of wild boar samples emphasizes the needof further studies in these animals whose byproducts aremanufactured and marketed at retail Lastly bioserotypesand virulence traits characterizing our Y enterocolitica iso-lates were related to null or low pathogenicity for humanshowever a wide field of knowledge remains unexplored aboutY enterocoliticaB1A virulence Our results suggest that a closemicrobiological monitoring might contribute to the know-ledge of prevalence and distribution of these enteropathogensin patients presumable animal reservoirs and foods in ourregion which would allow public health services to take pre-ventive measures

Conflict of Interests

The authors declare that there is no conflict of interests

Acknowledgments

This work was supported by Project 8803 Science andTechnologyDepartment NationalUniversity of San Luis SanLuis Argentina Authors wish to thankDr E Carniel Pasteur


100 90 80 70 60

Similarity ()

GTA1

GTA2

GTA3

GTB1

GTB2

182Bp

182Cp

182Dp

184Ep

196Ac

195Ec

197Bc

246Ap

246Bp

247Bp

W

234p

236p

A

B

182Ap

195Ec bis

197Bc bis

Figure 2 Fingerprints and dendrogram obtained by PFGE of fifteen Yersinia isolates GT genomic type Y enterocolitica B1A O1225-1226(182Ap 182Bp 182Cp 182Dp 184Ep) isolates from two chicken carcasses Y enterocolitica B1A O78-8-819 isolates from three (196Ac 195Ec195Ec bis 197Bc 197Bc bis) porcine skinbones and two (246Ap 246Bp 247Bp) chicken carcasses Y enterocolitica W1024 reference strain(W) Y intermedia B6 O17 (234p) and Y intermedia B4 O40 (236p)

Institute Paris for the classification of local Yersinia strainsDr G Cornelis of Louvain Catholic University Belgium andDr G Kapperud (Norwegian Institute of Public Health OsloNorway) for providing Y enterocolitica reference strainsand Drs M I Caffer and I Chinen (INEI-ANLIS BuenosAires Argentina) for kindly serotyping Salmonella strainsand providing reference STEC strain respectively

References

[1] M Rivas S Sosa-Estani J Rangel et al ldquoRisk factors forsporadic Shiga toxin-producing Escherichia coli infections in

children Argentinardquo Emerging Infectious Diseases vol 14 no5 pp 763ndash771 2008

[2] C R Hale E Scallan A B Cronquist et al ldquoEstimates ofenteric illness attributable to contact with animals and theirenvironments in the United Statesrdquo Clinical Infectious Diseasesvol 54 supplement 5 pp S472ndashS479 2012

[3] A V Page and W C Liles ldquoEnterohemorrhagic Escherichiacoli infections and the haemolytic-uremic syndromerdquo MedicalClinics of North America vol 97 no 4 pp 681ndash695 2013

[4] V Brusa V Aliverti F Aliverti et al ldquoShiga toxin-producingEscherichia coli in beef retail markets fromArgentinardquo Frontiersin Cellular and Infection Microbiology vol 2 article 171 2013


[5] K Murakami T Noda D Onozuka and N Sera ldquoSalmonellain liquid eggs and other foods in Fukuoka Prefecture JapanrdquoInternational Journal of Microbiology vol 2013 Article ID463095 5 pages 2013

[6] G I Favier C S M Lucero Estrada V Lazarte Otero andM EEscudero ldquoPrevalence antimicrobial susceptibility and mole-cular characterization by PCR and pulsed field gel electrophore-sis (PFGE) of Salmonella spp isolated from foods of animalorigin in San Luis Argentinardquo Food Control vol 29 no 1 pp49ndash54 2013

[7] A Rahman T S Bonny S Stonsaovapak and C Ananchaipat-tana ldquoYersinia enterocolitica epidemiological studies and out-breaksrdquo Journal of Pathogens vol 2011 Article ID 239391 11pages 2011

[8] C SM Lucero Estrada L D C Velazquez G I FavierM S DiGenaro andM E Escudero ldquoDetection of Yersinia spp inmeatproducts by enrichment culture immunomagnetic separationand nested PCRrdquo Food Microbiology vol 30 no 1 pp 157ndash1632012

[9] G A Leotta I Chinen S Epszteyn et al ldquoValidacion de unatecnica de PCR multiple para la deteccion de Escherichia coliproductor de toxina Shigardquo Revista Argentina de Microbiologiavol 37 no 1 pp 1ndash10 2005

[10] E M Ribot M A Fair R Gautom et al ldquoStandardization ofpulsed-field gel electrophoresis protocols for the subtyping ofEscherichia coliO157H7 Salmonella and Shigella for PulseNetrdquoFoodborne Pathogens and Disease vol 3 no 1 pp 59ndash67 2006

[11] C S M Lucero Estrada L D G Velazquez S D Genaro andA M S de Guzman ldquoComparison of DNA extraction methodsfor pathogenic Yersinia enterocolitica detection from meat foodby nested PCRrdquo Food Research International vol 40 no 5 pp637ndash642 2007

[12] N Bhagat and J S Virdi ldquoDistribution of virulence-associatedgenes in Yersinia enterocolitica biovar 1A correlates with clonalgroups and not the source of isolationrdquo FEMS MicrobiologyLetters vol 266 no 2 pp 177ndash183 2007

[13] Clinical and Laboratory Standards Institute (CLSI) ldquoPerfor-mance standards for antimicrobial susceptibility testing M100-S16rdquo in Clinical and Laboratory Standards Guidelines 2009

[14] R Capita C Alonso-Calleja M T Garcıa-Arias B Morenoand M del Camino Garcıa-Fernandez ldquoMethods to detect theoccurrence of various indicator bacteria on the surface of retailpoultry in Spainrdquo Journal of Food Science vol 67 no 2 pp 765ndash771 2002

[15] M A Jure S Condorı G A Leotta et al ldquoDetection isolationand characterization of Shiga toxin-producing Escherichia coli(STEC) in fresh groundbeef frombutcher shops inConcepcionTucuman ProvincerdquoRevista Argentina deMicrobiologıa vol 42no 4 pp 284ndash287 2010

[16] L Meichtri E Miliwebsky A Gioffre et al ldquoShiga toxin-pro-ducing Escherichia coli in healthy young beef steers fromArgentina prevalence and virulence propertiesrdquo InternationalJournal of Food Microbiology vol 96 no 2 pp 189ndash198 2004

[17] M E Sanz M R Vinas and A E Parma ldquoPrevalence of bovineverotoxin-producing Escherichia coli in Argentinardquo EuropeanJournal of Epidemiology vol 14 no 4 pp 399ndash403 1998

[18] C Farrokh K Jordan F Auvray et al ldquoReview of Shiga-toxin-producing Escherichia coli (STEC) and their significance indairy productionrdquo International Journal of Food Microbiologyvol 162 no 2 pp 190ndash212 2013

[19] H S Hussein and L M Bollinger ldquoInfluence of selective mediaon successful detection of shiga toxin-producing Escherichia

coli in food fecal and environmental samplesrdquo Foodborne Path-ogens and Disease vol 5 no 3 pp 227ndash244 2008

[20] A Vimont C Vernozy-Rozand M P Montet C Lazizzera CBavai and M-L Delignette-Muller ldquoModeling and predictingthe simultaneous growth of Escherichia coli O157H7 andground beef background microflora for various enrichmentprotocolsrdquoApplied and Environmental Microbiology vol 72 no1 pp 261ndash268 2006

[21] C R Hermos M Janineh L L Han and A J McAdam ldquoShigatoxin-producing Escherichia coli in children diagnosis andclinical manifestations of O157H7 and non-O157H7 infectionrdquoJournal of Clinical Microbiology vol 49 no 3 pp 955ndash959 2011

[22] N L Padola M E Sanz J E Blanco et al ldquoSerotypes and vir-ulence genes of bovine Shigatoxigenic Escherichia coli (STEC)isolated from a feedlot in Argentinardquo Veterinary Microbiologyvol 100 no 1-2 pp 3ndash9 2004

[23] O E Ojo A T P Ajuwape E B Otesile A A OwoadeM A Oyekunle and A I Adetosoye ldquoPotentially zoonoticshiga toxin-producing Escherichia coli serogroups in the faecesand meat of food-producing animals in Ibadan Nigeriardquo Inter-national Journal of Food Microbiology vol 142 no 1-2 pp 214ndash221 2010

[24] M I Caffer A Alcain M Panagopulo M Moroni S Brengiand R Terragno ldquoSalmonella serovars in Argentina 2007ndash2009rdquo Revista Argentina de Microbiologıa vol 42 p 80 2010

[25] M E Parish ldquoColiforms Escherichia coli and Salmonella sero-vars associated with a citrus-processing facility implicated in asalmonellosis outbreakrdquo Journal of Food Protection vol 61 no3 pp 280ndash284 1998

[26] J Durango G Arrieta and S Mattar ldquoPresence of Salmonellaas a risk to public health in the Caribbean zone of ColombiardquoBiomedica vol 24 no 1 pp 89ndash96 2004

[27] M E Floccari M M Carranza and J L Parada ldquoYersinia ent-erocolitica biogroup 1A serotypeO5 in chicken carcassesrdquo Jour-nal of Food Protection vol 63 no 11 pp 1591ndash1593 2000

[28] M Paz H Muzio S Teves and P Santini ldquoAnalysis of aYersinia enterocolitica strain isolated from human diarreic fecesin Argentinardquo Revista Argentina de Microbiologia vol 36 no 4pp 164ndash169 2004

[29] M E Escudero LVelazquez andAM S deGuzman ldquoYersiniaenterocolitica and related species isolated from animals slaugh-tered for human consumptionrdquo Food Microbiology vol 13 no3 pp 201ndash204 1996

[30] H Zheng Y Sun Z Mao and B Jiang ldquoInvestigation of viru-lence genes in clinical isolates of Yersinia enterocoliticardquo FEMSImmunology and Medical Microbiology vol 53 no 3 pp 368ndash374 2008

[31] S Bonardi L Bassi F Brindani et al ldquoPrevalence characteriza-tion and antimicrobial susceptibility of Salmonella enterica andYersinia enterocolitica in pigs at slaughter in Italyrdquo InternationalJournal of FoodMicrobiology vol 163 no 2-3 pp 248ndash257 2013

[32] J Scheiring A Rosales and L B Zimmerhackl ldquoClinical prac-tice todayrsquos understanding of the haemolytic uraemic syn-dromerdquo European Journal of Pediatrics vol 169 no 1 pp 7ndash132010

[33] J T Kielstein G Beutel S Fleig et al ldquoBest supportive careand therapeutic plasma exchange with or without eculizumabin Shiga-toxin-producing E coli O104H4 induced haemolytic-uraemic syndrome an analysis of the German STEC-HUSregistryrdquoNephrology Dialysis Transplantation vol 27 no 10 pp3807ndash3815 2012


[34] M P Ibar G Vigo P Pineyro et al ldquoSerovars of Salmonellaenterica subspecies enterica and its antimicrobial resistance inslaughterhouse pigsrdquoRevistaArgentina deMicrobiologia vol 41no 3 pp 156ndash162 2009

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of


Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology


Molecular Biology International

GenomicsInternational Journal of


The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014


BioinformaticsAdvances in

Marine BiologyJournal of



Signal TransductionJournal of


BioMed Research International

Evolutionary BiologyInternational Journal of



Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014


Genetics Research International


Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational



Enzyme Research



Microbiology


the clinical laboratories of Argentina a systematic epidemi-ological study of Y enterocolitica in environment reservoirsfoods and humans is still pending

For contributing to the knowledge of prevalence anddistribution of these enteropathogens in patients presumableanimal reservoirs and foods of our region this study wasaimed (i) to detect STEC Salmonella spp and Y enterocol-itica in human and animal feces and foods of animal originintended for human consumption (ii) to assess the patho-genic potential of Y enterocolitica strains through phenotypicand molecular virulence markers (iii) to test the antimi-crobial susceptibility of all strains and (iv) to determinepossible genetic relationships among isolates of each speciesby subtyping using pulsed field gel electrophoresis (PFGE) Inaddition counts of total coliformswere performed in samplesof foods

2 Materials and Methods

21 Bacterial Strains Y enterocolitica O9 W1024 pYV+(Belgium)Y enterocoliticaMCH700 B4O3 pYV+ (Canada)Y enterocolitica 29C-43 B4 O3 pYV+ (Norway) and Yenterocolitica B2 O9 pYV+ (Argentina) were used as pos-itive controls in phenotypic virulence assays assessmentof genetic virulence markers by PCR and comparison ofDNA profiles by PFGE Escherichia coli O157H7 EDL933Sor-120573glu-E-Hly+eae+ biotype C producer of Stx1 andStx2 was employed as positive control in PCR targetingSTEC stx1stx2 genes and Salmonella Braenderup H9812 wasused as molecular size marker in PFGE Y enterocoliticaB1A O5 CLO229 and Y enterocolitica B1A O630 CLO225local strains isolated from sausages were utilized as positivecontrols in ystB gene PCR [8] These strains were maintainedat 4∘C on trypticase soy agar slants (TSA Merck DarmstadtGermany) For determining culture purity isolations onMac Conkey agar (MC Merck) Sorbitol Mac Conkey agar(SMAC Merck) and Salmonella Shigella agar (SS Merck)for Y enterocolitica E coli O157H7 and S Braenderuprespectively were made prior to each experiment Plates wereincubated 48 h at 25∘C forY enterocolitica and 24 h at 37∘C forE coli O157H7 and S Braenderup

22 Samples A total of 453 samples from human and animalsources in San Luis city and upcoming rural areas Argentinawere analyzed during the period June 2008ndashNovember 2011They included 70 samples of human stools from patients withenterocolitis symptoms attending a local clinical laboratory167 stool samples obtained from feedlot bovines (119899 = 61)grazing bovines (119899 = 68) porcine (119899 = 20) ovine (119899 =10) goats (119899 = 6) and equines (119899 = 2) and 216 samplesof animal origin intended for human consumption such aschicken carcasses (119899 = 80) porcine skin and bones (119899 = 10)goat cheeses (119899 = 30) fresh sausages ldquochorizosrdquo (119899 = 90) andsix samples of three wild boars tongues (119899 = 3) and tonsils(119899 = 3) Samples of animal stools were randomly collected inregional cattle markets and farms immediately after defeca-tion Samples of wild boars were hunt products from ourregion Samples of foods of animal origin intended for humanconsumptionwere purchased at five retailmarkets in San Luis

city All samples were packed in individual sterile plastic bagsand stored at 4∘C for up to 6 h before processing

23 Investigation of Total Coliforms Thepresence of total col-iforms was investigated in samples of animal origin intendedfor human consumption except tonsils and tongues of wildboars Decimal dilutions of each sample were prepared in01 peptone water pH 72 (PW Merck) one milliliter ofeach dilution was seeded in violet-lactose-neutral red-bileagar (VLRB Merck) and incubated at 37∘C for 24 h Countsof characteristic colonies were performed and results wereexpressed as log

10CFUg

24 Investigation of Shiga Toxin-Producing Escherichia coli(STEC) Salmonella spp and Y enterocolitica

241 STEC Samples of 25 g were homogenized in stomacher(IULMasticator Germany) for 90 s and incubated in 225mLof EC broth (Merck) for 24 h at 37∘C For avoiding falsenegative PCR results produced by interference of organiccompounds and background microflora present in samplesstx1stx2 PCR was not immediately performed Instead ECenriched samples were streaked on SMAC incubating 24 h at37∘C [9] Each plate showed confluent growth in the initialstreaking zone where bacteria were clustered and individualcolonies in the final streaking zone where bacteria wereisolatedThen DNAwas extracted from confluent growth forscreening stx1stx2 genes by PCR [9] if amplified five sorbitolnonfermenting and five fermenting colonies were randomlyselected from SMAC plates and their DNA were individuallytested by PCR targeted to stx1 and stx2 genes When posi-tive results were observed Gram staining and biotyping ofeach colony was performed Sorbitol nonfermenting E colicolonies were challenged against O157 antiserum (NationalInstitute of Infectious Diseases-INEI ANLIS-ldquoDr Carlos GMalbranrdquo Buenos Aires Argentina) by a slide agglutinationtest

242 Salmonella spp This microorganism was investigatedaccording to US FDA Bacteriological Analytical Manual(httpwwwfdagovFoodFoodScienceResearchLaborato-ryMethodsucm2006949htm accessed in May 2008)Twenty five grams of each homogenized sample wasenriched in 225mL of lactose broth (LB Merck) at 37∘Cfor 24 h then one-mL LB aliquots was transferred intotwo tubes containing 9mL of tetrathionate broth (Merck)and two tubes with 9mL of Rappaport-Vassiliadis broth(Merck) One tube of each selective broth was incubatedat 37∘C for 24 h and the other one was incubated at 42∘Cfor 24 h Isolations were done on SS agar (Merck) for 24 hat 37∘C and suspect lactose nonfermenting colonies wereexamined by Gram staining and biochemical assays Sero-typing was performed in the National Reference Center forEnterobacteria INEI-ANLIS Buenos Aires Argentina

243 Y enterocolitica This species was searched by enrich-ment of 25-g homogenized sample in 225mL of phos-phate buffered saline pH 76 added with 1 sorbitol and 015bile salts and incubated 21 days at 4∘C Isolations were made




2 2 pmol120583L of



















2














3 Results


10CFUg respectively


10CFUg) and





Table2Frequencyof

detectionof

STEC


ofdiverseo

rigin

analyzed

inthisstu

dy

Source

Num

bero

fsamples

STEC

Salm

onellaspp

Yersiniaspecies

Positives

amples

(plusmn19

6SE)lowast

Positives

amples

(plusmn19

6SE)lowast

Serovar

(num

bero

fstrains)

Positives

amples

(plusmn19

6SE)lowast

Species

Bioserovar

(Nostr

ains)

Cultu

rePC

RHum

ansto

ols

701(14plusmn28)

mdash1(14plusmn28)

STy

phim

urium

(1)

mdashmdash

Animalsto

ols

167

feedlotb

ovines

61mdash

2(33plusmn46)

mdashmdash

grazingbo

vines

68mdash

1(15plusmn30)

mdashmdash

porcines

20mdash

mdashmdash

mdashovines

10mdash

mdashmdash

mdashgoats

6mdash

mdashmdash

mdashequines

2mdash

mdashmdash

mdashSamples

ofanim

alorigin

forh

uman

consum

ption

216

chickencarcasses

80mdash

mdashmdash

6(75plusmn60)

Yenterocolitica

Yinterm

edia

B1AO12

25-1226(2)

B1AO78-8-819

(2)

B6O17

(1)

B4O40(1)

porcines

kinandbo

nes

10mdash

mdashmdash

3(30plusmn35)

Yenterocolitica

B1AO78-8-819

(3)

goatcheeses

30mdash

mdashmdash

mdashfre

shsausages

90mdash

mdashmdash

mdashwild

boars

3

tonsils

3mdash

mdash1(33plusmn66)

SNew

port(1)

SGam

inara(

1)mdash

tong

ues

3mdash

mdash1(33plusmn66)

SGam

inara(

1)mdash

Total

453

4(09plusmn09)dagger

3(07plusmn08)dagger

9(20plusmn13

)daggerlowast(

)percentage

correspo

ndingto

positives

amplesto

talsam

ples

ofthes

ametypedagger(

)percentage

correspo

ndingto

totalp

ositive

samples

fore

achpathogento

talofsam

plesInbo

thcases19

6SEisthe119905

value

(120572005)m

ultip

liedby

thes

tand

arderror















4 Discussion













GTB2

GTB1

127A

127B

127G

127H

127I

127P

127Q

128B

128I

128O

128P

OSA

100 95 90 85 80 75 70 65

Similarity ()

A

B



5 Conclusions





Acknowledgments



100 90 80 70 60

Similarity ()

GTA1

GTA2

GTA3

GTB1

GTB2

182Bp

182Cp

182Dp

184Ep

196Ac

195Ec

197Bc

246Ap

246Bp

247Bp

W

234p

236p

A

B

182Ap

195Ec bis

197Bc bis



References














































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology




2 2 pmol120583L of



















2














3 Results


10CFUg respectively


10CFUg) and





Table2Frequencyof

detectionof

STEC


ofdiverseo

rigin

analyzed

inthisstu

dy

Source

Num

bero

fsamples

STEC

Salm

onellaspp

Yersiniaspecies

Positives

amples

(plusmn19

6SE)lowast

Positives

amples

(plusmn19

6SE)lowast

Serovar

(num

bero

fstrains)

Positives

amples

(plusmn19

6SE)lowast

Species

Bioserovar

(Nostr

ains)

Cultu

rePC

RHum

ansto

ols

701(14plusmn28)

mdash1(14plusmn28)

STy

phim

urium

(1)

mdashmdash

Animalsto

ols

167

feedlotb

ovines

61mdash

2(33plusmn46)

mdashmdash

grazingbo

vines

68mdash

1(15plusmn30)

mdashmdash

porcines

20mdash

mdashmdash

mdashovines

10mdash

mdashmdash

mdashgoats

6mdash

mdashmdash

mdashequines

2mdash

mdashmdash

mdashSamples

ofanim

alorigin

forh

uman

consum

ption

216

chickencarcasses

80mdash

mdashmdash

6(75plusmn60)

Yenterocolitica

Yinterm

edia

B1AO12

25-1226(2)

B1AO78-8-819

(2)

B6O17

(1)

B4O40(1)

porcines

kinandbo

nes

10mdash

mdashmdash

3(30plusmn35)

Yenterocolitica

B1AO78-8-819

(3)

goatcheeses

30mdash

mdashmdash

mdashfre

shsausages

90mdash

mdashmdash

mdashwild

boars

3

tonsils

3mdash

mdash1(33plusmn66)

SNew

port(1)

SGam

inara(

1)mdash

tong

ues

3mdash

mdash1(33plusmn66)

SGam

inara(

1)mdash

Total

453

4(09plusmn09)dagger

3(07plusmn08)dagger

9(20plusmn13

)daggerlowast(

)percentage

correspo

ndingto

positives

amplesto

talsam

ples

ofthes

ametypedagger(

)percentage

correspo

ndingto

totalp

ositive

samples

fore

achpathogento

talofsam

plesInbo

thcases19

6SEisthe119905

value

(120572005)m

ultip

liedby

thes

tand

arderror















4 Discussion













GTB2

GTB1

127A

127B

127G

127H

127I

127P

127Q

128B

128I

128O

128P

OSA

100 95 90 85 80 75 70 65

Similarity ()

A

B



5 Conclusions





Acknowledgments



100 90 80 70 60

Similarity ()

GTA1

GTA2

GTA3

GTB1

GTB2

182Bp

182Cp

182Dp

184Ep

196Ac

195Ec

197Bc

246Ap

246Bp

247Bp

W

234p

236p

A

B

182Ap

195Ec bis

197Bc bis



References














































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology








2














3 Results


10CFUg respectively


10CFUg) and





Table2Frequencyof

detectionof

STEC


ofdiverseo

rigin

analyzed

inthisstu

dy

Source

Num

bero

fsamples

STEC

Salm

onellaspp

Yersiniaspecies

Positives

amples

(plusmn19

6SE)lowast

Positives

amples

(plusmn19

6SE)lowast

Serovar

(num

bero

fstrains)

Positives

amples

(plusmn19

6SE)lowast

Species

Bioserovar

(Nostr

ains)

Cultu

rePC

RHum

ansto

ols

701(14plusmn28)

mdash1(14plusmn28)

STy

phim

urium

(1)

mdashmdash

Animalsto

ols

167

feedlotb

ovines

61mdash

2(33plusmn46)

mdashmdash

grazingbo

vines

68mdash

1(15plusmn30)

mdashmdash

porcines

20mdash

mdashmdash

mdashovines

10mdash

mdashmdash

mdashgoats

6mdash

mdashmdash

mdashequines

2mdash

mdashmdash

mdashSamples

ofanim

alorigin

forh

uman

consum

ption

216

chickencarcasses

80mdash

mdashmdash

6(75plusmn60)

Yenterocolitica

Yinterm

edia

B1AO12

25-1226(2)

B1AO78-8-819

(2)

B6O17

(1)

B4O40(1)

porcines

kinandbo

nes

10mdash

mdashmdash

3(30plusmn35)

Yenterocolitica

B1AO78-8-819

(3)

goatcheeses

30mdash

mdashmdash

mdashfre

shsausages

90mdash

mdashmdash

mdashwild

boars

3

tonsils

3mdash

mdash1(33plusmn66)

SNew

port(1)

SGam

inara(

1)mdash

tong

ues

3mdash

mdash1(33plusmn66)

SGam

inara(

1)mdash

Total

453

4(09plusmn09)dagger

3(07plusmn08)dagger

9(20plusmn13

)daggerlowast(

)percentage

correspo

ndingto

positives

amplesto

talsam

ples

ofthes

ametypedagger(

)percentage

correspo

ndingto

totalp

ositive

samples

fore

achpathogento

talofsam

plesInbo

thcases19

6SEisthe119905

value

(120572005)m

ultip

liedby

thes

tand

arderror















4 Discussion













GTB2

GTB1

127A

127B

127G

127H

127I

127P

127Q

128B

128I

128O

128P

OSA

100 95 90 85 80 75 70 65

Similarity ()

A

B



5 Conclusions





Acknowledgments



100 90 80 70 60

Similarity ()

GTA1

GTA2

GTA3

GTB1

GTB2

182Bp

182Cp

182Dp

184Ep

196Ac

195Ec

197Bc

246Ap

246Bp

247Bp

W

234p

236p

A

B

182Ap

195Ec bis

197Bc bis



References














































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


Table2Frequencyof

detectionof

STEC


ofdiverseo

rigin

analyzed

inthisstu

dy

Source

Num

bero

fsamples

STEC

Salm

onellaspp

Yersiniaspecies

Positives

amples

(plusmn19

6SE)lowast

Positives

amples

(plusmn19

6SE)lowast

Serovar

(num

bero

fstrains)

Positives

amples

(plusmn19

6SE)lowast

Species

Bioserovar

(Nostr

ains)

Cultu

rePC

RHum

ansto

ols

701(14plusmn28)

mdash1(14plusmn28)

STy

phim

urium

(1)

mdashmdash

Animalsto

ols

167

feedlotb

ovines

61mdash

2(33plusmn46)

mdashmdash

grazingbo

vines

68mdash

1(15plusmn30)

mdashmdash

porcines

20mdash

mdashmdash

mdashovines

10mdash

mdashmdash

mdashgoats

6mdash

mdashmdash

mdashequines

2mdash

mdashmdash

mdashSamples

ofanim

alorigin

forh

uman

consum

ption

216

chickencarcasses

80mdash

mdashmdash

6(75plusmn60)

Yenterocolitica

Yinterm

edia

B1AO12

25-1226(2)

B1AO78-8-819

(2)

B6O17

(1)

B4O40(1)

porcines

kinandbo

nes

10mdash

mdashmdash

3(30plusmn35)

Yenterocolitica

B1AO78-8-819

(3)

goatcheeses

30mdash

mdashmdash

mdashfre

shsausages

90mdash

mdashmdash

mdashwild

boars

3

tonsils

3mdash

mdash1(33plusmn66)

SNew

port(1)

SGam

inara(

1)mdash

tong

ues

3mdash

mdash1(33plusmn66)

SGam

inara(

1)mdash

Total

453

4(09plusmn09)dagger

3(07plusmn08)dagger

9(20plusmn13

)daggerlowast(

)percentage

correspo

ndingto

positives

amplesto

talsam

ples

ofthes

ametypedagger(

)percentage

correspo

ndingto

totalp

ositive

samples

fore

achpathogento

talofsam

plesInbo

thcases19

6SEisthe119905

value

(120572005)m

ultip

liedby

thes

tand

arderror















4 Discussion













GTB2

GTB1

127A

127B

127G

127H

127I

127P

127Q

128B

128I

128O

128P

OSA

100 95 90 85 80 75 70 65

Similarity ()

A

B



5 Conclusions





Acknowledgments



100 90 80 70 60

Similarity ()

GTA1

GTA2

GTA3

GTB1

GTB2

182Bp

182Cp

182Dp

184Ep

196Ac

195Ec

197Bc

246Ap

246Bp

247Bp

W

234p

236p

A

B

182Ap

195Ec bis

197Bc bis



References














































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology















4 Discussion













GTB2

GTB1

127A

127B

127G

127H

127I

127P

127Q

128B

128I

128O

128P

OSA

100 95 90 85 80 75 70 65

Similarity ()

A

B



5 Conclusions





Acknowledgments



100 90 80 70 60

Similarity ()

GTA1

GTA2

GTA3

GTB1

GTB2

182Bp

182Cp

182Dp

184Ep

196Ac

195Ec

197Bc

246Ap

246Bp

247Bp

W

234p

236p

A

B

182Ap

195Ec bis

197Bc bis



References














































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology












GTB2

GTB1

127A

127B

127G

127H

127I

127P

127Q

128B

128I

128O

128P

OSA

100 95 90 85 80 75 70 65

Similarity ()

A

B



5 Conclusions





Acknowledgments



100 90 80 70 60

Similarity ()

GTA1

GTA2

GTA3

GTB1

GTB2

182Bp

182Cp

182Dp

184Ep

196Ac

195Ec

197Bc

246Ap

246Bp

247Bp

W

234p

236p

A

B

182Ap

195Ec bis

197Bc bis



References














































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


GTB2

GTB1

127A

127B

127G

127H

127I

127P

127Q

128B

128I

128O

128P

OSA

100 95 90 85 80 75 70 65

Similarity ()

A

B



5 Conclusions





Acknowledgments



100 90 80 70 60

Similarity ()

GTA1

GTA2

GTA3

GTB1

GTB2

182Bp

182Cp

182Dp

184Ep

196Ac

195Ec

197Bc

246Ap

246Bp

247Bp

W

234p

236p

A

B

182Ap

195Ec bis

197Bc bis



References














































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


100 90 80 70 60

Similarity ()

GTA1

GTA2

GTA3

GTB1

GTB2

182Bp

182Cp

182Dp

184Ep

196Ac

195Ec

197Bc

246Ap

246Bp

247Bp

W

234p

236p

A

B

182Ap

195Ec bis

197Bc bis



References














































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology









































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology










Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology








Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology

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