Wide spread of carbapenemase-producing bacterial isolates in a Nigerian environment

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Short

Communication

Wide

spread

of

carbapenemase-producing

bacterial

isolates

in

a

Nigerian

environment

Christophe

Le

Terrier

a

,

Amandine

Masseron

a

,

Nkolika

Stella

Uwaezuoke

b

,

Chinagozi

P. Edwin

c

,

Agantem

E. Ekuma

d

,

Folake

Olugbeminiyi

e

,

Shuwaram

Shettima

f

,

Simon

Ushie

g

,

Laurent

Poirel

a,h,i,

*

,

Patrice

Nordmann

a,h,i,j

a

MedicalandMolecularMicrobiology,SectionofMedicine,FacultyofScienceandMedicine,UniversityofFribourg,Switzerland

b

DepartmentofMedicalMicrobiologyandParasitology,NationalHospitalAbuja,Abuja,Nigeria

c

DeptofMedicalMicrobiology&Parasitology,AminuKanoTeaching,Hospital,Kano,Nigeria

d

DepartmentofMedicalMicrobiology,UniversityofCalabarTeachingHospital,Calabar,Nigeria

e

DepartmentofMedicalMicrobiology,NationalOrthopaedicHospital,Igbobi,Lagos,Nigeria

f

DepartmentofMedicalMicrobiology,FederalMedicalCenter,Yola,Nigeria

g_Department_of_Medical_Microbiology_&_{Parasitology,}_Nnamdi_Azikiwe_University,_Nnewi_Campus,_Nnewi,_Nigeria h

INSERMEuropeanUnit(IAME,France),UniversityofFribourg,Switzerland

i

SwissNationalReferenceCenterforEmergingAntibioticResistance(NARA),UniversityofFribourg,Switzerland

j

InstituteforMicrobiology,UniversityofLausanneandUniversityHospitalCentre,Lausanne,Switzerland

ARTICLE INFO Articlehistory: Received11July2019

Receivedinrevisedform11October2019 Accepted11October2019

Availableonline19October2019 Keywords: Carbapenemase Environment Gram-negativebacteria Nigeria ABSTRACT

Objectives:Thepresenceofcarbapenemase-producingbacterialisolatesisfoundnotonlyinhospitaland

communitysettingsbutalsointheenvironment.Carbapenemaseproductionmayberelatedtoacquired,

usuallyplasmid-borne,β-lactamasegenesortochromosomalgenesintrinsictovariousspecies.Theaim

ofthisstudywastoevaluatetheoccurrenceofsuchcarbapenemase-producingbacterialisolatesamong

environmentalsamplesfromNigeria.

Methods:Atotalof122environmentalsampleswereplatedoncarbapenem-containingmedia.Atotalof

259isolateswererecovered,amongwhich124werecarbapenemase-producersaccordingtotheresults

oftheRapidec1CarbaNPtest.

Results:Themajorityofisolates(n=112)recoveredcorrespondedtonaturalproducersof

carbapene-mases,i.e.Stenotrophomonasmaltophilia(n=108),Burkholderiacepacia(n=1),Shewanellasp.(n=1),

Sphingobacteriumsp.(n=1)andChryseobacteriumgleum(n=1).Tenisolates(mainlyEnterobacteriaceae

andAcinetobacterbaumannii)producedanacquiredcarbapenemase,mostcommonlyoftheNDMtype.In

addition, two Pseudomonas otitidis isolates were identiﬁed as producing the Ambler class B

carbapenemase POM-1,further conﬁrming that this carbapenemase is naturallyproduced in this

environmentalspecies.Finally,severalisolatesco-producing16SrRNAmethylases(ArmA,RmtC)and/or

extended-spectrumβ-lactamases(CTX-M-9,CTX-M-15)werealsoidentiﬁed.

Conclusion:Thisstudyrevealedthepresenceanddiversityofclinically-relevantantimicrobial-resistant

bacteriaintheenvironmentinNigeria.

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

1.Introduction

Carbapenemsare last-resort antibiotics for managing multi-drug-resistantbacterialinfectionsbuttheireffectivenessmaybe compromisedbytheproductionofcarbapenemases[1].Knowing

the extent of the reservoir of carbapenemases is becoming important. The most clinically-relevant carbapenemases are of theKPCtype(serinecarbapenemaseofAmblerclassA),VIM-, IMP-and NDM-types (metallo-β-lactamases of Ambler class B) and OXA-48-type(oxacillinasesofAmblerclassD).Althoughextensive surveillancestudieshavebeenperformedinEurope,Americaand Australia, data from Africaremain scarce. In Nigeria, the most populatedcountryofAfrica(ca.200millioninhabitants),antibiotic useiswidespreadandlargelyunregulated[2].Owingtoalackof sanitation and poor control of public health in many African countries, large amounts of antibiotics may end up in the

* Correspondingauthor.Presentaddress:MedicalandMolecularMicrobiology Unit,DepartmentofMedicine,FacultyofScience,UniversityofFribourg,Chemindu Musée18,CH-1700Fribourg,Switzerland.

E-mailaddress:laurent.poirel@unifr.ch(L.Poirel).

http://dx.doi.org/10.1016/j.jgar.2019.10.014

JournalofGlobalAntimicrobialResistance21(2020)321–323

ContentslistsavailableatScienceDirect

Journal

of

Global

Antimicrobial

Resistance

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environmentandthuscontributetotheselectionandpersistence of antimicrobial resistance. Therefore, the primary aim of this studywastoevaluatethespreadofmultidrug-resistantbacterial strainsamongenvironmentalsamplescollectedinNigeria,witha particularfocusoncarbapenemase-producers.

2.Materialsandmethods 2.1.Soilandwatersamples

A total of122 environmental samples(waterandsoil) were collectedduringthesametimeperiodin2017fromsixdifferent locationsinNigeria, namelyAbuja(NorthCentral),Kano(North West),Yola(NorthEast),Nnewi(SouthEast),AkwaIbom(South) and Ibadan (SouthWest). Water samples wererecoveredfrom differentbodiesoflakes.

2.2.Selectiveisolationofcarbapenem-resistantGram-negative bacteria

Selection of carbapenem-resistant isolates was performed followinga selectiveenrichmentstepbyculturein brain–heart infusion broth supplemented with ertapenem (0.25

m

g/mL) overnight at 30C. Following enrichment,100

m

L of pure and diluted(1/100;1:1000)cultureswasplatedontoselectivemedia, including SuperCarba1 (CHROMagar, Paris, France) [3] and Drigalski agar medium supplemented with imipenem (0.75

m

g/mL),daptomycin(10

m

g/mL)andamphotericinB(5

m

g/ mL).Plateswerethenincubatedat30Cfor24h.

2.3.Phenotypicandgenotypiccharacterisationof carbapenem-resistantbacteria

Antimicrobialsusceptibilitytestingwasinitiallyperformedby the disk diffusion method and was interpreted according to European Committee on Antimicrobial Susceptibility Testing (EUCAST)recommendations (http://www.eucast.org), except for colistinfor which susceptibilitywas evaluatedby broth micro-dilution,asrecommended.

Carbapenemaseactivitywas assessedbytheRapidec1Carba NPtest(bioMérieux)foreachcolonytypegrowingontheselective media[4].Identiﬁcationofcarbapenemasegeneswasperformed by PCR using previously published primers [5]. The following carbapenemasegenesweresearchedfor:AmblerclassAgenesof

blaKPC, blaGES, blaIMI and blaFRI types; Ambler class B genes of

blaNDM,blaIMP,blaVIM,blaGIMandblaPOMtypes;andAmblerclassD

genes of blaOXA-48, -181, -23, -40 and -58 types. In addition, the

following genesweresearched for:extended-spectrum β-lacta-mases(ESBLs),includingblaCTX-M-1,-2,-3,-8,-9and-15,blaPER,blaTEM

and blaSHV [6]; aminoglycoside resistance 16S rRNA methylase

genes (armA, rmtA–rmtH and npmA) [7]; and plasmid-borne colistinresistancemcr-likegenes(mcr-1tomcr-9)[8].Allpositive PCRampliconsweresentforsequencing(MicrosynthAG,Balgach, Switzerland). Bacterialidentiﬁcationwas performed by matrix-assistedlaserdesorption/ionisationtime-of-ﬂightmass spectrom-etry(MALDI-TOF/MS)(VITEK1_MS;_{bioMérieux).}

Whole-genome sequencing was performed for the two Pseudomonasotitidisstrainsforwhichidentiﬁcationwasuncertain. DNAwasextractedfromisolatesusingaQIAamp1DNAMiniKit andQIAcubeWorkstation(QIAGEN,Courtaboeuf,France), accord-ing to the manufacturer's instructions. Genomic libraries were generated using a Nextera XT DNA Sample Preparation Kit (IlluminaSwitzerlandGmbH,Zurich,Switzerland)andsequencing wasperformedonanIlluminaMiSeqbenchtopsequencer.Reads fromsequencingwereassembledusingCLCGenomicsWorkbench 7 (QIAGEN).Putative β-lactamase genes werethen detected by ResFinder(https://cge.cbs.dtu.dk/services/ResFinder/).

3.Resultsanddiscussion

Fromthe122environmentalsamples,259distinctisolateswere isolatedonselectivecarbapenem-containingmedia.All carbape-nem-resistantisolateswereidentiﬁedonbothscreeningmedia.Of the259isolates,124werefoundtobecarbapenemase-producers according to the results of the Rapidec1 Carba NP test. The carbapenemase-negativeisolatescorrespondedto135 Pseudomo-nas spp. isolates (no acquired resistance genes) that were recovered only on the SuperCarba1 medium owing tonatural ertapenemresistanceinthisgenus.Themajority(n=112)ofthe carbapenemase-producers identiﬁed corresponded to bacterial species that naturally produce carbapenemases such as Steno-trophomonasmaltophilia(L1-likeβ-lactamase[9])(n=108), Chrys-eobacterium gleum (CGB-like carbapenemase [10]) (n=1), Burkholderia cepacia (PenA chromosomal A penicillinase [11]) (n=1),Sphingobacteriumsp.(n=1)andShewanellasp. (OXA-181-likecarbapenemase[12])(n=1).

Tenisolatesharbouringanacquiredcarbapenemasegenewere also identiﬁed (Table 1). A series of enterobacterial strains

Table1

InformationandgeneticfeaturesofisolateswithanacquiredcarbapenemasegenefromenvironmentsamplesfromvariousgeographicallocationsinNigeria. Strainno. Typeof

samplea

Geographical locationb

Species Carbapenemase ESBL 16SrRNAmethyltransferase (s)

Non-susceptiblephenotypec

1 SoilUO Abuja(NC) Acinetobacter baumannii

OXA-23 None None CIP,TET,SXT,CHL 2 SoilHE Nnewi(SE) A.baumannii OXA-40 None ArmA GEN,AMK,CIP,SXT,CHL 3 Water Nnewi(SE) A.baumannii OXA-40 None ArmA GEN,AMK,CIP,SXT,CHL 4 SoilHE Abuja(NC) Aeromonascaviae NDM-1 CTX-M-9 ArmA,RmtC GEN,AMK,SXT 5 SoilHE Abuja(NC) Citrobacterfreundii NDM-5 None None GEN,CIP,TET,SXT,CHL 6 SoilHE Abuja(NC) Enterobactercloacae NDM-5

CTX-M-15

None GEN,CIP,TET,SXT 7 SoilHE AkwaIbom(S) E.cloacae NDM-7 None None SXT

8 SoilHE AkwaIbom(S) Klebsiellapneumoniae NDM-1 CTX-M-15

ArmA GEN,AMK,CIP,TET,SXT 9 SoilHE AkwaIbom(S) K.pneumoniae NDM-1

CTX-M-15

ArmA GEN,AMK,CIP,TET,SXT 10 SoilHE Kano(NW) Pseudomonasputida VIM-5 None None CIP,TET,SXT,CHL ESBL,extended-spectrumβ-lactamase.

a

UO,urbanoutskirt;HE,hospitalenvironment.

b

NC,NorthCentral;SE,SouthEast;S,South;SW,SouthWes;NW,NorthWest.

c

CIP,ciproﬂoxacin;TET,tetracycline;SXT,trimethoprim/sulfamethoxazole;CHL,chloramphenicol;GEN,gentamicin;AMK,amikacin. 322 C.LeTerrieretal./JournalofGlobalAntimicrobialResistance21(2020)321–323

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producing different NDM-type carbapenemases was found in differentlocationsinNigeria.Thisﬁndinghighlightsthe environ-mentalspreadof theNDM-type carbapenemaseinNigeria[13]. ThelargespreadofNDM-producershasalreadybeenidentiﬁedin manyotherAfricancountries[14],includingAngola[15]andTogo

[16],indicatingthatthiscontinentmayactasasecondaryreservoir after the Indian subcontinent for spreading NDM-producing isolatestoEurope.TheAcinetobacterbaumanniiisolatesproduced eitherOXA-23orOXA-40,whichareextensivelyreportedinthat species[17].In addition,a blaVIM-5-positive Pseudomonasputida

isolate was identified, as previously identified from polluted wetlands in Nigeria [18]. Genome sequencing allowed the identification of a Sphingobacteriumsp. isolatethat produced a naturallyoccurringAmblerclassBcarbapenemase[19]andtwoP. otitidisisolatesthatharbouredtheblaPOM-1gene.Thislattergene

encodes a class B carbapenemase, with no associated mobile geneticelement,whichhasbeenpreviouslyidentiﬁedasintrinsic tothatbacterialspecies[20].

Regardingthenon-β-lactamaseresistancedeterminants,it is worth mentioning that the 16S rRNA methylase determinants conferringpan-aminoglycosideresistancewerefrequently associ-atedwithNDM-producers,aspreviouslyshown[21].Noteworthy, thespreadof16SrRNAmethylasesinGram-negativebacteriain Africaappearstorepresentamajorproblem,withrecentreports showingtheiroccurrencein,e.g.,EnterobacteralesinAngola,Libya, Egyptand Algeriaaswell asA.baumannii inAlgeria andEgypt. Finally,threecolistin-resistantisolateswererecoveredamongthe acquiredcarbapenem-resistantisolates(A.baumanniiand Enter-obactercloacae)andnaturalcarbapenem-resistantisolates (Sphin-gobacteriumspp.)butwerenotpositiveforMCR-encodinggenes, suggestingthattheresistancewasmediatedbyothermechanisms ofresistancesuchaschromosomalmutations.Theseresultsmay indicatethatacquiredcolistinresistancehasnotyetspreadamong environmentalisolatesinNigeria.

Overall, this study revealed the presence and diversity of clinically-relevantantimicrobial-resistantbacteriainthe environ-mentanddemonstratestheneedforfurtherinvestigation. Competinginterests

Nonedeclared. Funding

ThisworkwasfundedbytheUniversityofFribourg(Fribourg, Switzerland),theSwissNationalScienceFoundation[projects FNS-31003A_163432 and FNS-407240_177381] and INSERM (Paris, France).

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