Genomic characterization of multidrug-resistant ESBL-producing Klebsiella pneumoniae isolated from a Ghanaian teaching hospital

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Genomic characterization of multidrug-resistant ESBL-producing Klebsiella pneumoniae isolated from a Ghanaian teaching hospital

Nicholas Agyepong

^a,

*, Usha Govinden

^a

, Alex Owusu-Ofori

^b

, Daniel Gyam ﬁ Amoako

^a,c

, Mushal Allam

^d

, Jessin Janice

^e

, Torunn Pedersen

^e

, Arn ﬁ nn Sundsfjord

^e,f

, Sabiha Essack

^a

aAntimicrobialResearchUnit,CollegeofHealthSciences,UniversityofKwaZulu-Natal,Durban,SouthAfrica

bSchoolofMedicalSciences,KwameNkrumahUniversityofScienceandTechnology,Kumasi,Ghana

cInfectionGenomicsandAppliedBioinformaticsDivision,AntimicrobialResearchUnit,CollegeofHealthSciences,UniversityofKwaZulu-Natal,Durban,South Africa

dSequencingCoreFacility,NationalInstituteforCommunicableDisease,NationalHealthLaboratoryService,SouthAfrica

eNorwegianNationalAdvisoryUnitonDetectionofAntimicrobialResistance,DepartmentofMicrobiologyandInfectionControl,UniversityHospitalofNorth Norway,Tromso,Norway

fDepartmentofMedicalBiology,FacultyofHealthSciences,UiT–TheArcticUniversityofNorway,Norway

ARTICLE INFO Articlehistory:

Received13March2019

Receivedinrevisedform13May2019 Accepted20May2019

CorrespondingEditor:EskildPetersen,Aar- hus,Denmark

Keywords:

Wholegenomesequencing Multidrug-resistant ESBL

Klebsiellapneumoniae Resistome

Ghana

ABSTRACT

Objectives:Thisstudydelineatedtheclonallineages,antibioticresistomeandplasmidreplicontypesin multidrug-resistantK.pneumoniaeisolatesfromateachinghospitalinGhana.

Methods:IdentiﬁcationandantibioticsusceptibilitytestingweredoneusingtheMALDI-TOFMSand Vitek-2automatedsystem.GenomicDNAextractionwascarriedoutusingtheNucliSenseasyMAG1 (BioMérieux)kitsandtheDNAwassubjectedtowholegenomesequencing(WGS)usingtheIllumina MiSeqplatform.

Results:Ofthe200isolatesobtained,37wereidentiﬁedasK.pneumoniaeofwhich9wereresistanttoall secondandthird-generationcephalosporins.These9isolatesselectedforfurthergenomicanalysiswere characterizedbythepresenceof8diversesequencetypes(STs),capsularpolysaccharideserotypes(K typesandwziallelictypes)andmultiplegenesencodingresistancetoβ-lactams(blaCTX-M-15,blaSHV-11,

blaTEM-1B,blaOXA-1),aminoglycosides(aac(3)-IIa,strB,strA,aadA16),fluoroquinolones/quinolones(qnrB66, oqxA,oqxB)andotherantibioticclasses.Resistancegeneswereassociatedwithplasmids,predominantly IncFIB(K)andColRNAI.Multipleanddiversemutationsinquinoloneresistance-determiningregionsof gyrA(S83Y,D87A)andparC(S80I,N304S)inisolatesresistanttociprofloxacin(MIC4mg/mL)were found.Globalphylogenomicanalysisaffirmedthediverseclonalclusteringandoriginoftheseisolates.

Conclusions:Thevariedclonalclustersandresistomeidentiﬁedinthemultidrug-resistantK.pneumoniae isolatesisamajorthreattothemanagementofinfectionsinGhana.Themolecularcharacterizationof antibioticresistanceisthusimperativetoinformstrategiesforcontainment.

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

Introduction

Themechanismsofβ-lactamantibioticresistanceemployedby Gram-negative bacteria including K. pneumoniae involve the expression of β-lactamases with/without other non-enzymatic resistance mechanisms such as efﬂux and/or, outer protein membrane or porin reduction rendering the agents ineffective (Alekshun and Levy,2007; Lau etal., 2014; Wilson, 2014).The resistancemaybeintrinsicallyexpressedoracquired.Saravanan

andcolleagues2018reportedhighratesofresistancetoβ-lactam antibiotics,particularlysecondandthirdgenerationcephalospor- ins among Enterobacteriaceae, ranging from 12% to 82.8% in hospitalsettingsinasystematicreviewonprevalenceanddrug resistance pattern of extended spectrum β–lactamases (ESBLs) producing Enterobacteriaceae inAfrica (Saravananet al.,2018).

ESBL encoding plasmids are commonly associated with genes mediating resistance to other antibiotic classes including ﬂuo- roquinolonesandaminoglycosides,facilitatingthedissemination of multidrug-resistant bacteria in hospital settings. The swift acquisition of plasmid-borne extended-spectrum β-lactamases (ESBLs),especiallythose belongingtotheTEM, SHVandCTX-M β-lactamase families produced by Enterobacteriaceae including

* Correspondingauthor.

E-mailaddress:[email protected](N.Agyepong).

https://doi.org/10.1016/j.ijid.2019.05.025

1201-9712/©2019TheAuthors.PublishedbyElsevierLtdonbehalfofInternationalSocietyforInfectiousDiseases.ThisisanopenaccessarticleundertheCCBY-NC-ND license(http://creativecommons.org/licenses/by-nc-nd/4.0/).

ContentslistsavailableatScienceDirect

International Journal of Infectious Diseases

j o u r n a l h o m ep a g e : w w w . e l s e v i e r . c o m / l o c a te / i j i d

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K.pneumoniaewithhighpreferenceforoxyimino-cephalosporin hydrolysisisincreasingglobally(Bonnet,2004;Giboldetal.,2014;

ZhaoandHu,2013).PlasmidsoftheIncFgrouprepresentoneofthe most common plasmid types contributing to the spread of antibiotic resistance genes in Enterobacteriaceae with CTX-M- 15-positive IncFIIK plasmids commonly characterized in K.pneumoniae(Dolejskaetal.,2012; JohnsonandNolan,2009).

Thespreadoftheseresistantbacteriahascompromisedtheuseof β-lactams, considered as safest and most easily available anti- bioticsfor treatment of infections in many parts of the world, includingGhana.

StudiesconductedinGhanahavereportedK.pneumoniaeasa majorpathogenresponsibleforUTI(Gyasi-Sarpongetal.,2014).A laboratory-basednationwidesurveillanceof antimicrobialresis- tance in Ghana by Opintan and co-workers reported that K.

pneumoniaerepresented1.06%ofallbacterialinfectionsand1.4%of Gram-negativebacilli(Opintanetal.,2015).Agyepongetal.(2018) indicatedanincreasedK.pneumoniaeresistanceof19%(37/200)of Gram-negativebacteriaintheirstudyonMDRbacterialinfections inateachinghospital inGhana.Inspiteof thethreatposedby multidrugresistantGram-negativebacteriainhealthcaresettings inGhana,thereispaucityofmolecularepidemiologystudies.This study,whichformspartofabroaderstudyonthemolecularproﬁle of Gram-negative ESKAPE pathogens in a Ghanaian teaching hospital,delineatestheclonallineages,antibioticresistomeand plasmidrepliconsofasub-setofK.pneumoniaewithresistanceto the second and third-generation cephalosporins using whole genomesequencing(WGS).

Materialsandmethods

Ethicalapprovalandvoluntaryinformedconsent

EthicalclearancewasgrantedbytheJointCommitteeofHuman Research Publications and Ethics, School of Medical Sciences, KwameNkrumahUniversityofTechnology,ResearchandDevel- opmentUnitoftheHospitalAdministration(ref:CHRPE/AP/015/

15)andtheBiomedicalResearchEthicsCommitteeofUniversityof Kwa-ZuluNatal(ref:BE494/14).Voluntary,informedconsentwas obtainedfromallparticipantsandfromparentsorguardiansfor minorsinwrittenformeither signedor bya thumb printafter explaining the procedure and purpose of the study, using an interpreterasappropriate.

Studysetting

ThestudywasconductedbetweenFebruaryandAugust2015in KomfoAnokyeTeachingHospital(KATH)inKumasi,intheAshanti region of Ghana. The facility is a 1000-bed tertiary care governmenthospital.Theaveragedailyprimarycareandspecialist outpatient attendance was 169 and 954 patients respectively during the period of study. The population of the region is concentrated in a few districts, with the Kumasi metropolis accounting for nearly one-third of the region’s population of 4,780,380 (Owusu and Oteng-Ababio, 2015). KATH is the only regionalandreferralhospitalthattakescareofabout80%ofboth emergenciesandregularmedicalcasesintheregionandservesas referralhospitalforpartsofBrongAhafo,Western,Easternandthe NorthernregionsofGhana.

Bacterialselectionandidentiﬁcation

TheK.pneumoniaeisolatesincludedinthisstudywasasubset from a larger collection of 200 clinical, non-duplicate Gram- negativebacterial samples (Agyepong et al., 2018).Of the 200 isolates,37wereidentiﬁedasK.pneumoniae,ninewereresistantto

all second and third-generation cephalosporins and thereby selectedforgenomiccharacterizationbywholegenomesequenc- ing(WGS).Sevenoftheseisolateswereobtainedfromurineand oneeachfromgastriclavageandtrachealaspirate.Eightofthenine isolates were obtained from in-patients and one from an outpatient.Dateofcollection,diagnosis,sex,ageandwardtypewere obtained from patient records (Information on all 37 isolates appearintheTableS1).

Bacterial identiﬁcation and antibiotic susceptibilities were determined by the Vitek-2 (BioMérieux, France) automated system. Identity and MICs were further conﬁrmed by using MALDI-TOFMS(BrukerDaltonicGmbh,Bremen,Germany)broth micro-dilution in accordance with European Committee on AntimicrobialSusceptibilityTesting(EUCAST)guidelinesrespec- tively (Testing). K. pneumoniae ATCC700603 was used as the controlstrain.

DNAextractionandgenomesequencing

DNAextractionwascarriedoutusingtheNucliSenseasyMAG1 (BioMérieux) kits according to themanufacturers’ instructions.

ThegenomicDNAlibrariesweregeneratedusingtheNextera1kit (Illumina)followedbysequencingonanIlluminaMiSeqplatform attheGenomicsResourceCenterattheUniversityofTromso,the ArcticUniversityofNorway.Rawsequencereadswereadaptorand quality-trimmedusingTrimmomatic(Bolgeretal.,2014).Assem- blyusingSPAdes3.9.1(Bankevichetal.,2012),qualityscoreswere assessedbyQUASTversion4.6.0software(Bankevichetal.,2012).

TheassembledreadswereannotatedusingtheBacterialAnalysis Pipeline (BAP)of software revision4.2 and NationalCenterfor BiotechnologyInformation(NCBI) BasicLocal Alignment Search Tool (BLAST) searches (https://www.ncbi.nlm.nih.gov/genome/

annotation_prok/).Theantibioticresistancegenesandplasmids were identiﬁed by mapping the sequence data to an online databaseusingResFinder(Zankarietal.,2012)andPlasmidFinder (Carattolietal.,2014)respectively.Comparativegenomicanalysis was further performed using K. pneumoniae ATCC 13883 (PRJNA244567)asreferencestraintoelucidatethechromosomal mutationresultinginquinoloneresistance.Multi-locusSequence Typing(MLST)wasalsodeterminedfromtheassembledgenomes (https://github.com/tseemann/mlst) which also predicted the allelicproﬁlesofthe7housekeepinggenes,gapE,infB,mdh,pgi, phoE, rpoB,and tnoB of K.pneumoniae.ThereferenceKlebsiella WGS data online platform tool, Kaptive-web (http://kaptive.

holtlab.net/) was used to infer the serotypes (K types and wzi allelictypes)oftheisolates.

Phylogeneticanalyses

Agenome-widegene-by-genecomparisonapproachwasused to assess the genetic relatedness between isolates within and acrosswards.Thecoregenesweredeterminedfromtheannotated genomeassemblies,predictedcodingregionswereextractedand converted into protein sequences. A phylogeny was drawn for K. pneumoniae using Rapid large-scale prokaryote pangenome analysis (Roary; https://sanger-pathogens.github.io/Roary/) to estimate the tree for the core genome. The genome of K. quasipneumoniae strain P27-02 (accession number:

NXHG00000000.1) served as the outgroup to root the tree to enableeasyconﬁgurationof thephylogeneticdistancebetween thestrainson thebranches. Altogether,3,492core geneswere extractedwithanalignmentlengthof3,484,711bpsharedbythe nineK.pneumoniaegenomes.TheallelicdistancefromthecgMLST was visualized using Figtree v1.4.3 (https://tree.bio.ed.ac.uk/

sofware/ﬁgtree/)inamaximumlikelihoodphylogenetictreeusing optimizedparametersasfollows:nucleotidesubstitutionmodel,

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Jukes-Cantor;transition/transversionratio,2;estimatesubstitu- tionrate,yes;numberofsubstitutionrate,4;performbootstrap analysis,yes;replicates,1,000.Ametadata(includingisolatename, ward,ST,Ktypeandwziallelictype)wereimportedtoprovidea comprehensiveanalysisofthegeneratedphylogenetictree.

To investigate the global phylogeny of the K. pneumoniae isolates, genome assembly datasets including metadata were downloadedfromthePathosystemsResourceIntegrationCenter (PATRIC) database (https://www.patricbrc.org/). Genomes with less than 400 contigs and with MLST and isolation country availablewereselectedandrunthroughparsnp(softwaredesigned for intraspecific or core genome alignment for high quality assemblies)v.1.2(Treangenetal.,2014)with“–c”-flagsenabled toincludealltheselectedgenomesinthephylogenetictree,and randomreferenceselectionamongtheincludedsamples.FigTree (https://tree.bio.ed.ac.uk/software/figtree/) and R-ape package (v5.1)wereusedtovisualizeandeditthephylogenetictrees.

Accessionnumbers

The raw read sequences and the assembled whole genome contigs have beendeposited in GenBank. The data is available underprojectnumberPRJNA411997.

Results

TheclinicaldataindicatedthatK.pneumoniaewasfrequently implicatedinUTI.Antibioticsusceptibilityprofilesshowedthatall theisolateswereresistanttocefuroxime,cefotaximeandceftazi- dimebutsensitivetoimipenem,ertapenem,meropenem,amikacin andcolistin.Sevenofthenineisolateswereadditionallyresistantto gentamicin, nitrofurantoin and trimethoprim-sulfamethoxazole and six weresensitive tociprofloxacin (Table1).WGS analysis revealedthatalltheisolateswerepredominantlycharacterizedby thepresenceofmultipleresistomeencodingforresistancewithin andbetweenantibioticclasses.Theisolatescarried3-5β-lactamase genes,3-6aminoglycosideresistancegenes,2-5fluoroquinolone resistance genes in different permutations and combinations (Table2).blaCTX-M-15andblaTEM-1B),(aac(3)-IIa-like,aph(3')-Iaand aac(6')Ib-cr)and(oqxA-like,oqxB-like,qnrB10-likeandqnrB2)were themostcommonβ-lactam,aminoglycosideandfluoroquinolone

resistance genesobservedrespectively.Theisolates alsocarried resistance genesforotherantibioticclasses includingsul2, fosA, dfrA14 and catB7-like encoding resistance for sulphonamide, fosfomycin,trimethoprimandphenicolrespectively.MLSTanalysis showedahighvariationamongthestrainsidentifying8different sequence types including; ST2171, ST2816, ST17, ST152, ST397, ST1788,ST798and ST101(Table2 andFigure1)evidentbythe differentallelicproﬁlesofthe7housekeepinggenesbetweenthe isolates (Table 3). This indicates the circulation of multiple K. pneumoniae sequence types in a single hospital. The high diversitywasconﬁrmedbytheepidemiologicaltypingschemevia the Kaptive databasewhich also predicted 8 different capsular polysaccharide serotypes (KL15-wzi50, KL30-wzi122, KL155- wzi173, KL149-wzi110, KL158-wzi475, KL2-wzi2, KL18-wzi18 and KL17-wzi137)(Table2andFigure1)fortheisolates.IncFIB(K)and ColRNAIwerethemostprevalentplasmidreplicontypesamong K.pneumoniaeisolates(Table2).

cgMLSTanalysisusingK.quasipneumoniaestrainP27-02asthe outgroupcollaboratedthehighgeneticvariationinthestrains.The treewasdividedintomanysubcladesshowingthedifferentiation of theisolates(Figure1).Phylogeneticscoupledwithmetadata analysisprovidedadeeperinsightintothediversityoftheclones betweenthewardsinthehospital(Figure1).Speciﬁcally,three differentclonaltypes(ST101-KL17-wzi137,ST17-KL155-wzi173and ST397-KL158-wzi475)werefoundinthechildhealthwardwhiles two otherclones (ST152-KL149-wzi110 and ST2171-KL15-wzi50) wereidentiﬁedintheobstetricsandgynaecologyward.Phyloge- nomicanalysesincludingthegenomesfromthisstudy(n=9)and fromaglobalstraincollection(n=1158,including20SouthAfrican isolates) wereperformed to ascertain clustering and the likely originstheisolatesfromourstudyshowedadispersedorwider distributionontheglobaltree(Figure1).Asvisualized,thetwo ST101-isolatesfromGhana(P26-75andP26-81)arecloselyrelated toeachotherandbelongtothesamedistinctphylogeneticcluster astheSouthAfricanlineageofST101.Moreover,isolatesP26-71 (ST397)andP26-66(ST152)arecloselyrelatedtotheSouthAfrican ST14 and South African ST323 isolates, respectively. As shown, mostbranchesconstituteisolatesfromdiversegeographicorigin.

ThebranchAisolates,P27-01 andP26-63arecloselyrelatedto eachotherandtoST17-isolatesfromtheUSA.InbranchB,P26-71 clusterswithisolates(mainlyST14)fromeightcountries,including

Table1

AntibioticsusceptibilityproﬁlesoftheMDRK.pneumoniae(n=9).

Isolate code

Demographics Susceptibilityproﬁle-MICs(mg/L)

Date SPM Diagnosis WT AMC TZP CXM FOX CTX CAZ CFP ETP IMP MEM AMK GEN CIP TET NIT COL SXT

P27-01 [20]

06/06/

2015

Urine UTI O&G 32 64 64 4 64 16 8 0.5 0.25 0.25 2 16 0.2 1 64 0.5 320

P26-62 [70]

13/06/

2015

Urine UTI Surg. 32 16 64 16 64 16 16 0.5 0.25 0.25 2 16 0.25 2 64 0.5 320

P26-63 [76]

02/06/

2015

Urine UTI CH 32 32 64 4 32 16 2 0.5 0.25 0.25 2 16 0.25 1 128 0.5 320

P26-66 [117]

11/03/

2015

Urine UTI O&G 16 32 64 4 64 16 2 0.5 0.25 0.25 2 16 4 4 128 0.5 320

P26-71 [155]

14/09/

2015

Gastric lavage

Gastritis CH 16 64 64 64 16 16 2 0.5 0.25 0.25 2 16 0.25 1 16 0.5 320

P26-75 [183]

01/07/

2015

Urine UTI CH 16 64 64 16 64 16 2 0.5 0.25 0.25 2 16 4 4 >256 0.5 320

P26-78 [201]

19/03/

2015

Aspirate Sinusitis ICU 32 32 64 4 64 16 2 .5 .25 .25 2 16 .25 1 16 0.5 320

P26-79 [202]

11/03/

2015

Urine UTI OPD 32 128 64 4 64 16 2 0.5 0.25 0.25 2 16 0.25 0.5 32 0.5 320

P26-81 [206]

14/03/

2015

Urine UTI Med 16 32 64 16 6 16 2 0.5 0.25 0.2 2 16 4 0.5 512 0.5 320

ResistantMICsbreakpoint(EUCAST,2017) >8 >16 >8 NA >2 >4 >4 >1 >8 >8 >16 >4 >0.5 >2 >64 >2 >4 AMC-Amoxicillin-Clavulanate,TZP-PiperacillinTazobactam,CXM-Cefuroxime,FOX-CefoxitinCTX-Cefotaxime,CAZ-Ceftazidime,CFP-Cefepime,ETP-Ertapenem,IMP- Imipenem,MRP-Meropenem,AMK-Amikacin,GEN-Gentamicin,CIP-Ciproﬂoxacin,TET-Tetracycline,NIT-Nitrofurantoin,COL-Colistin,SXT-Trimethoprim-sulfamethoxazole,Fluoro-Fluoroquinolones,Tet-Tetracycline,CH-ChildHealth,O&G-ObstetricsandGynaecology,Med-Medicine,Surg-Surgery,WT-Wardtype,SPM-Specimen.

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Table2

Genomiccharacterizationsofmultidrug-resistantK.pneumoniaeisolatesfromWGSAnalysis.

Isolate code

WGSin-silicotyping Plasmidreplicons Antibioticclasses/resistancegenes Chromosomal

mutation MLST K

type Wzi type

β-lactamases Aminoglycosides Fluoroquinolones/

Quinolones

Otherresistance gyrA gyrB parC P27-01

[20]

ST2171 KL15 wzi50 IncFIA(HI1),IncFIB(K),IncFII (K),IncR

blaTEM-1B, blaCTX-M-15, blaSHV-11

aac(3)-IIa,strB, strA

oqxA,oqxB fosA,sul2,tet(D), dfrA14

-¹ - -

P26-62 [70]

ST2816 KL30 wzi22 IncFIA(HI1),IncFIB(K),IncFII (K),IncR,ColRNAI

blaTEM-1B, blaCTX-M-15,

strA,aac(3)-IIa, strB,

oqxA,oqxB fosA,catA2,sul2, tet(D),dfrA14

- - -

P26-63 [76]

ST17 KL155 wzi173 IncFIA(HI1),IncFIB(K),IncFII (K),Col(MGD2),IncR, ColRNAI

blaCTX-M-15, blaTEM-1B

strA,aac(3)-IIa, strB,

oqxA,oqxB, fosA,catA2,sul2, tet(D),dfrA14,

- - -

P26-66 [117]

ST152 KL149 wzi110 IncFIB(K),IncFII(K),ColRNAI blaCTX-M-15, blaOXA-1,

aac(6')Ib-cr,aac (3)-IIa,aac(6')Ib- cr,

oqxB,oqxA,qnrB66 fosA,catB4,sul2, tet(A),dfrA14

S83F D87A

NM² S80I

P26-71 [155]

ST397 KL158 wzi475 IncFIB(pKPHS1),IncFIB(K), IncFII(K),ColRNAI

blaTEM-1B, blaCTX-M-15

aac(6')Ib-cr,aac (3)-IIa,strB,strA,

oqxB,oqxA fosA,catB4,sul2, dfrA14

- - -

P26-75 [183]

ST101 KL17 wzi137 IncFIA(HI1),IncFII,IncFIB(K), ColRNAI

blaCTX-M-15, blaOXA-1

aac(6')Ib-cr,aac (3)-IIa,strA,strB

qnrB66,oqxA,oqxB, fosA,catB4,sul1, sul2,dfrA14, dfrA5

S83Y D87A

NM S80I, N304S P26-78

[201]

ST1788 KL2 wzi2 IncFIB(pKPHS1),IncFIB(K), IncFII(K),ColRNAI

blaCTX-M-15,like, blaTEM-1B

aac(3)-IIa,strA, strB

oqxA,oqxB FosAsul2dfrA14 - - -

P26-79 [202]

ST789 KL18 wzi18 IncFIA(HI1),IncFIB(K),IncFII (K),Col(MGD2),IncR, ColRNAI

blaCTX-M-15, blaTEM-1B

aac(3)-IIa, aadA16-strA,strB

oqxA,oqxB fosAsul1,sul2tet (D)dfrA14catA2

- - -

P26-81 [206]

ST101 KL17 wzi137 IncFIA(HI1),IncFII,IncFIB(K), ColRNAI

blaCTX-M-15, blaOXA-1, blaTEM-1B

aac(3)-IIa-strA, strB

oqxA-like,oqxB- like,qnrB66aac(6') Ib-cr

fosAsul1,sul2 dfrA14dfrA5

S83Y D87A

NM S80I, N304S

Unlessotherwisestatedinthefootnote,K.pneumoniaeATCC13883(PRJNA244567)wasusedasreferencestraininthecomparativegenomicanalysis.MLST-multilocus sequencetyping,Ktyping-Klebsiellasurfacepolysaccharidecapsulecharacterizationandwzitype-wziallelictypingscheme.

1 Susceptibletociproﬂoxacin.

2 NM-Nomutation.

Figure1.Aphylogenybasedoncoregenomemultilocussequencetypinggenesofthe9K.pneumoniaegenomes.TheK.quasipneumoniaestrainP27-02(accessionnumber:

NXHG00000000.1)wasrootedandusedastheoutgroupinthetree.Thefollowinginformationisprovidedforeachisolate:name/reference,ward,MLST(STtypes),Ktypeand wziallelictype.Thetreewasdividedintomanysubcladesshowingthedifferentiationoftheisolatesinthephylogenetictree.Thecolourcodesdepictthediversityofthe isolatesonthephylogenetictree.Thebootstrapvalues(%)forthenodeshasbeenindicatedonthetree.Thescalebarrepresentsonenucleotidesubstitutionper1000sequence positions.

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SouthAfrica.InbranchC,P26-79relatestoisolatesfromUKand Norway(diverse STs), and in branchD, P26-78 is mostclosely relatedtoisolatesofST493fromtheNetherlandsandtheUSA.In branchE,P26-66islocatedtogetherwithisolatesofdiverseorigin andSTs,whileisolatesfromfourcountries(diverseSTs)collocate withP26-62inbranchF.TheST101-clustersituatesonbranchG, which in addition to the Ghanaian and South African isolates includesisolatesfromUKandPakistan.

Discussion

WereportonthecomplexityofmultidrugresistanceinESBL- producingK.pneumoniaeisolatesfromareferralhospitalinGhana.

The isolates were phylogenetically diverse in terms of their alignmenttogeographicaldistinctclusters.Theywerecharacter- izedwithdiverseandmultiplepermutationsandcombinationsof antibiotic resistance genes. A high prevalence of CTX-M-15 β- lactamaseswasobserved,whichmediatedhigh-levelphenotypic resistancetothesecond-andthird-generationcephalosporinsas indicatedintheMICsproﬁle(Tables1and2).Theresistancegenes weremainlycorrelatedwithIncFIB(K)plasmids,withColRNAIalso beingcommonamongtheisolates.

Theisolateswereresistanttocefuroxime,cefotaximeceftazi- dime,amoxicillin-clavulanate,piperacillin-tazobactam,gentamicin, nitrofurantoin and trimethoprim-sulfamethoxazole. This poses a serious challengeto antibiotic therapy asthese agents arecommonlyusedasempiricaltreatmentinGhana(Hackman etal.,2014).Thephenotypicproﬁlewascorroboratedbythewhole genomesequencingresultsasevidentfromTables1and2.Thisis comparable to studies from many parts of the world, which reported CTX-M class of β-lactamases as a major resistance mechanism among Gram-negative bacteria tooxyimino-cephalosporins,particularlycefotaxime(Bonnet,2004;Toftelandetal., 2007), withCTX-M-15being themostcommonallele inEnter- obacteriaceaeinAfrica(Ahmedetal.,2012;Breurecetal.,2013), includingGram-negativeESKAPEbacteriaand(Breurecetal.,2013;

Rodriguesetal.,2014;SantajitandIndrawattana,2016),particu- larlyinK.pneumoniae(Baraniaketal.,2013).

MultipleK.pneumoniaeSTswereidentiﬁedinlineagewithother isolatesfrom a globalstraincollection, although fromdifferent geographicalsourcesandgeneticexchangesuggesthighdiversity andclonalexpansionofthisspecies,asreportedinotherstudies (Breurecetal.,2013;Brisseetal.,2009).Thehighvariationbetween the isolates was in concordance with the Klebsiella capsular serotypeswhichalsopredicted8differentKtypesandwziallelic typesforthestrains.ThishighlightstheabilityofWGStoaccurately predictdifferentepidemiologicaltypingtechniques.TheCTX-M- 15-producingK.pneumoniaeST type101wasﬁrstreportedinGreece in an ICU infections outbreak caused by ertapenem-resistant K.pneumoniae(Poulouetal.,2013)andthen inothercountries includingSpain, Italy,Franceand Tunisia in hospitaloutbreaks

associatedwithcarbapenemresistance(Marcadeetal.,2013).In contrast,ourisolatesweresensitivetocarbapenemsastheseagents have only recently been introduced into the Ghanaian clinical practice, are comparatively more expensive than the mainstay antibiotics and used as last-resort agents in treating serious infections.Thus,thereisrelativelylowselectionpressureforthe developmentofcarbapenemresistance.Globalphylogenyinvesti- gationindicatedthattwooftheisolates(P26-75andP26-81)were of the same sequence type (ST101) as the main cluster of carbapenemase-producingK.pneumoniaefoundinDurban,South Africa.Interestingly,thecgMLSTanalysiscoupledwithmetadata conﬁrmedthatthetwoisolates;P26-75(child-health)andP26-81 (Medicine) of ST101 clone were genetically related with 100%

identity, anallelic distanceofzero (Figure1 and Table 3).This reiteratestheimportanceofvisualizingphylogeneticstructuresin relation totheirmetadata as it offersvaluableinsight intothe identiﬁcation,characterizationspreadandevolutionofpathogens.

However,thelineagesofST101werethesameinourisolatesbut have evolved in different local environments and resistance patterns includingcarbapenemresistance. AlsotheP26-66 and P26-71isolateswerephylogeneticallyrelatedtotheSouthAfrican ST323isolateandST14isolaterespectively(SekyereandAmoako, 2017).Thiscouldindicateregionaltransmission,perhapsdueto international travel between the two countries facilitating the disseminationofspeciﬁcK.pneumoniaeSTs(Figure2).

ThepredominanceofCTX-M-15anddifferentTEM-typesfoundin thisstudyisassociatedwithmultidrug-resistanceinEnterobacteriaceae.

TheCTX-M-15andTEM-,SHV-andOXA-typesofβ-lactamasesare plasmidencodedwiththetendencytodisseminateamongvarious speciestoconferresistancetoβ-lactamsandothernon-β-lactam antibiotics including quinolones, chloramphenicol,tetracyclines andaminoglycosides(Shaikhetal.,2015)asreﬂectedinthisstudy.

The IncFIB(K) and ColRNAI plasmids were found in all the isolatesandwereassociatedwithCTX-M-15andotherresistance genes.Thisﬁndingis consistentwithstudiesthatreportedthat CTX-M-15 is mainly harboured on IncFII(K) plasmids in ESBL- producing K.pneumoniae isolates (Coelho et al.,2010; Tokajian etal.,2015).ReportsfromothersstudieshavedescribedIncFIB(K) plasmidsasdynamicinnature,withthecapacitytodisseminate antibiotic resistance genes among Enterobacteriaceae (Carattoli, 2013;Coelhoetal.,2010;Dolejskaetal.,2013).

OurstudyfoundqnrB66variantoftheqnrBgeneintheisolates (P26-66, P26-75 and P26-81)which mediated quinoloneresistance, consistent with a study that reported this gene as predominantlyencodingforfluoroquinolone/quinoloneresistance amongK.pneumoniaeinAfrica(Breurecetal.,2013).oqxAandoqxB geneswerefoundtogetherinalltheisolates,suggestingthatthe oqxAandoqxBgenescannotbeamajormechanism,particularlyas theyweredetectedinisolatesofbothsusceptibleandmuchhigher MICs.PerhapsoqxABinsynergywithothermechanismsincreased fluoroquinolone resistance intheisolates asother studieshave indicatedoqxAandoqxBgenesencodingoqxABproteintomediate high fluoroquinolone resistance commonly in K. pneumoniae (El-Badawy et al., 2017; Sekyere and Amoako, 2017). We also identified multiple aminoglycoside (aac(6')Ib-cr, StrA, StrB) and quinolone(qnrB66)resistancegeneswhichhavebeenreportedin otherstudiestomediateresistancetogentamicinandciprofloxa- cin(Breurecetal.,2013).

Analysisofquinoloneresistance-determiningregionsofgyrAand parCgenesrevealedthepresenceofmultipleanddiversemutations ingyrA(S83Y,S83F,D87A)andparC(S80I,N304S)inisolatesthat wereclonallydistinct.MutationsingyrAandparCgeneshavebeen reported as major mechanisms of ﬂuoroquinolone/quinolone resistance associated with DNA gyrase and topoisomerase IV alterationsinEnterobacteriaceae(Alvietal.,2018;Piekarskaetal., 2015; Sekyere and Amoako, 2017), as the plasmid-mediated Table3

AtableshowingthediversityofMLST(STtypes)andallelicproﬁlesofthe7 housekeepinggenesinthemultidrug-resistantK.pneumoniaeisolates(n=9).

Isolate MLST gapE infB mdh pgi phoE rpoB tnoB

P27-01[20] ST2171 2 6 1 1 4 4 4

P26-62[70] ST2816 7 40 169 26 1 1 398

P26-63[76] ST17 2 1 1 1 4 4 4

P26-66[117] ST152 2 3 2 1 1 4 56

P26-71[155] ST397 2 1 1 1 21 44 9

P26-75[183] ST101 2 6 1 5 4 1 6

P26-78[201] ST1788 2 6 160 1 226 111 299

P26-79[202] ST789 25 10 1 1 20 1 22

P26-81[206] ST101 2 6 1 5 4 1 6

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quinoloneresistancegenes(PMQR)andextrusionbyintrinsicefflux pumpscommonly mediatelow-levelfluoroquinolone/quinolone resistance(Sekyere andAmoako,2017).However,mutationsinboth gyrAandparCareoftencommonandassociatedwithhigh-level quinoloneresistanceinEnterobacteriaceaecomparedwithalter- ationsingyrB(Piekarskaetal.,2015)asevidentinthisstudy.ST101 (P26-75,P26-81)andST152(P26-66)isolateswereofthesame mutationcodons83and87ofthegyrAandat80inparCgeneswith nomutationingyrBgeneamongisolateswithaciprofloxacinMICof 4mg/L.Mutationsat83and87ingyrAand80inparCgeneshave beenreportedasthemostcommonmutationpointswhichdisplay majoralterationsamongclinicalisolates,associatedwithfluoro- quinoloneresistance(SekyereandAmoako,2017),withcodon83 commonlyidentifiedinfluoroquinoloneresistantK.pneumoniae (SekyereandAmoako,2017).ThecombinedeffectofS83Y/F,D87A andS80IdetectedingyrAandparCgenesintheisolates,P26-66, P26-75andP26-81(MIC4mg/L)inourstudycouldbeassociated with increased level of ciprofloxacin resistance as previously reported(MinariniandDarini,2012).Thisisbecausefluoroquino- lones have been the main stay in the management of UTIs in Ghanaian teaching hospitals including KomfoAnokye Teaching Hospital (KATH) as levofloxacin and ciprofloxacin remain the commonestfluoroquinolonesprescribedforUTIsleadingtorecent reportsoftheemergenceofresistanceinthesesettings(Agyepong etal.,2018;Afriyieetal.,2015;Feglo etal.,2013).Thecomplexity and diversity of resistance gene combinations detected among K. pneumoniae strains in this study and their potential for dissemination poses a serious threat to the management of infectionsbythisspeciesinGhana.

Conclusion

Thisstudyidentiﬁedgenesencodingresistanceforβ-lactams, ﬂuoroquinolones,aminoglycosidesandotherantibioticsindiverse

permutations and combinations among multidrug-resistant K.pneumoniaebacteriainKomfoAnokyeTeachingHospital.There isthusanurgentneedforepidemiologicalandmolecularstudiesto understandthedynamicsofantibioticresistancetransmissionto informstrategiesforcontainment.

Funding

ThisstudywassupportedbytheNationalResearchFoundation IncentiveFundingforRatedResearchers(GrantNo.:85595)anda PhDScholarshipawardedtoNAgyepongbytheCollegeofHealth Sciences,University of KwaZulu-Natal.Any opinion,ﬁndingand conclusionorrecommendationexpressedinthismaterialisthatof theauthor(s)andtheNRFdoesnotacceptanyliabilityinthisregard.

Authors’contribution

The study was co-conceptualizedand jointlydesigned by NA, UG, AO, DGA, MA, JJ, TP, AS and SE.NA collected the dataundertook the preliminarylaboratoryworkandpresentresultastables.AOsupervised thesampling andpreliminarylaboratorywork. UG supervisedthe isolatesphenotypicscreeningandanalysis.DGAandMAcontributedto thebioinformaticsdataanalysis.TPcontributedtoisolationofhigh- quality DNA and illumina bioinformatics analyses. JJ, TP and AS contributedtodataanalysesandassuringthequalityoftheresult.SE contributedto assuringthequalityoftheﬁnalmanuscript. Allthe authorscontributedinpreparationandsubmissionofmanuscript.

Competinginterests

ProfessorEssackisamemberoftheGlobalRespiratoryInfection Partnershipsponsoredbyanunrestrictededucationalgrantfrom ReckittandBenckiser,UK.Theotherauthorshavenocompeting interesttodeclare.

Figure2.Klebsiellapneumoniaeglobalphylogenyasrevealedbyrapidcoregenomemulti-alignment(https://github.com/marbl/parsnpAssemblydatasetfromthisstudywas analyzedtogetherwithdatasetsfromthePATRICdatabase(n=1158)andfromSouthAfrica(ref.35).SubtreesshowingthedistributionoftheGhanaianisolatesineightin distinctphylogeneticbranches(A-G)asindicatedbythecolorcodesintheglobaltree.Thesubtreesrooted(includesbranchlengths)andscaleof0.005(forallthetrees)using (https://cran.r-project.org/web/packages/ape/ape.pdf).Fortheisolatesincludedineachofthesebranches,assemblyID,isolationcountryandMLSTareshown.

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Acknowledgements

WewouldliketothanktheKomfoAnokyeTeachingHospital staff,especiallythetechnicalstaffintheMicrobiologylaboratory for their support during sample collection and preliminary identificationaswellasthephysiciansandnursingstaffinvarious wardsfortheirassistance.WethankthestaffofNationalHealth LaboratoryServices(NHLS),Albert LuthuliTeaching Hospitalin SouthAfricaforfurtherbacteriaidentificationandtheantibiotics susceptibilityprofiletesting.WealsothankJessinJanice,Bettina AasnæsandEllenJosefsenofUniversityHospitalofNorthNorway forthetechnicalassistance.

AppendixA.Supplementarydata

Supplementarymaterialrelatedtothisarticlecanbefound,inthe onlineversion,atdoi:https://doi.org/10.1016/j.ijid.2019.05.025.

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