Occurrence and antimicrobial resistance patterns of Listeria monocytogenes isolated from vegetables

h ttp : / / w w w . b j m i c r o b i o l . c o m . b r /

Food

Microbiology

Occurrence

and

antimicrobial

resistance

patterns

of

Listeria

monocytogenes

isolated

from

vegetables

Vanessa

de

Vasconcelos

Byrne

_,

_Ernesto

_Hofer

_,

_Deyse

_Christina

_Vallim

_,

Rogeria

Comastri

de

Castro

Almeida

a_{PharmacyFaculty,FederalUniversityofBahia,RuaBarãodeGeremoabo,Salvador,BA,Brazil}

b_{LaboratoryofBacterialZoonoses,OswaldoCruzInstitute,PavilhãoRochaLima,RiodeJaneiro,RJ,Brazil} c_{NutritionSchool,FederalUniversityofBahia,Salvador,BA,Brazil}

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Articlehistory:

Received16March2015 Accepted12November2015 Availableonline2March2016 AssociateEditor:ElaineCristina PereiraDeMartinis Keywords: Vegetables Foodsafety Listeriamonocytogenes Antimicrobialresistance

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Althoughthe consumption offresh and minimallyprocessed vegetables is considered healthy,outbreaksrelatedtothecontaminationoftheseproductsarefrequentlyreported. Amongthefood-bornepathogensthatcontaminatevegetablesisListeriamonocytogenes,a ubiquitousorganismthatexhibitstheabilitytosurviveandmultiplyatrefrigerated tem-peratures.ThisstudyaimedtoevaluatetheoccurrenceofL.monocytogenesinvegetables aswellastheantimicrobialresistanceofisolates.Theresultsshowedthat3.03%of sam-pleswerecontaminatedwithL.monocytogenes,comprising2.22%ofrawvegetables and 5.56%ofready-to-eatvegetables.MultiplexPCRconfirmedthevirulencepotentialofthe isolates.Antimicrobialresistanceprofilingshowedthat50%oftheisolatesweresusceptible totheantibioticsused.TheresistanceofoneisolatetopenicillinG,acommonlyemployed therapeuticagent,andthepresenceofserotype4b,aserotypecommonlyassociatedwith food-borneoutbreaks,couldbepotentialhealthhazardsforconsumers.

©2016SociedadeBrasileiradeMicrobiologia.PublishedbyElsevierEditoraLtda.Thisis anopenaccessarticleundertheCCBY-NC-NDlicense

(http://creativecommons.org/licenses/by-nc-nd/4.0/).

Introduction

Foodsafetyisanincreasinglyrelevantissueinthedailylives ofconsumers.Thesearchforahealthierdietand,atthesame time,fasterpreparationhasfavoredtheconsumptionoffresh andready-to-eatvegetables.Becausetheyarenotsubjectedto treatmentsthatconsiderablyreducemicrobiologicalhazards, theseessentialfoodsarepotentialvehiclesforthe transmis-sionofpathogenicmicroorganisms.1

∗ _{Correspondingauthor.}

E-mail:rogeriac@ufba.br(R.C.deCastroAlmeida).

Toinhibit microbial multiplicationand ensure adequate conservation, certainvegetablesare storedandtransported atcooltemperatures.However,theseconditionsfacilitatethe growthofsomemicrobialpathogens,suchasListeria monocyto-genes,apsychotropicmicroorganismthatishighlyrelevantto publichealth.1,2

L. monocytogenes is a ubiquitous bacterium that can be foundintheirrigationwater,soilandfertilizerusedonfarms and indecaying plantmatter,making the presenceofthis bacteriuminvegetablesacontinualrisk.

http://dx.doi.org/10.1016/j.bjm.2015.11.033

1517-8382/©2016SociedadeBrasileiradeMicrobiologia.PublishedbyElsevierEditoraLtda.ThisisanopenaccessarticleundertheCC BY-NC-NDlicense(http://creativecommons.org/licenses/by-nc-nd/4.0/).

Thedisease causedbyL.monocytogenes, knownas liste-riosis,isparticularlytroublesomeforvulnerablepopulations. Thisgroupofpeople,including pregnantwomen andtheir fetuses,the veryyoungand theelderly, isparticularly sus-ceptibletoinvasive listeriosis,withmortalityratesranging between20and40%.3

Outbreaks and sporadic cases of listeriosis have been associated with the contamination of various food items, including milk;soft cheese; meat and meat products; veg-etables;seafoodproducts;ready-to-eatfoods4_{;and,recently,}

cantaloupes.5

Itiswellestablishedthatfoodproductcontaminationis associatedwithfood-processingenvironments harboringL. monocytogenesandsubsequentpost-processingtransferto fin-ishedproducts.6,7

InBrazil,food-bornelisteriosis outbreakshavenotbeen documented,butinrecentstudies,thepresenceofL. monocyto-geneshasbeendescribedinseveralproducts,including ready-to-eat vegetables, such as watercress and escarole8_{; leafy}

salads9_{;choppedkaleandamixtureofspringonion/parsley}10_;

saladmix,lettuce,collardgreens,amixforyakisoba, water-cress,escarole,cabbage,spinach,andamixforsukiyaki.11

L.monocytogenesisnaturallysusceptibletoarangeof antibi-otics that act on Gram-positive bacteria.12 _{Human strains}

of L. monocytogenes are sensitive to a group of antibiotics that includes penicillin, ampicillin, amoxicillin, gentami-cin, erythromycin, tetracycline, rifampicin, co-trimoxazole, vancomycin and imipenem.13,14 _{However, most strains of}

L. monocytogenes show natural resistance to current fluo-roquinolones and cephalosporins, and especially those of the third and fourth generations, such as cefotaxime and cefepime,andtofosfomycin,oxacillinandlincosamides.13

Clinicians usually treatlisteriosis with aminopenicillins incombinationwithanaminoglycoside,suchasgentamicin. Additionally,incasesinwhichreducedsensitivityor resis-tancetobeta-lactamsisencountered,anumberofagentsthat areactiveagainstGram-positivebacteriamaybeused.14

Studies performed bythe Clinical and Laboratory Stan-dardsInstitute(CLSI)15 usinghumanstrainsand,toalesser extent, foodstuff strains have not revealed an increase in resistancetoantibioticsamongthe circulating strainsofL. monocytogenes.16,17 _{However, since the isolation of the first}

multi-resistantstrainofL.monocytogenesin1988,interspecies variationinantimicrobialsusceptibilitieshasbeenreported amongListeriaspecies.12,18

ThepurposeofthisstudywastoverifytheoccurrenceofL. monocytogenesinraw,frozenandready-to-eatvegetables com-mercializedindifferentlocalesinSalvador,BA,Brazil,andto characterizeL.monocytogenesstrainusingthemultiplexPCR method.Furthermore,theresistance/susceptibilityoftheL. monocytogenesstrainstoeightantibioticsusedinhumanand veterinarymedicinewasinvestigated.

Materials

and

methods

Samplecollection

Raw,frozenandready-to-eatvegetableswerepurchasedata localsupermarketandatfastfoodoutletsinSalvador,BA,in

northeasternBrazil,fromOctober2013throughJanuary2014. Overall,132sampleswerecollected,comprising45raw veg-etables,33frozenvegetablesand54ready-to-eatvegetables (salads).Therawvegetablesincludedlettuce,broccoli,white cabbage,purplecabbageandarugula(ninesamplesofeach). Thefrozenvegetables consistedofmixedvegetables (peas, carrots,greenbeansandmaize),peasandbroccoli(11samples of each).The ready-to-eat vegetables included salads con-tainingcarrot,purplecabbageandlettuce;saladscontaining lettuce,beetandpurplecabbage;andsaladscontainingpurple cabbage,whitecabbage,lettuceandbeets(18sampleseach). Representativesamplingwasensuredbytakingsamplesfrom themostconsumedbrandsthatcontainedatleastoneofthe mostconsumedvegetablesinBrazil.19

DetectionofL.monocytogenes

FortheisolationofL.monocytogenes,approximately25gofeach samplewashomogenizedwith225mLofhalf-concentrated Fraserbroth(FB;Merck,Darmstadt,Germany)inastomacher (240bpm;ITRmodel1204,series126;SãoPaulo,SP,Brazil)for 2mininaclassIIbiosafety cabinet(LabconcoPurifierClass IIb, Total Exhaust, model 36210-04,certified ISO 9002; Lab-concoCorporation,KansasCity,MO,USA).Thishomogenate wasthenincubatedat30◦Cfor24h.Analiquotof1mLwas transferredtotubescontainingFBsupplementedwithFraser selectivesupplementandincubatedat37◦Cfor48h.The cul-tureswere streakedontoplates containingtheListeriaagar Ottaviani&Agosti(ALOATM_{,Laborclin,Pinhais,PR,Brazil)and}

incubatedat37◦Cfor24h.20_{Afterward,3–5suspectcolonies}

(blue, diameter less than 3mm and with a regular white halo)wereselectedforconfirmation.TheconfirmationofL. monocytogenescoloniesinisolationmediawasbasedon sev-eralmethods,includingGramstainingandmeasurementof hemolyticactivityonsheepbloodagar(Columbiaagar supple-mentedwith5%defibrinatedhorseblood;HiMedia,SãoPaulo, SP,Brazil),thecarbohydrateutilizationpattern(0.5% manni-tol,0.5%rhamnoseand0.5%xylose),thecatalasereactionand tumblingmotility.21_{OneL.monocytogenesScottA(serotype4b;}

ATCC15313)positivecontrolandoneuninoculated-medium negativecontrolwereusedforeachsetofconcurrently ana-lyzedsamples.

Antigenic characterization was performed in the Labo-ratory of Bacterial Zoonoses (LABZOO/IOC/FIOCRUZ) using somatic and flagellar serotyping antisera produced in the same laboratory, asdescribed bySeeliger and Höhne.22 _All

strainsaredepositedintheCollectionofListeria(CLIST)from LABZOO andmaintainedin BHIbrothwith20%glycerolat −20◦_C.

MultiplexPCRconfirmationofisolates

MultiplexPCRwasperformedtoconfirmgenus,species, lin-eageandserotypes,andtheconditionsandthesetofprimers aresummarizedinTable1.TheDNAwasextractedusingthe DNeasy Blood& Tissuekit® _{(Qiagen,Hilden, Germany)}

fol-lowingthemanufacturer’sinstructions.ThePCRassaysina finalvolumeof50␮Lweredoneusingthefollowing:1× reac-tionbuffer,MgCl21.5mM,0.4mMofeachdNTP,10pmol/␮Lof

Table1–Primers,ampliconsizeandamplificationconditionsforthePCRassays.

Genetarget Sequence(5–3) Determinant Amplicon Amplification

conditions

Reference

23SrRNA F=GGGGAACCCACTATCTTTAGTC R=GGGCCTTTCCAGACCGCTTCA

GenusListeria 239bp 95◦C(1min),62◦C (1min),72◦C (1min) Hudsonetal.26 D1 F=CGATATTTTATCTACTTTGTCA R=TTGCTCCAAAGCAGGGCAT DivisionIorIII 214bp 95◦C(30s),56◦C (30s),72◦C(1min) Boruckiand Call27 D2 F=GCGGAGAAAGCTATCGCA R=TTGTTCAAACATAGGGCTA DivisionII 140bp 95◦C(30s),56◦C (30s),72◦C(1min) Boruckiand Call27 ORF2110 F=AGTGGACAATTGATTGGTGAA R=CATCCATCCCTTACTTTGGAC Serotype4b 597bp 95◦C(1min),60◦C (1min),72◦C (1min) Doumithetal.28 lmo0737 F=AGGGCTTCAAGGACTTACCC R=ACGATTTCTGCTTGCCATTC L.monocytogenes serovars1/2a, 1/2c,3aand3c 691bp 95◦C(1min),60◦C (1min), 72◦C(1min) Doumithetal.28 lmo1118 F=AGGGGTCTTAAATCCTGGAA R=CGGCTTGTTCGGCATACTTA L.monocytogenes serovars1/2cand 3c 906bp 95◦C(1min),60◦C (1min), 72◦C(1min) Doumithetal.28 ORF2819 F=AGCAAAATGCCAAAACTCGT R=CATCACTAAAGCCTCCCATTG L.monocytogenes serovars1/2b,3b, 4b,4d,and4e 471bp 95◦C(1min),60◦C (1min), 72◦C(1min) Doumithetal.28 Hly F=GCCTGCAAGTCCTAAGACGCCAATC R=CTTGCAACTGCTCTTTAGTAACAGC ListeriolysinO 706bp 95◦C(1min),62◦C (1min), 72◦C(1min) Hudsonetal.26

Hilden,Germany).StandardstrainsofL.monocytogenesATCC 19115(serotype4b),ATCC19111(serotype1/2a),ATCC19112 (serotype1/2c)andCDCF4976(serotype1/2b),wereusedas positivecontrols,andListeriainnocuaATCC12612wasusedas anegativecontrol.

Theamplifiedfragmentsweresubjectedto electrophore-sisona2%agarosegel(inTBEbuffer),stainedwithethidium bromidesolution (10mg/mL) (Sigma, SãoPaulo, Brazil)and visualizedwithaUVtransilluminatorcoupledwithadigital gelimagingsystem(KodakEDAS290).

Antimicrobialresistanceoftheisolates

Antimicrobial resistance was assessed by a disk diffusion assay accordingto CLSIguidelines15 _{using the breakpoints}

of Staphylococcus species resistance because no resistance criteria exist for Listeria susceptibility testing in the CLSI guidelines.23–25

Cellsweregrownat35◦Cfor24hintrypticsoybroth(TSB; HiMedia,São Paulo,SP,Brazil), suspendedinasaline solu-tion and dilutedto 0.5 points on the McFarland scale (ca. 108_{cfu).Thesuspensionofcellswasinoculatedin}

Mueller-Hinton (MH) agar (HiMedia, São Paulo, SP, Brazil) using a swab.Bacterialgrowthwasrecordedafter24hofincubation at35◦C.OneStaphylococcusaureus(ATCC33591)positive con-trolwasusedforeachsetofanalyzedsamples.Theisolates were tested against a panel of eightantimicrobial agents: penicillinG(PEN)(10IU),erythromycin(ERY)(15␮g), tetracy-cline(TET)(30␮g),oxacillin(OXA)(1␮g),cefoxitin(CEF)(30␮g), vancomycin (VCM) (30␮g), streptomycin (STR) (10␮g) and ciprofloxacin(CIP)(5␮g).Theantibioticdiscswerepurchased

from Laborclin(Pinhais, PR,Brazil).Thezonesofinhibition weremeasured(mm)at24h.

Results

and

discussion

PrevalenceofL.monocytogenesinvegetables

Inthiswork,wedetectedandidentifiedL.monocytogenesin rawandready-to-eatvegetablespurchasedatsupermarkets locatedinthecityofSalvador,Brazil,toshowtheexistingrisk toconsumers.

Ofthe132samplesanalyzed,L.monocytogeneswasisolated from four(3.03%)samples,ofwhichone(2.22%)camefrom rawvegetablesandthree(5.56%)camefromready-to-eat veg-etables(salads)(Table2).

Lettuce was the onlyraw vegetable contaminated byL. monocytogenes,whereastwotypesofmixedvegetablesalads presentedthemicroorganism.Nosampleoffrozenvegetables wascontaminatedbyL.monocytogenes.

The four obtained isolates recovered from the raw and ready-to-eat vegetable samples were identified as L. mono-cytogenes serotype 4b by both the conventional antigenic characterization and molecular serotyping methodologies. MultiplexPCRtargetingthelisteriolysinOgenes(hly)ofthe

L.monocytogenesrecoveredfromthevegetablesamples con-firmedthevirulencepotentialoftheisolates.

ThepresenceofL.monocytogenesinmanytypesofrawand ready-to-eatvegetablesintendedforhumanconsumptionhas beenclearlydemonstratedinmanycountries.InBrazil,the incidenceofL.monocytogenesinvegetableshasbeenreported inmanystudiesbyanumberofresearchers.Theresultsfound inthepresentworkdemonstratedhigherlevelsofdetectionof

Table2–OccurrenceofL.monocytogenesinraw,frozenandready-to-eatvegetables.

Vegetablegroup Samples Positivesamples Origin

n n %

Raw 45 1 2.22 Lettuce(1sample)

Frozen 33 ND 0.00

Ready-to-eat(salads) 54 3 5.56 Lettuce,beetsandpurple

cabbage(1sample)

Lettuce,beets,purplecabbage andwhitecabbage(2samples)

Total 132 4 3.03%

ND,notdetected.

L.monocytogenesinsaladsthanthelevelspreviouslyreported byFroderetal.(0.55%),9_{Oliveiraetal.(3.7%),}10_{andSant’ana}

etal.(3.1%).11

Incountries other than Brazil,variousresultshave also beenreportedfortheoccurrenceofL.monocytogenesin veg-etables.InSantiago,Chile,thebacteriumwasisolatedfrom 10.2%ofsalad samples29_{; inMalaysia, from 22.5%ofsalad}

samples30_{; inSpain,from 4.18%ofvegetable samples}31_{; in}

Marmara,Turkey,from13.6%offreshvegetablesamples32_and

inGermany,fromonlyfoursamplesof1001investigated veg-etablesamples.33

Work performed by Moreno et al.31 _{in Valencia, Spain,}

showedthatninelettuce sampleswerecontaminatedbyL. monocytogenes,withcountsbetween2.85and3.55log10viable

cells/goffood.Additionally,onlyonepositivesample,found inadishcalleda‘FourSeasonsSalad,’yieldedahighnumber ofviablecells,withavalueof4.35log10cells/goffood.

The presenceof L. monocytogenes in vegetables,verified inthe present study and in other studies reportedby var-iousauthors, is cause for concern,as listeriosis cases are increasing at the global level, in many cases due to the cross-contamination ofprocessed foods.31 _{Therefore, Good}

AgriculturalPracticesandGoodManufacturingPracticesmust beaddressedtoguaranteethesafetyoffoodforconsumers. Antimicrobialresistance

Inthecurrentstudy,allL.monocytogenesisolateswere suscep-tibletoerythromycin(ERY),oxacillin (OXA),cefoxitin(CEF),

vancomycin(VCM),streptomycin(STR)andciprofloxacin(CIP)

(Table3).

Two L. monocytogenes isolates from ready-to-eat vegeta-blesexhibitedresistancetopenicillinG(PEN)andtetracycline (TET).

Incontrast,Kovacevicetal.34_{foundthatL.monocytogenes}

recoveredfromready-to-eatfishpossessedreduced suscep-tibility to ciprofloxacinand two ofthe three clonal1/2bL. monocytogenesisolateswere resistanttostreptomycin. How-ever,liketheresultsofpresentstudy,theauthorsobservedno resistancetoerythromycin.

StudiesperformedbyDavisandJackson25_{withstrainsof}

L.monocytogenesrecoveredfromdiverse origins(i.e.clinical, animal, food,andenvironmental)showedsimilarresultsto thoseinthepresentworkwithregardtoCIPandTET resis-tance. Also,Troxleretal.35 _{inGermanytested87strainsof}

Listeriaspp.(19L.monocytogenesfromhumanandsheepand avianorigin),predominantlyisolatedintheUSAand differ-entEuropeancountries,andgroupedL.monocytogenesandL. welshimeriasnaturallysensitivetoCIP.

However,accordingtoKovacevicetal.34_,theextentof

com-parison betweenstudies ishamperedbydifferences inthe methodsusedtoverifyresistance.

In general, antibiotic therapy is required for the treat-ment of listeriosis infections, with clinicians commonly using ampicillin in combination with gentamycin or using trimethoprim-sulfamethoxazole.36_{Inthepresentstudy,}

resis-tancetopenicillinG,acommonlyemployedtherapeuticagent, wasobservedforoneisolateofL.monocytogenes.

Table3–Resistance/susceptibilityofL.monocytogenesisolatedfromvegetables.

Antimicrobial Breakpoints(mm) Isolates(number)

Resistant Intermediate Susceptible Resistant Intermediate Susceptible

Cefoxitin ≤19 – ≥20 ND – 4 Ciprofloxacin ≤15 16–20 ≥21 ND ND 4 Erythromycin ≤13 14–22 ≥22 ND ND 4 Streptomycin ≤19 – ≥23 ND – 4 Oxacillin ≤17 – ≥18 ND – 4 PenicillinG ≤28 – ≥29 1 – 3 Tetracycline ≤14 15–18 ≥19 1 ND 3 Vancomycin – – ≥15 ND – 4 ND,notdetected.

Accordingto Kovacevicet al.,34 _{onlythreeisolates ofL.}

monocytogenesrecoveredfrom4668clinicalsamplesinFrance wereresistanttostreptomycin(STR).Twoisolateswere resis-tanttolower concentrations(4and 6mg/mL),whereas one exhibitedresistanceat256mg/mL.17_{Regardingfoodand}

envi-ronmentalstrains, none ofthe 49L. monocytogenesisolates fromfoodand environmentalsamplestested inthe U.S.in 2009showedresistanceto STR(1mg/mL).In 2010,another studyperformedinU.S.withL.monocytogenesisolatedfrom catfish filets and their respective processingenvironments reported reduced susceptibility to STR(10mg) in 2% (2/80) oftheisolates).23_{Hasbeenreportedthatsublethalexposure}

totriclosan,abroad-spectrumbiocideusedintoavarietyof commercialproducts,promotesresistancetovarious amino-glycosides,includinggentamycin,kanamycin,streptomycin and tobramycin.34 _{In addition, resistanceto low and high}

concentrations of the antibiotic has been suggested to be associated with possible ribosomal mutations and/or the productionof6-N-streptomycinadenylyltransferase,encoded by the aad6 gene.17,34 _{These finds is a cause for concern,}

consideringthatclinicianstypicallyuseaminopenicillins(e.g., ampicillinoramoxicillin)incombinationwithan aminogly-coside, such as gentamicin, for the treatment of invasive infections.13,36

Similartotheresultspresentedinourstudy,Gómezetal.13

found that one strain (0.5%) ofL. monocytogenes recovered fromastainlesssteelsurfaceinSpanishindustrywas resis-tanttotetracyclineand eightstrains(3.9%) recoveredfrom meatproductsshowedreducedsusceptibilitytopenicillinG. According to the authors, the effectiveness of tetracycline diminishedinthelastdecadesowingtothewidespread exist-enceofresistancegenes,probablybecauseoftheextensive andprolongeduseoftheseantimicrobialsinhumanmedicine andasgrowthpromotersinanimals. Inaddition, regarding fluoroquinolones,intermediatesusceptibilitytociprofloxacin was found in the mentioned work for one strain of the pathogenic species. Theauthors concluded that all Listeria

strainswerehighlysensitivetothepreferredantibioticsused totreatlisteriosis,althoughspecialmentionwasmadeofthe reducedsusceptibilityofeightstrainsofL.monocytogenes(3.9%) andofsevenstrainsofL.innocua(5.4%)topenicillinG.

TheisolatesofL.monocytogenesinvestigatedinthepresent studydidnotshowmulti-resistance.

WorkperformedbyKorsaketal.37 _{on471samplesfrom}

different typesof food and food-related sources in Poland demonstrated that one L. monocytogenes strain, isolated in 2005fromicedgreenbeans,wasresistanttotetracycline(MIC 16␮g/ml).Chenetal.,23_{whoinvestigatedtensamplesoffoods}

commercializedinChina,reportedsimilarresults,with2.7% oftheisolatesresistanttotetracyclineand8.1%resistantto penicillin.Theseresultsshowthatantimicrobialresistancein

L.monocytogenesoccursatalowprevalence.

Conclusion

Thepresenceof L.monocytogenesin freshand ready-to-eat vegetablesandtheresistanceofisolatestopenicillinG,a com-monlyemployedtherapeuticagent,asverifiedinthepresent study,arecauseforconcern,aslisteriosiscasesareincreasing

atthegloballevel.Inaddition, thepresenceofserotype4b, morecommonlyassociatedwithoutbreaks,couldbea poten-tialhealthhazardforconsumers.

Conflicts

of

interest

Thecontentofthisreportsolelyreflectstheopinionsofthe authors,andwereportnoconflictsofinterest.

Acknowledgements

This research was supported in part by Coordenac¸ão de Aperfeic¸oamento de Pessoalde Nível Superior(CAPES). We wishtothankstudentsRodrigodeCastroLisboaandVanessa deSouzaRodrigues,from theLaboratóriodeZoonoses Bac-terianas(LABZOO/IOC/FIOCRUZ),RiodeJaneiro,RJ,Brazil,for helpinPCRassay.

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