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Food
Microbiology
Occurrence
and
antimicrobial
resistance
patterns
of
Listeria
monocytogenes
isolated
from
vegetables
Vanessa
de
Vasconcelos
Byrne
a,
Ernesto
Hofer
b,
Deyse
Christina
Vallim
b,
Rogeria
Comastri
de
Castro
Almeida
c,∗aPharmacyFaculty,FederalUniversityofBahia,RuaBarãodeGeremoabo,Salvador,BA,Brazil
bLaboratoryofBacterialZoonoses,OswaldoCruzInstitute,PavilhãoRochaLima,RiodeJaneiro,RJ,Brazil cNutritionSchool,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:[email protected](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/parsley10;
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.20Afterward,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.21OneL.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 finalvolumeof50Lweredoneusingthefollowing: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. 108cfu).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)(15g), tetracy-cline(TET)(30g),oxacillin(OXA)(1g),cefoxitin(CEF)(30g), vancomycin (VCM) (30g), streptomycin (STR) (10g) and ciprofloxacin(CIP)(5g).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%),9Oliveiraetal.(3.7%),10andSant’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 samples31; in
Marmara,Turkey,from13.6%offreshvegetablesamples32and
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.34foundthatL.monocytogenes
recoveredfromready-to-eatfishpossessedreduced suscep-tibility to ciprofloxacinand two ofthe three clonal1/2bL. monocytogenesisolateswere resistanttostreptomycin. How-ever,liketheresultsofpresentstudy,theauthorsobservedno resistancetoerythromycin.
StudiesperformedbyDavisandJackson25withstrainsof
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.36Inthepresentstudy,
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.17Regardingfoodand
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).23Hasbeenreportedthatsublethalexposure
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 16g/ml).Chenetal.,23whoinvestigatedtensamplesoffoods
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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