Food handlers as potential sources of dissemination of virulent strains of Staphylococcus aureus in the community

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Food

handlers

as

potential

sources

of

dissemination

of

virulent

strains

of

Staphylococcus

aureus

in

the

community

Ana

Castro,

Carla

Santos,

Helena

Meireles,

Joana

Silva,

Paula

Teixeira

∗

CBQF—CentrodeBiotecnologiaeQuímicaFina—LaboratórioAssociado,Escola SuperiordeBiotecnologia,UniversidadeCatólicaPortuguesa,Porto,Portugal Received10April2015 ;receivedinrevisedform26June2015;accepted29August2015

KEYWORDS

Staphylococcusaureus;

Foodhandlers; Handsandnose carriage; Antimicrobial resistance; Enterotoxingenes

Summary Foodhandlersmayconstituteareservoirofvirulentstrainsof Staphy-lococcusaureusandmaybevehiclesoftheirtransmissiontofood.

Onehundredandsixty-twovolunteerswereassessedforthepresenceofS.aureus

onthehandsandinthenose.S.aureuswasisolatedbyroutineprocedures,andthe isolatesweretestedforsusceptibilityagainstapanelofnineantimicrobialagents. TheisolateswerefurthercharacterizedbySmaI-PFGEproﬁlingandthepresenceof virulencefactors.

Results:TheprevalenceofS.aureuswas19.8%inthenoseand11.1%onthehands; 6.2%oftheindividualscarriedS.aureusbothin theirnosesandhands,andthree individualshadthesamestrain(PFGEtype)inthenoseandonthehands.Although 82%oftheisolateswereresistanttoatleastoneantibiotic,nonedemonstratedthe presenceofeithermecAgeneorresistancetooxacillin(noneidentiﬁedasMRSA). Sixty-eightpercentoftheisolatesfromthenoseandhandspossessedenterotoxin genes.

Thisstudyrevealedahighprevalenceofantibioticresistanceandvirulence deter-minantsamongtheisolates,includingnotonlyclassicalandnovelenterotoxingenes butalsomajorvirulencefactorssuchastst.Potentialdisseminationofthesestrains inthecommunityisamatterofconcern.

∗_{Corresponding}_author_at:_Escola_Superior_de_{Biotecnologia,}

Rua Arquiteto Lobão Vital, Apartado 2511, 4202-401 Porto,

Portugal.Tel.:+351225580095.

E-mailaddress:pcteixeira@porto.ucp.pt(P.Teixeira).

Introduction

Staphylococcus aureus is one of the most impor-tantspecies in the ﬁeldof foodmicrobiology and has been considered a foodborne hazard for a http://dx.doi.org/10.1016/j.jiph.2015.08.001

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long time. In 2013, 386 staphylococcal outbreaks were reported by the EFSA, representing 7.4% of all outbreaksreported in the European Union [1]. Staphylococcalfoodpoisoning,gastroenteritiswith emesisandwithorwithoutdiarrhea[2] character-ized by a short incubationperiod, typically 2—4h [3], is caused by the ingestion of foodcontaining preformed enterotoxins. Not all strains are capa-ble of producing staphylococcal enterotoxins [4], butupuntilnow,22SEshavebeendescribed,11of themwithknownemeticaction[5].

S.aureuscancolonizetheskinandtheanterior nares of individuals and is carried by a signiﬁ-cant proportion of the population [6]. As found by Kluytmans and Wertheim [6], S. aureus colo-nizes the nares of approximately 50% of healthy adults, either persistently or intermittently. In a study by Lues and Van Tonder [7], S. aureus was isolated from the hands of 88% of the popula-tion sampled. Human nasal or hand carriage of enterotoxigenic S. aureus during food process-ing is an important source of food contamination with S. aureus [5,8]. In fact, food poisoning outbreaks associated with post-process contami-nation of foods with S. aureus are in part the responsibility of food handlers who carry entero-toxigenic staphylococci in their nares or on their skin[7].

In recent decades, the increasing prevalence of antimicrobial-resistant S. aureus is receiv-ing widespread attention. Strains of methicillin-resistantS.aureus(MRSA)areofparticularconcern given that they represent a signiﬁcant cause of morbidity and mortality throughout the world. Methicillin-resistant S. aureus are resistant to all availablepenicillinsandother␤-lactam antimicro-bial drugs [9]. Trends for the period 2009—2012 were calculated for28 countries. Statistically sig-niﬁcant increasing trends were observed for four countries, including Portugal, where in 2012, the percentage ofMRSAisolateswas greaterthan 50% [10].

Since Kluytmans et al. [11] described the ﬁrst fatalfoodborneoutbreakofMRSA,food microbiolo-gistsnowconsiderthepossibilityoffoodsasvectors ofantimicrobial-resistantstrains.

To identify MRSAs, the detection of the pres-enceofthe mecAgeneand consequentresistance tomethicillinisimportantnotonlyinfoodisolates but also on food handlers who contribute to the cross-contaminationoffoodproducts.

The combination of enterotoxin genes and the mecAgenecouldprovideuswithinformationabout the presence ofresistantstrains infoodborne dis-easesandalsotheimportanceoffoodasavehicle forantimicrobialresistance.

The purpose of this study was to evaluate the prevalenceofS.aureusamonghealthyindividuals workinginafoodcompanyandtocharacterize iso-lates regarding their resistance to antibiotics and virulence factors. A potential clonal relationship between isolates from the nose and hands of the sameindividualswasalsoinvestigated.

Material

and

methods

Staphylococcus

aureus

sampling

Onehundredandsixty-twovolunteersfromafood company were assessed for the presence of S. aureus on their hands and in their nose (a total of324 sampleswere recovered).Thedeﬁnitionof the sample was one of convenience and included 103 women and 59 men. This company sells food tonumerousclients alloverPortugal;rawmeatis choppedand usedwithin thecompanyfor further processedmeat-containingfoodsorissoldtolocal shops.

The specimens were collected using a cotton-tipped swab previously moistened with sterile Ringerssolution.Theanteriornaresweresampled by rotating the swab tip in both nostrils. Swabs were then spread onto Baird-Parker Egg Yolk Tel-lurite Medium (LabM, Bury, United Kingdom) and incubatedaerobicallyat37◦Cfor 48h. Character-istic colonies were sub-cultured on Mannitol Salt Agar(MSA;Pronadisa,Madrid,Spain)incubated aer-obically at 37◦C for 24h. Presumptive S. aureus coloniesonMSA(yellowcolonieswithyellowzones, Gram-positive, catalase positive, coagulase posi-tiveandDNasepositive)werestreakedonTryptone Soy Agar (TSA; Pronadisa) before being stored at −80◦_C _in _Brain _Heart _Infusion _(BHI; _LabM) _broth containing30%(v/v)glycerol.

DNA

extraction

DNAwasextractedfromsinglecoloniesonTSAusing theguanidine-isothiocyanatemethod[12].DNAwas quantiﬁed spectrophotometrically at 260nm and 280nm.

Identiﬁcation

of

isolates

by

multiplex

PCR

PCRmultiplextodetectthesimultaneouspresence of 16S rRNA (Staphylococcus genus speciﬁc), nuc (S. aureus species speciﬁc) and mecA (determi-nantofmethicillinresistance)geneswasperformed according to Zhang et al. [13]. Staphylococcus aureus DSM 11729 was used as a positive control forthegenemecA,StaphylococcusepidermidisDSM

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20044asanegativecontrolforthegenenucandS. aureusATCC29213asapositivecontrolfor target-ing 16S rRNA and the nuc gene and as anegative controlforthegenemecA.

Detection

of

staphylococcal

enterotoxin

genes

by

multiplex

PCR

Thedetectionofenterotoxingenes,seatosejand tst,was performedbymultiplexPCR accordingto Løvseth, Loncarevic and Berdal [14]. The ampliﬁ-cationofthetarget16SrRNAgenewasincludedas theinternalcontrol.Aspositivecontrols,different strainsofS.aureuskindlysuppliedbyProf.Løvseth (NationalVeterinaryInstitute,Norway)wereused: R2102/00forthesec,seg,andseigenes;R4571/00 forthesecgene,FRI572forthesegandseigenes; 3169forthesec-bovine,sed,andsejgenes;FRI472 for the sed,seg, sei,and sejgenes; R5371/00for the sea, seg, seh, and seigenes; R963/00for the sed,seg,sei,andsejgenes;R5460/00fortheseb, seg, seh, and sei genes; FRI913 for the sea, sec, and see genes; FRI445 for the seg and sei genes; R4071/00forthesebgene;andR4774/00asa neg-ativecontrol.

A5-␮l aliquotofDNAwasaddedtoa20-␮l PCR mixture containing 0.3␮M of each primer except 16SrRNAandsei(0.1␮M)and12.5␮lKapa2GFast master mix (Grisp,Porto, Portugal). Ampliﬁcation wascarriedout asfollows:an initialdenaturation step at95◦Cfor 3min; 30cyclesat95◦Cfor 15s, 62◦Cfor30s and72◦Cfor 30s;and aﬁnal exten-sionstepat72◦Cfor1min.ThePCRproductswere resolved in 2% (w/v) agarose gels (1× Tris Acetic Acid, EDTA) at60V(constant voltage) for 3h and visualizedinatransilluminator.

Antibiotic

susceptibility

testing

by

agar

dilution

The minimal inhibitory concentrations (MICs; ␮g/mL)forS.aureusisolatesweredeterminedby theagardilutionmethoddescribedintheguidelines of the Clinical and Laboratory Standards Insti-tute[15]. Theinoculum waspreparedfroma24h culture on TSA by suspension in sterile Ringer’s solution to obtain turbidity equivalent to the 0.5 McFarland standard. The antibiotics investigated werepenicillinG,chloramphenicol(bothobtained from Sigma, Steinheim, Germany), oxacillin (Bio-Chemica, Billingham, UK), rifampin, gentamicin, tetracycline, erythromycin and ciproﬂoxacin (all kindly supplied by Labesfal, Tondela, Portugal) andVancomycin(Fluka,Steinheim,Germany).The MICsweredeterminedinMueller-Hintonagar(MH;

bioMérieux,Marcy l’Etoile, France) plus 2% (w/v) NaCl in the case of oxacillin, in cation-adjusted MH for penicillin and ampicillin and in MH for the other antibiotics investigated. Staphylococcus aureusATCC29213andEnterococcusfaecalisATCC 29212 were used as controls. For each antibiotic susceptibilitydetermination,atleasttwo indepen-dentexperimentswereperformed.

Detection

of

other

of

virulence

factors

The production of hemolysin was evaluated on blood agar plates (COS, Columbia agar plus 5% (v/v)sheep’sblood;bioMérieux).Theisolateswere streakedontotheplatesandincubatedat37◦Cfor onetotwodays.Thepresenceorabsenceofzones ofclearingaroundthecolonies wasinterpretedas ␤-hemolysis (positive) or gamma-hemolysis (nega-tive)activity, respectively.Greenish zones around thecolonieswereinterpretedas␣-hemolysis[16]. Lipase activity was assessed as described by Tiagoetal.[17]. Apositivereactionwasindicated byopacityaroundthecolonies.

Gelatinase activity was assessed according to Tiagoetal.[17].

For each virulence factor tested, at least two independentexperimentswereperformed.

DNA-macrorestriction

by

pulsed-ﬁeld

gel

electrophoresis

(PFGE)

PFGEtypingofthe isolateswasperformedas pre-viously described by Chung et al. [18] using the restriction enzyme SmaI (ThermoScientiﬁc, New York, USA) and Salmonella enterica ser. Braen-derupH9812asastandard and aCHEFMapperXA (Bio-Rad, Laboratories, Hercules, CA, USA). PFGE image analysisand similarity clustering were per-formed with GelCompar software (Applied Maths, Sint-Martens-Latem,Belgium).Clusteranalysiswas done by the unweighted pair group method with average linkages (UPGMA), using the Dice coefﬁ-cient to analyze the similarities of the banding pulsotypes.

Results

Fifty S. aureus isolates were recovered from the hand and nose samples of 162 individuals (Sup-plementary Table). Nearly one-quarter (24.7%, 40/162)oftheindividuals wereS.aureuscarriers; 60%ofthese(24/40)werefemale,and40%(16/40) were male (Supplementary Table). Nasal carriage was found in 19.8% and hand carriage was found

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Table 1 HandandnasalcarriageofStaphylococcus aureusamongfoodhandlers.

Numberof positive(%)

Hands 18(11.1)

Nose 32(19.8)

Handsandnose(simultaneously) 10(6.2)

Handsbutnotnose 8(4.9)

in 11.1% ofthe individuals; 6.2% had S. aureus in both theirhandsandnose.Eightindividuals(4.9%) had S.aureusontheir handsbutnot intheir nose (Table1).

Supplementary Table related to this arti-cle can be found, in the online version, at http://dx.doi.org/10.1016/j.jiph.2015.08.001.

PFGEanalysiswasperformedonlyforthose iso-lates collected from the nose and hands of the same individual. Eighteen of these isolates were distributed among 15 PFGE types, and two were non-typeable (2095H and 2095N; Supplementary Table). In three individuals, the same strain was found in the nose and on the hands (Fig. 1).seg and seiwere the most prevalentgenes in the iso-lates recovered from both the nose (82.6%) and hands (70%),followed bythe tstgene, which was recovered from 39.1% to 40%of noses and hands, respectively(SupplementaryTableandTable2).For

Table2 Arrangementsofenterotoxingenesproﬁles ofStaphylococcusaureusisolatedfromhandsandnose offoodhandlers.

N(%) Nasalisolates

secbov,seg,sei 6(30.4)

seg,sei 3(13.0)

sea,seg,sei,tst 2(8.7)

seh,seg,sei,tst 2(8.7)

tst 2(8.7)

sej,tst 1(4.3)

sea,seh,seg,sei 1(4.3)

seg,sei,tst 1(4.3)

sea,tst 1(4.3)

sea,sej,seg,sei,sed 1(4.3)

sec,seg,sei 1(4.3)

sea,sec,seg,sei 1(4.3)

sea,seh 1(4.3)

Handsisolates

secbov,seg,sei 3(33.3)

sej,tst 2(22.2)

seg,sei 2(22.2)

seh,seg,sei,tst 1(11.1)

seb 1(11.1)

sec,seg,sei,tst 1(11.1)

Table3 Antibioticsensitivitypatternofthe Staphy-lococcus aureus isolates recovered from hands and noseoffoodhandlers.

Antibiotic Sensitive Intermediate Resistant

N(%) Penicillin 26(52.0) a ₂₄_(48.0) Oxacillin 50(100.0) a ₀_(0.0) Tetracycline 48(96.0) 0(0.0) 2(4.0) Ciproﬂoxacin 37(74.0) 3(6.0) 10(20.0) Erythromycin 34(68.0) 0(0.0) 16(32.0) Gentamicin 49(98.0) 0(0.0) 1(2.0) Rifampin 46(92.0) 0(0.0) 4(8.0) Chloramphenicol 41(82.0) 8(16.0) 1(2.0) Vancomycin (100) 0(0.0) (0.0)

a_Antibiotic_with_no_described_value_for_the_intermediate

MIC.

allofthe isolates,anassociationbetweentheseg andseigeneswasobserved(Table2).

TheantibioticresistanceprofileoftheS.aureus isolates is presented in Table 3. Eighteen percent ofthe isolates were sensitive to allof the antibi-oticsinvestigated. Forty-eight percentand 26%of the isolateswereresistant totwo orthree antibi-oticsofdifferentclasses,respectively.Oneisolate was resistant to five antibiotics, and another was resistanttosix.Forty-eightpercentofisolateswere resistant to penicillin, 32.0% to erythromycinand 20.0%tociprofloxacin.Fourisolateswereresistant torifampin,twowereresistanttotetracycline,one wasresistanttogentamicin,andanotherwas resis-tant to chloramphenicol. All of the isolates were sensitivetovancomycinandoxacillin(Table3).The gene mecA was absent among all of the isolates (datanotshown).

ˇ-Hemolysin, -hemolysin, ˛-hemolysin, lipase andgelatinasewereidentiﬁedin66%,12%,22%,82% and 88% of isolates, respectively (Supplementary Table).

Discussion

The prevalence of S. aureus in the nose of the workers atthe foodcompanyinvestigated(19.8%) is in accordance with the mean nasal coloniza-tionofhealthyadults(20—30%)whoarepersistent carriers [6]. Similarvalues have beenreportedby otherauthors[19,20].Nevertheless,otherauthors hadreportedhigher[21—24]andlowerprevalences [25]. A lower prevalence of S. aureus was found on thehands(11.1%) thanin the noseofthe food handlers. This is consistent with the ﬁndings of previous studies[21,22]. Thereportedprevalence of S. aureus on the hands of food handlers is highly variable. While values similar to or lower

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Figure1 DendrogramofStaphylococcusaureusisolatesrecoveredfromthehandsandnoseofthesameindividual.

thanthose foundinthis studyhavebeenreported [21,26],prevalenceshigherthan50%havealsobeen reported [7,24,27,28]. A high carriage level had beenfound for food handlersworking in hospitals [24,27]whereS.aureusishighlydisseminated.

Ten individuals carried S. aureus both nasally and on their hands, and three of these individ-uals had the same strain in their nose and hands. ThepresenceofS.aureuson thehandsbutnotin noseand thedetection ofisolatesin thenoseand hands ofthe same individual with different PFGE typesindicatethathandcontaminationmayresult fromsourcesotherthantheindividual[21].Foodis naturallycontaminated,atleasttemporarily,when raworeventhroughexposureduringprocessingor refrigerationtotemperaturesthatallowthegrowth of S. aureus. Although food handlers are usually the main source of food contamination in food poisoning outbreaks, equipment and environmen-tal surfaces can also be sources ofcontamination withS.aureus[29].

Eighty-two percent of the isolates were resis-tant to at least one antibiotic, and 48%, 26%and

8%were resistant to one, two and three or more antibioticsofdifferentclasses, respectively.None demonstrated the presence of either the mecA gene or resistance to oxacillin. MRSAs have fre-quently been isolated from health professionals [30], although the isolation of MRSAs from food handlers is rare [22,23,25]. The rate of penicillin resistancewas lowerin ourstudy relative tothat found for isolates recovered from foods commer-cializedinPortugal[31]andalsofromfoodhandlers inotherstudies[22,24,25].Incontrast,higher per-centages ofisolatesresistant to ciproﬂoxacin and toerythromycinwere observed.Working in hospi-tals seems to be a risk factorfor the carriage of resistantstrainsbyfoodhandlers,asdemonstrated inthestudybyFerreiraetal.[24].Thehigh preva-lenceofantibioticresistanceamongfoodhandlers andthecarriageofmultidrug-resistantstrains high-lightsthegrowingproblemofantibiotic resistance inthe‘‘healthy’’community.

S. aureus produces a wide array of cell sur-faceandextracellular proteins(proteases,lipases and cytotoxins suchas hemolysins — alpha, beta,

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gammaanddelta)involvedinvirulencethatenable it toinvadeand destroyhosttissuesand metasta-size to other sites [2]. Staphylococcus aureus Hlb (gene of beta-hemolysin) also plays an important roleinskincolonizationbydamagingkeratinocytes, inadditiontoitswell-knownhemolyticactivityfor erythrocytes [32]. The results fromthis studyare consistent with those observed in previous stud-ies. Among the S. aureus isolated from the oral cavity, 100% producedgelatinase, 77%lipase, 59% beta-hemolysins and 41% alpha-hemolysins [33]. Saisingetal.[34]reportedthat65.6%ofthestrains isolated from acne lesions were lipase positive, and Wu et al. [35] showed that 80% of S. aureus isolated from corneal ulcers producedgelatinase. Beta-hemolysin is secreted by certain strains of S. aureus,especially strains isolatedfromcorneal infections[33].

S. aureus produces a wide variety of toxins including staphylococcalenterotoxins(SEs; SEAto SEE, SEG to SEI, SER to SET) with demonstrated emetic activity and staphylococcal-like (SEl) pro-teins,whicharenotemeticinaprimatemodel(SElL andSElQ)orhaveyettobetested(SElJ,SElK,SElM to SElP, SElU, SElU2 and SElV). SEs and SEls have beentraditionallysubdividedinto classical(SEAto SEE) and new (SEGto SElU2)types. Each ofthese toxinsisknowntohavepotent effectsonthecells oftheimmunesystem,butmanyofthemhaveother biologicaleffectsaswell.Theirprimaryfunctionin vivomaybetoinhibithostimmuneresponsestoS. aureus[2].SEsarethecausativeagentsof staphylo-coccal food poisoningresulting fromthe ingestion of contaminated food. Due to their extraordinary stabilityindenaturingconditions,suchasheatand lowpHlevels,SEsarenotcompletelydestroyedby mild cookingorthe digestionoffoodin the stom-ach. Nausea, emesis,abdominal painorcramping anddiarrheaensueafterashortincubationperiod. Thediseaseisusuallyself-limiting[36].

The majority (71.9% and 55.6%, respectively) of isolates recovered from the nose and hand samplespossessedenterotoxingenes.Ahigh preva-lence of enterotoxigenic isolates recovered from food handlers has been reportedby several stud-ies [19,22,37].Althoughseaisthemostcommonly reported enterotoxin gene [19,37], this was not observed in the present study in which seg and seiwerethemostprevalententerotoxingenesand were associatedwith alloftheisolates.This asso-ciation has been previously reported [19] and is justiﬁedbythefactthattheybelongtoanoperon oftheegcenterotoxingenecluster,whichcontains ﬁveenterotoxingenes(seg,sei,sem,sen,andseo) [38].Thetstgeneswerealsohighlyprevalent.tst genes have been previously detected in isolates

recoveredfromfoodhandlers[22,37], though nor-mally in lower percentages. TSST-1-producing S. aureus was detected for the first time on a food service worker’s hand by Sospedra et al. [39]. TSST-1 was the first markeridentified for Staphy-lococcal Toxic Shock Syndrome (TSS), which is an acute and potentially fatal illness that is charac-terizedbyahighfever,diffuseerythematousrash, desquamation ofthe skin one to two weeks after onset (if not fatalbeforethis time), hypotension, andtheinvolvementofthreeormoreorgansystems [36].

Foodhastomeethighfoodsafetyandfood qual-itystandards.GoodManufacturingPractices(GMP) andHazardAnalysisCriticalControlPoints(HACCP) systemsareappliedtoimprovethemicrobialsafety and quality offood. However, even with the best controlmeasuresinplace,afoodproductmaystill pose a risk to the consumer. The presented data have certain limitations, as the population sam-pledinthisstudyisnotrepresentativeofthefood handler population asawhole in Portugal. Never-theless,thefoodcompanyanalyzedfollowedallof themeasuresreferredtoabove,andyet,thefood handlers’ hands were contaminated with entero-toxigenicandantibiotic-resistantS.aureusstrains.

Conclusion

Thisstudyrevealedahighprevalenceofantibiotic resistance and virulence determinants, including notonlyclassicaland novelenterotoxingenesbut alsomajorvirulencefactorssuchastst,inthe stud-iedpopulation, whichis one ofthe majorsources ofcontamination/recontaminationoffoodwith S. aureus during processing. Potential dissemination of these strains in the community is a matter of concern.

Funding

Nofundingsources.

Competing

interests

Nonedeclared.

Ethical

approval

Notrequired.

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Acknowledgments

This work was supported by National Funds from the Fundac¸ão para aCiência e aTecnologia(FCT) throughprojectPest-OE/EQB/LA0016/2013. Finan-cial support for Ana Castro was provided by FCT throughadoctoralfellowshipBD/39315/2007. Edit-ing of this paper by Dr. P.A. Gibbs is gratefully acknowledged.

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