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Czech J. Food Sci. Vol. 31, 2013, No. 1: 2732

Antimicrobial Resistance of Lactobacilli Isolated from Food
M DUKOV
1
and R KARPKOV
1,2
1
Department of Milk Hygiene and Technology, Faculty of Veterinary Hygiene and Ecology,
University of Veterinary and Pharmaceutical Sciences Brno, Brno, Czech Republic;
2
Veterinary Research Institute, Brno, Czech Republic
Abstract
DusovXM.,KvvIsovXR.(2013): Antimicrobial resistance of lactobacilli isolated from food.CzechJ.
FoodSci.,31:2732.
BacteriaofthegenusLactobacillusareofgreatbenefitinmanyareasoflife.Theyarewidelyusedinfoodindustry,in
particularaspartofstarterculturesforfermenteddairyandmeatproducts,butalsoinhumanandveterinarymedicine
asprobiotics.Theincreasingglobalproblemofantimicrobialresistancemayalsoinvolvelacticacidbacteriabecauseof
thepossibleriskofresistancegenestransfer.Wedeterminedtheantimicrobialsusceptibilityoflactobacilliisolatedfrom
food.Ninetyfacultativelyheterofermentativelactobacilliisolatedfromretaildairyandmeatproductsweretested.The
resistancetoantimicrobialswasscreenedbythediskdiffusionmethodandtheminimuminhibitoryconcentrations
weredeterminedbythebrothmicrodilutionmethod.Fifteenstrains(17)wereresistanttoatleastoneantimicrobial
agentandonestrainwasmultiresistant.
Keywords:Lactobacillusspp.,diskdiffusionmethod,brothmicrodilutionmethod,minimuminhibitoryconcentration
(MIC),susceptibilitytoantibiotics,dairyproducts,meatproducts
Bacterialresistancetoantimicrobialagentsisa
majorglobalpublichealthproblem,aectingnot
onlyhumanandveterinarymedicine(Arrovet
al.2008)butalsofoodproduction.Tefoodchain
isbecomingapossiblewayofdisseminationofan-
tibioticresistanceamongbacterialpopulationsof
animalsandhumans(Wi::v2000).Manyspeciesof
lactobacilli,previouslygenerallyrecognisedassafe
(GRAS),maybecomevectorsofantibioticresistance
genes.Tesebacteriaareusuallyconsumedinhigh
quantitiesandclosecontactwithotherbacteriainthe
humangastrointestinaltractprovidesperfectcondi-
tionsforhorizontaltransferofconjugativeplasmids
andtransposonswithgenesencodingresistance
toantimicrobialagents(M:nuv&Sion2005,
Jconsvet al.2007,Arrovet al.2008,Nzet
al.2011).Teabsenceoftheacquiredantimicrobial
resistancehasbecomeanimportantcriterionfor
evaluatingthesafetyoflactobacilliusedasstarter
culturesorprobiotics(Mvvnorvvet al.2008).
Althoughtheminimuminhibitoryconcentrations
(MIC)aredefinedforclinicallyimportantmicroor-
ganisms,internationallyvalidMICsforlactobacilli
havenotbeendeterminedyet.Todistinguishthe
strainswiththeacquiredantimicrobialresistance
fromthesusceptibleones,thePanelonAdditives
andProductsorSubstancesusedinAnimalFeed
(FEEDAP)oftheEuropeanFoodSafetyAuthority
(EFSA) defined the microbiological breakpoints
usedintheassessmentofbacterialresistanceto
antibioticsofhumanorveterinaryimportance.The
breakpointdatawerederivedfromthepublished
bodyofresearchandfromnationalandEuropean
monitoringprogrammes(EFSA2008).
Following a request from EFSA, the Panel on
BiologicalHazards(BIOHAZ)wasaskedtoreview
the list of the Qualified Presumption of Safety
(QPS)microorganismsandtoupdatetheantimi-
crobialresistancecriteriausedtojudgethesafety
of foodifeed use microorganisms. If a defined
SupportedbytheMinistryofEducation,YouthandSportsoftheCzechRepublic,ProjectNo.MSM6215712402.
28
Vol. 31, 2013, No. 1: 2732 Czech J. Food Sci.
taxonomicunitdoesnotraisesafetyconcernsor
ifanypossibleconcernscanbeexcluded,theQPS
approachcanbeappliedandthetaxonomicunit
canberecommendedtobeincludedintheQPS
list.TheQPSlistisreviewedandupdatedannually
bythePanelonBiologicalHazards.ThisQPSlist
includes36speciesofLactobacillus(EFSA2011).
Teaimofthisstudywastodeterminetheantimi-
crobialsusceptibilityoflactobacilliisolatedfromfood.
MATERIAL AND METHODS
Bacterial strains.Thesusceptibilitytoantimi-
crobial agents was monitored in 90facultatively
heterofermentativelactobacilliisolates.Theyorigi-
natedfrom68foodsamplescollectedintheCzech
Republicfromretailmeatproducts(n=11)and
dairy products (n = 57), and seven strains were
obtainedfromtheCzechCollectionofMicroorgan-
isms(CCM,Brno,CzechRepublic).Theisolates
werecultivatedonMRSagar(Oxoid,Basingstoke,
UK)at30Cfor4872hmicroaerophilically.Cell
morphologyinthesuspectcolonieswasstudied
microscopically(Gramstaining),whilethecultures
werealsotestedforthepresenceofcatalaseand
oxidase ( JK Trading, Prague, Czech Republic).
Genotypeconfirmationoflactobacilliisolateswas
performedwiththeuseofpolymerasechainreac-
tion (PCR) with genus-specific primers LbLMA
1-revandR16-1(Dunvvv:et al.2002).DNAwas
extracted from bacterial cultures by the boiling
procedurewithChelex

100(Bio-Rad,Hercules,
USA).Amplificationtookplaceina PTC-200ther-
mocycler(MJResearch,Watertown,USA).Atwo-
stepmultiplexPCRmethodwasusedforspecies
identificationintheselectedisolatesandsubse-
quently,followingtheclassificationoflactobacilli
into groups, PCR with species-specific primers
wasalsousedbasedonthedetectionofnucleotide
sequencesofthe16S-23SrRNAintergenicspacer
regionandadjacent23SrRNAgenedifferingfor
individual species of lactobacilli (Bvv:nivv &
Enviicn1998,Wvo&Tirris1999,Sooet
al. 2000, Wi:vv et al. 2000). Amplicons were
detected by agarose gel electrophoresis, stained
withethidiumbromide,andvisualisedusingaUV
transilluminator ( = 305 nm). The distribution
of90lactobacilliisolatesbyspeciesandoriginis
summarisedinTable1.
Antimicrobial susceptibility testing.Theresist-
ance was determined using two methods, broth
microdilution and disk diffusion. Inocula of the
isolates tested were prepared in a sterile saline
bysuspendingthecoloniesfromMRSagarplates
(Oxoid, Basingstoke, UK) incubated at 30C for
24hundermicroaerophilicconditions.
Tebrothmicrodilutionmethod(Trios,Prague,
CzechRepublic)wastherstmethodofchoice.Te
followingantimicrobialsweretested(inMRSbroth):
ampicillin(AMP,0.0152mgil),trimethoprim(TRI,
0.2532mgil),gentamicin(GEN,1128 mgil),chlo-
ramphenicol(CMP,0.564mgil),oxacillin(OXA,
0.2532mgil),streptomycin(STR,2256 mgil),
tetracycline(TET,2256mgil),erythromycin(ERY,
0.0314mgil),andvancomycin(VAN,2256mgil).
Microtiterplatesinoculatedwiththebacterialsus-
pension with a McFarland standard turbidity of
0.5wereincubatedat30Cfor24hundermicro-
aerophilic conditions. The minimum inhibitory
concentrationswereestablished.Testrainswere
classiedassusceptibleorresistantbasedonthe
minimum inhibitory concentration required to
inhibitthegrowthof90oforganisms(MIC
90
).
Bacterialsuspensionswithaturbidityequivalent
toaMcFarlandstandardof1wereswabbedevenly
ontoMRSagarplateswithasterilecottonswab
for the disk diffusion method. Antibiotic disks
containing 10 g ampicillin, 5 g trimethoprim,
10 g gentamicin, 30 g chloramphenicol, 5 g
Table1.Distributionof90lactobacilliisolatesbyspeciesandorigin
Species No.ofisolates
Originofisolates
CCM meatproducts BIOmilkproducts cheeses
L. casei 2 2
L. curvatus 18 2 1 14 1
L. paracasei 16 - 8 8
L. plantarum 10 1 5 4
L. rhamnosus 38 2 35 1
L. sakei 6 6 -
Total 90 7 12 61 10
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Czech J. Food Sci. Vol. 31, 2013, No. 1: 2732
oxacillin,10gstreptomycin,30gtetracycline,
15gerythromycin,and30gvancomycin(Oxoid,
Basingstoke,UK)wereplacedonMRSagarplates.
Theplateswereincubatedat30Cfor24 hunder
microaerophilicconditionsandthentheinhibition
zonediameters(IZ
D
),includingthediameterofthe
disk,weremeasured.
Statistical analysis.Tothecorrelationbetween
thebrothmicrodilutionmethod(MICsingiml)
andthediskdiffusionmethod(IZ
D
sinmm),lo-
gisticorlinear(forAMP)regressionanalysiswas
appliedafterlogarithmicconversion(log
2
)ofthe
MICs.Overallstatisticalsignificancewasassessed
using
2
-testforlinearregressionand F-testfor
logistic regression. P values of < 0.05 were con-
sideredstatisticallysignificant.
RESULTS AND DISCUSSION
Teminimuminhibitoryconcentrationsof90fac-
ultativelyheterofermentativelactobacilliandMIC
90

rangesareshowninTable2.BasedontheMIC
90
,
15isolates(17)weredeterminedasresistanttoat
leastoneantibiotic.Tesestrainsandtheirresist-
ancephenotypesarelistedinTable3.Lactobacillus
plantarumA54isolatedfromfermentedsausage
wasresistanttoveantibioticsoffourantimicrobial
groups.Teresistancetogentamicinwasfoundtobe
themostfrequent(in7.8ofisolates).Divisv
andWio(2003)andNzet al.(2011)havealso
shownahighresistancetogentamicin.Frequent
resistanceoflactobacillitoaminoglycosideshas
beenreportedbyK:iet al.(2001).
In this study, the resistance to trimethoprim
andvancomycinwasnotevaluatedbythebroth
microdilutionmethod.TheMIC
90
valuesforthese
antibiotics were above the range determined in
themicrodilutionplateused.
BasedonthestatisticalanalysisoftheMICre-
sults,thediscriminatoryinhibitionzonesIZ
D
(in
mm)forthediskdiffusionmethodwereestablished
tocategorisethelactobacilliisolatesassuscepti-
ble(IZ
isolate
>IZ
D
)orresistant(IZ
isolate
IZ
D
)to
antimicrobials. The rate of complete agreement
betweenthetwomethodsrangedfrom63.3to
97.8.Thesensitivityofthediskdiffusionmethod
fortheassessmentofresistantisolatesrangedbe-
tween98.1and100anditsspecificity(ability
of correct detection of susceptible isolates) was
55.097.7,exceptforvancomycinwithonly2.9.
This low specificity was caused by the value of
MIC
90
250mgiland98.9ofisolatesshowedan
inhibitionzoneof6mm.TheIZ
D
,percentageof
theresistantisolatesandratesofcompleteagree-
mentbetweenthetwoantimicrobialsusceptibility
testingmethodsarepresentedinTable4.
According to the discriminatory inhibition
zones, a high percentage of isolates resistant to
vancomycin and trimethoprim was determined.
Theresistanceoffacultativelyheterofermentative
lactobacillitovancomycinisintrinsic,duetothe
presenceofo-Ala-o-lactateintheirpeptidogly-
caninsteadofthenormaldipeptideo-Ala-o-Ala
(Arrovet al.2008).Althoughrelativelyrarein
Gram-positivebacteria,theacquiredtrimethoprim
resistancehasbeenoccasionallydetected(Youo
et al. 1987, Cnvvv:ivv & Couvvii 1997).
Thedataavailable(Kovnovet al. 2007)indicate
that within the species of lactobacilli, the range
of the apparent trimethoprim resistance can be
widewithnoclearbreakpointvalues.Therefore,
Table2.Minimuminhibitoryconcentratio(MIC)rangesdeterminedbythemicrodilutionmethod
Antimicrobial MICrange(mgil) MIC
90
(mgil) No.ofresistantisolates Breakpoints(mgil)
AMP <0.0150.5 0.5 0(0) 4
TRI 0.25>32 >32 ND ND
GEN <164 16 7(7.8) 16
CMP <0.54 2 3(3.3) 4
OXA 232 16 3(3.3) ND
STR 4256 128 4(4.4) 64
TET <232 8 3(3.3) 8
ERY <0.0310.5 0.25 2(2.2) 1
VAN 16>256 >256 ND ND
MIC
90
numberofresistantisolatesaccordingtoMIC
90
andEFSAbreakpoints,AMPampicillin,TRItrimethoprim,
GENgentamicin,CMPchloramphenicol,OXAoxacillin,STRstreptomycin,TETtetracycline,ERYerythromycin,
VANvancomycin,ND notdetermined
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Vol. 31, 2013, No. 1: 2732 Czech J. Food Sci.
the MIC testing of trimethoprim for lactic acid
bacteriawasnotconsideredrelevant(EFSA2008).
Theresultsoftrimethoprimsusceptibilitytest-
ingmaybedistortedbyreadingat80inhibition
ofthegrowth(brothmicrodilutionmethod)and
ignoring the slight growth within the inhibition
zones (disk diffusion method). The difficulty in
readingat80inhibitionofgrowthhasalsobeen
reportedbyMvvnorvvet al.(2008).
The resistance to chloramphenicol was 3.3,
similarresultshavebeenreportedalsobyK:i
et al. (2001) and Arrov et al. (2008). Testing
forchloramphenicolresistancewouldefficiently
cover for the hazard of acquiring the resistance
to linezolid since non-mutational resistance to
linezolid is encoded by the cfr gene, which also
conferstheresistancetochloramphenicol(Ton
et al.2007,Aviset al.2008,EFSA2008).
Table3.ResistantisolatesaccordingtoMIC
90
andresistancephenotypes
Strain Species Originofisolate Resistancephenotype
A54 L. plantarum meatproduct GEN,CMP,STR,TET,ERY
C16 L. plantarum meatproduct GEN
BIOI16 L. plantarum BIOmilkproduct TET
CCM7039T L. plantarum collectionstrain CMP,ERY
D16 L. sakei meatproduct TET
MI9 L. sakei meatproduct OXA
MI13 L. sakei meatproduct OXA
MI19 L. sakei meatproduct OXA
US27 L. curvatus cheese GEN, STR
BIOIII67 L. curvatus BIOmilkproduct STR
BIOIII70 L. curvatus BIOmilkproduct STR
CCM7558T L. curvatus collectionstrain GEN
BIOII65 L. paracasei BIOmilkproduct GEN
BIOIII53 L. paracasei BIOmilkproduct GEN
CCM7088T L. casei collectionstrain GEN,CMP
inbold antibioticsbelongingtothesamegroup(aminoglycosides),AMPampicillin,TRItrimethoprim,GENgentamicin,
CMPchloramphenicol,OXAoxacillin,STRstreptomycin,TETtetracycline,ERYerythromycin,VANvancomycin
Table4.Inhibitionzonesfornineantimicrobials,percentageofresistant Lactobacillusisolates,andsensitivityand
specicityofthemethod
Antimicrobial
IZ
D

(mm)
Resistantisolates
()
Completeagreement
betweenBMandDD

()
DDsensitivity
a
()
DDspecity
()
AMP 19 22.2 77.8 77.8
TRI 8 87.8 90.0 100.0 55.0
GEN 8 76.7 82.2 98.2 57.1
CMP 27 24.4 78.9 100.0 78.2
OXA 8 5.6 97.8 100.0 97.7
STR 7 62.2 95.6 100.0 89.5
TET 22 11.1 92.2 100.0 92.0
ERY 25 8.9 93.3 100.0 93.2
VAN 6 98.9 63.3 100.0 2.9
AMPampicillin,TRItrimethoprim,GENgentamicin,CMPchloramphenicol,OXAoxacillin,STRstreptomycin,
TETtetracycline,ERYerythromycin,VANvancomycin,IZ
D
dicriminatoryinhibitionzone,BMbrothmicrodilution
method,DDdiskdiusionmethod
a
100.0allresistantisolates(basedontheMIC)weredeterminedasresistantbasedontheIZ
31
Czech J. Food Sci. Vol. 31, 2013, No. 1: 2732
Acrucialfactorforantimicrobialtestingoflacto-
bacilliistheselectionofsuitablecultivationmedium.
Lactobacillihavespecicnutritionalandatmospheric
requirementsfortheirgrowth,thereforestandardised
susceptibilitytestmediasuchasMueller-Hinton
brothandIso-Sensitest(IST)brothwerenotused.
Cnv:vviset al.(2001)andDvioooet al.(2005)
usedMRSmediumintheirstudies.Ocet al.
(2006)evaluatedMRSagarasasuitablemediumto
studyantimicrobialsusceptibilityofmicroaerophilic
oranaerobiclactobacilli.However,thereareindica-
tionsthatthemediumMRSmayexhibitantagonistic
eectswithsupplementalantimicrobialsinsuscepti-
bilitytesting(Huvset al.2002).Kivvet al.(2005)
developedlacticacidbacteriumsusceptibilitytest
medium(LSM),amixedformulationofISTbroth
(90viv)andMRSbroth(10viv).Tismedium
wasusedbyHuvset al.(2010)forantimicrobial
susceptibilitytestingofnon-enterococcallacticacid
bacteria(NELAB)andbidobacteriaineightEuro-
peancountriesandthisstudyhasfurthervalidated
thestandarduseofLSMandformedthebasisfor
theISO10932iIDF223from2010(standardfor
susceptibilitytestingofNELABandbidobacteria).
Ourstudyhadbegunbeforethepublicationofthe
InternationalStandard,sotheappliedmethodis
dierentfromtheISO10932:2010.
For the purpose of distinguishing the strains
harbouringacquiredantimicrobialresistancefrom
susceptiblestrains,theFEEDAPPaneldefinesthe
microbiologicalbreakpoints(orepidemiologicalor
cut-offvalues).Thesebreakpoints(Table2)have
beensetbystudyingthedistributionofMICsof
antimicrobialsinbacterialpopulationsbelonging
toasingletaxonomicalunit.Thedatausedforthe
definition of microbiological breakpoints have
beenderivedfromthepublishedbodyofresearch
andfromnationalandEuropeanmonitoringpro-
grammes.OurvaluesofMICs
90
wereobtainedby
testing90isolatesonly,sotheymaybedifferent
fromtheFEEDAPbreakpoints.
The isolates with MICs above the breakpoint
requirefurtherinvestigationtomakethedistinc-
tionbetweentheintrinsicandacquiredresistance
(throughthegainofexogenousDNAorthemutation
ofindigenousgenes).Thepresenceoftheacquired
antimicrobialresistancegenesonmobileelements
posesthehighestriskofantimicrobialresistance
dissemination.TheFEEDAPPanelconsidersthat
thestrainsofbacteriacarryingtheacquiredresist-
ancetoantimicrobialsshouldnotbeusedasfeed
additives,unlessitcanbedemonstratedthatitis
aresultofchromosomalmutation(EFSA2008).
CONCLUSIONS
Testudyresultsshowthatlactobacillimaybere-
sistanttoantimicrobialagents.Resistantstrainswere
detectedinallfoodcategoriesexamined.Although
considerednon-pathogenic,lactobacillicommonly
occurinlargenumbersinfoods,especiallyfermented
foods.Lactobacillienterintohumangastro-intestinal
tract in large numbers where they interact with
theintestinalmicroora.Whenabacterialstrain
demonstratestheresistancetoantimicrobialsby
phenotypicmethods,itisdesirabletomonitorthe
molecularbasisofthisresistanceandtodistinguish
whetheritisintrinsicoracquired.Testrainswith
themobilegeneticelementscarryinggenesencod-
ingresistanceshouldnotbeusedasstartercultures.
Teymightcontributenegativelytoanuncontrolled
horizontalspreadoftheresistancetoantibiotics
throughoutthehumanfoodchain.
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ReceivedforpublicationMarch9,2012
AcceptedaftercorrectionsMay15,2012
Corresponding author:
Mgr. Mv: DusovX, Ph.D., Veterinrn a farmaceutick universita Brno, Fakulta veterinrn hygieny a ekologie,
stavhygienyatechnologiemleka,Palackehot.1i3,61242Brno,eskrepublika,E-mail:duskovam@vfu.cz