GeneticcharacterisationofNDM-1andNDM-5 -producingEnterobacteralesfromretailchicken meatinEgypt
Sir,
Amongtheacquiredcarbapenemasesthathavebeenidentified worldwide,NDMtypesareoneofthemostclinicallysignificant sincetheyhydrolyzealmostallβ-lactamsatahighlevel.Theyhave been found in all continents and almost all clinically-relevant Gram-negativepathogens. NDMproducers areoften multidrug-resistant,havingaccumulateda significant number of acquired resistancegenes.TheblaNDM-1genewasfirstdetectedinKlebsiella
pneumoniaefromapatientwhopreviouslytraveledinNewDelhi, India,and the blaNDM-5 gene was identified from a
multidrug-resistantEscherichiacoliintheUK.NDM-5differsfromNDM-1by twoaminoacidsubstitutionsatpositions 88(Val3Leu)and154 (Met3Leu)involvedinincreasedcarbapenemaseactivity.Although NDM-producingEnterobacteralesareknowntobewidely dissem-inated among humans, animals, and the environment, limited studiesareavailableregardingtheiroccurrenceanddissemination inthefoodchain.
In this study, a total of 145 retail chicken carcasses were collected between June and December 2017 from different supermarkets, poultry slaughterhouses, and butcher shops in Egypt.Thesamples(neckskin)werehomogenizedandenrichedin a sterilebuffer peptone waterfor 24 hat 37 C with shaking, followedbydirect platingona selectiveplate(MacConkeyagar supplemented with 2
m
g/mL of meropenem (Sigma-Aldrich, Tokyo,Japan). Coloniesofdifferentmorphology, size,and color fromeachplatewereselectedandpurifiedforDNAextractionand PCRscreening.Atotalof155meropenem-resistantisolateswere obtainedand screenedbyPCRforthecarbapenemase-encoding genes blaVIM, blaIMP, blaKPC, blaNDM, blaOXA-48-like, blaBIC, blaAIM,blaSPM,blaDIM,blaGIM,andblaSIM.AsingleK.pneumoniaeKp38anda
singleE.coliEc34isolatewerefoundtoproducetheNDM-1and NDM-5enzymes,respectively.Bothisolatespossessedamultidrug resistance phenotype. MICs were determined using the broth microdilutiontechnique(Table1).ResultsshowedthatE.coliEC34 wasresistanttoampicillin,amoxicillin-clavulanicacid,aztreonam, cefotaxime,ceftazidime,ceftriaxone,doripenem,imipenem, mer-openem, nalidixic acid, ciprofloxacin, kanamycin, gentamicin, chloramphenicol,andtetracycline,andremainedsusceptibleonly toamikacin,colistin,tigecycline, andfosfomycin.K.pneumoniae isolateKp38was resistanttoallantimicrobialstested,except to gentamicin,tigecycline,and colistin.Phenotypiccarbapenemase productionwas confirmed by using theCarba NP test and the recentlydevelopedNitroSpeedCarbaNPtest[1,8].PCRandDNA sequencingwereusedtodetectESBLs,plasmid-mediated quino-lone-resistancedeterminants,and16SrRNAmethylasesconferring resistancetoallaminoglycosides.TheE.coliEc34isolatecarried
blaOXA-1,blaTEM-1,blaCTX-M-3,andaac(6)-Ib-cr,whiletheK.
pneumo-niaeKp38isolateharboredtheblaSHV-1,blaCTX-M-15,andaac(6)-Ib-cr
genes. In both isolates, the ISAba125 insertion sequence was identified immediately upstream of the blaNDM-1 and blaNDM-5
genes,whilethebleomycinresistancegenebleMBLwasidentified
downstreamofit.Thosegeneticstructurescorrespondedtothose usuallyfoundintheclosevicinityofmostoftheblaNDM-1genesin
Enterobacterales[2].
Conjugationandtransformationexperimentswereperformed toevaluatethetransferabilityoftheblaNDM-1andblaNDM-5genes
[3,9].Theazide-resistantE.colistrainJ53wasusedastherecipient andtransconjugantswereselectedonLBagarplatessupplemented withampicillin(100
m
g/mL)andsodiumazide(150m
g/mL).TheblaNDM-5genewassuccessfully transferredintotheE.colistrain
J53,indicatingthatitwaslocatedonatransferableplasmid.Onthe opposite, and despite repeated attempts, conjugation failed to transfertheblaNDM-1genefromK.pneumoniaeKp38toE.coliJ53.
Consequently, the plasmid extraction was performed from K. pneumoniaeKp38 and electroporatedintoE.coli DH10B. Trans-formantsweresuccessfullyselectedonLBagarplatescontaining ampicillin(100
m
g/mL)[9].TheE.colitransconjugantproducing NDM-5showed resistancetocarbapenems andbroad-spectrum cephalosporinsbutremainedsusceptibletoaminoglycosidesand aztreonam,indicatingthatthecorrespondingplasmiddidneither harboranESBLnoranaminoglycosideresistancedeterminant.The E.colitransformantproducingNDM-1showedresistanceto broad-spectrum cephalosporins, carbapenems, and aztreonam, but remainedsusceptibletoaminoglycosides,showingthatanESBL encoding gene was co-located onto the same plasmid as theblaNDM-1genewas.PCRandsequencingoftheampliconconfirmed
thattheblaNDM-1-positiveplasmidadditionallycarriedthebla
CTX-M-15gene.
MICsofimipenemandmeropenemfortheNDM-5-and NDM-1-producingE.colitransconjugantsandtransformantswere4–8 and4
m
g/mL,respectively,similarlytowhatiscommonlyobserved [4,5].ThoseMICvaluesweremuchlowerthanthosefoundforthe respective parental strains (Table 1), showing that a series of additional resistance mechanisms (permeability defects, efflux overexpression)alsooccurredinthoselatterclinicalisolates. PCR-based replicon typing(PBRT) methodwas used to identifythe incompatibility group of those NDM-encoding plasmids. TheblaNDM-5-positiveplasmidthatwasca.48kbin-sizebelongedto
the IncX3incompatibility group,whereas the blaNDM-1-positive
plasmidthatwasca.100kbin-sizebelongedtotheIncRgroup. Multilocussequencetyping(MLST)analysisrevealedthatE.coli EC34belongedtotheST648sequencetype,whileK.pneumoniae Kp38belongedtoST147.NDM-5-producingST648E.colihasbeen reportedinIndia,UK,andAustralia[6].NDM-1-producingST147K. pneumoniae clone hasbeen reportedin several Mediterranean countriesandbeyond,includinginIraq,Oman,Tunisia,andEgypt. In Egypt,NDM-1 was first reported in 2013 from one ST11 K. pneumoniaeisolaterecoveredfromahospitalizedpatient[7].The firstcaseofE.coliproducingNDM-5hasbeenidentifiedfromthe asciticfluidofa52-years-oldfemalepatientwithpost-hepatitis cirrhosis[4].Tothebestofourknowledge,thisisthefirstreportof
http://dx.doi.org/10.1016/j.jgar.2020.07.031
2213-7165/©2020TheAuthor(s).PublishedbyElsevierLtdonbehalfofInternationalSocietyforAntimicrobialChemotherapy.ThisisanopenaccessarticleundertheCC BY-NC-NDlicense(http://creativecommons.org/licenses/by-nc-nd/4.0/).
JournalofGlobalAntimicrobialResistance23(2020)70–71
ContentslistsavailableatScienceDirect
Journal
of
Global
Antimicrobial
Resistance
NDM-5fromchickenmeatinEgyptandtheMiddleEast.Itfurther indicatesthat NDMproducersmay bea true One-Health issue disseminatingintheenvironmentandtheninhumansthroughthe foodchain.
Funding
ThisworkwaspartlysupportedbytheUniversityofFribourg and by the Swiss National Science Foundation (projects FNS-31003A_163432).M.S.was supportedbyascholarshipfromthe Egyptiangovernment. Ethicalapproval Notrequired Competinginterests Nonedeclared References
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MustafaSadeka,b
LaurentPoirela,c,*
PatriceNordmanna,c,d
aMedicalandMolecularMicrobiology,FacultyofScienceand
Medicine,UniversityofFribourg,Fribourg,Switzerland
bDepartmentofFoodHygieneandControl,FacultyofVeterinary
Medicine,SouthValleyUniversity,Qena,Egypt
cSwissNationalReferenceCenterforEmergingAntibioticResistance
(NARA),UniversityofFribourg,Fribourg,Switzerland
dInstituteforMicrobiology,UniversityofLausanneandUniversity
HospitalCentre,Lausanne,Switzerland HirofumiNariya ToshiShimamoto TadashiShimamoto** LaboratoryofFoodMicrobiologyandHygiene,GraduateSchoolof BiosphereScience,HiroshimaUniversity,Higashi-Hiroshima 739-8528,Japan
* Correspondingauthorat:MedicalandMolecularMicrobiology, FacultyofScienceandMedicine,UniversityofFribourg,Fribourg, Switzerland.
** Correspondingauthorat:LaboratoryofFoodMicrobiologyand Hygiene,GraduateSchoolofBiosphereScience,Hiroshima University,1-4-4Kagamiyama,Higashi-Hiroshima739-8528, Japan. E-mailaddresses:Laurent.poirel@unifr.ch(L.Poirel), tadashis@hiroshima-u.ac.jp(T.Shimamoto).
Received27September2019
Availableonline1September2020
Table1
Minimuminhibitoryconcentrations(MICs)ofdifferentantibioticsforNDM-5-producingEscherichiacoliEc34,NDM-1-producingKlebsiellapneumoniaeKp38andderivedE. colitransconjugants(Tc)andtransformants(Tf).
Strain MIC(mg/mL)
CTX CRO MEM IPM KAN GEN NAL CIP NOR TET CHL COL
E.coliEc34 256 256 128 64 256 256 256 256 256 256 256 0.5
E.coliTc(NDM-5) 256 32 8 4 16 8 64 0.5 0.5 0.5 8 0.5
K.pneumoniaeKp38 256 256 128 64 256 8 256 256 256 16 256 0.5
E.coliTf(NDM-1) 256 256 4 4 256 8 8 0.5 0.5 4 4 0.5
CRO,ceftriaxone;NOR,norfloxacin;CHL,chloramphenicol;CIP,ciprofloxacin;TET,tetracycline;IPM,imipenem;MEM,meropenem;COL,colistin;KAN,kanamycin;GEN, gentamicin;NAL,nalidixicacid;CTX,cefotaxime.
