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Molecular

typing

of

drug-resistant

Mycobacterium

tuberculosis

strains

from

Turkey

Alper

Karagoz

a

,

Hidayet

Tutun

b

,

Levent

Altintas

c,

*

,

Umit

Alanbayi

d

,

Dilara

Yildirim

d

,

Nadir

Kocak

e

aDepartmentofMolecularBiologyandGenetics,UşakUniversity,Uşak,Turkey b

DepartmentofPharmacologyandToxicology,FacultyofVeterinaryMedicine,BurdurMehmetAkifErsoyUniversity,Burdur,Turkey

c

DepartmentofPharmacologyandToxicology,FacultyofVeterinaryMedicine,AnkaraUniversity,06110Diskapi,Ankara,Turkey

d

MicrobiologyLaboratory,SivasNumuneHospital,Sivas,Turkey

e

FacultyofMedicine,MedicalGenetics,SelçukUniversity,Konya,Turkey

ARTICLE INFO Articlehistory: Received31May2019

Receivedinrevisedform3August2020 Accepted18August2020

Availableonline18September2020 Keywords: Drugresistance Geneticdiversity MIRU-VNTR Mycobacteriumtuberculosis Spoligotyping Turkey ABSTRACT

Objectives:Appropriateantibiotictherapyandpreventionofcross-contaminationarethemostimportant subjectsintuberculosis(TB)control.Theaimofthisstudywastoinvestigatethemajorphylogenetic cladesandtransmissionrateofmultidrug-resistant(MDR)Mycobacteriumtuberculosisisolates(n=200) frompatientswithTBinSivasandKonyaProvincesofTurkey.

Methods:Thephylogeneticrelationshipamongtheisolateswasinvestigatedbyspoligotypingmethod.In addition, the 24-locus mycobacterial interspersed repetitive unit–variable-number tandem repeat (MIRU-VNTR)typingmethodwasusedtorevealcross-contamination.

Results:Spoligotypingrevealed13differentspoligotypes.Atotalof188strains(94.0%)wereincludedin thecluster.ThemostprominentspoligofamilywastheTfamily(43.0%ofstrains),followedbyLAM (26.0%),H(8.0%),XandS(both6.0%)andU(5.0%).Also,12strains(6.0%)belongedtotheBeijingprofile. MIRU-VNTRresultsshowed176(88.0%) differentgenotypesamongtheisolates.Intotal,24strains (12.0%)wereinthecluster.

Conclusions:Accordingtospoligotyping,thereisaheterogeneousM.tuberculosispopulationinTurkey. MIRU-VNTRresultsshowedthatcross-contaminationobservedbetweenMDRM.tuberculosisisolatesin Turkeyiscontrollable.

©2020TheAuthor(s).PublishedbyElsevierLtdonbehalfofInternationalSocietyforAntimicrobial Chemotherapy.ThisisanopenaccessarticleundertheCCBY-NC-NDlicense(http://creativecommons.

org/licenses/by-nc-nd/4.0/).

1.Introduction

Tuberculosis(TB),causedbyMycobacteriumtuberculosis,isone oftheoldestknowndiseasesandistheleadinginfectiouscauseof deathworldwide.DespiteeffortstopreventandcontrolglobalTB infections,itcontinuoustobeanimportantpublic-healthissuein many countries. Multidrug-resistant (MDR) TB occurs when bacteriabecomeresistanttoagentsusedinthetreatmentofthe infectionand isa severeproblemthatis becomingincreasingly prevalentworldwide. Thefactthat MDR-TBhasreacheda high prevalenceworldwidehasincreasedtheimportanceof epidemi-ologyandcontrolprogrammesforTB[1,2].AccordingtotheWorld HealthOrganization(WHO)2018report,itwasestimatedthatin 2017thenumberofTBpatientsintheworldwasapproximately11

millionandthat1.6millionTBdeathsoccurred;moreover,itwas estimatedthatapproximately458000oftheTBcaseswere MDR-TB.InTurkeyin2017,theTBmortalityrate(per100000people) andtheincidencerate(per100000people)werereportedas0.53 and17,respectively.ThetotalnumberofTBcaseswas12046in Turkey in 2017 [2]. Of the 6028 cases that underwent drug susceptibilitytesting,200(3.3%)werefoundtobeMDR-TB[3].

ProtectionfromTBispossiblewithbacillusCalmette–Guerin vaccinationandeffectivelybreakingthechainoftransmissionas well as the use of isoniazid preventive therapy. Molecular epidemiological methods are the most preferred methods for breaking the infection chain, detecting sources and routes of transmission[4,5].Detectionofsourceand transmissionroutes, identificationofcross-contaminationinthelaboratory, differenti-ationofre-infectionandre-activation,understandingof transmis-siondynamicsandcharacteristics,thedynamicsofepidemics,the spreadof thediseasein thecommunity,andinformation about epidemiology can be achieved by molecular typing of strains

*Correspondingauthor.

E-mailaddress:lantintas@ankara.edu.tr(L.Altintas).

http://dx.doi.org/10.1016/j.jgar.2020.08.012

2213-7165/©2020TheAuthor(s).PublishedbyElsevierLtdonbehalfofInternationalSocietyforAntimicrobialChemotherapy.ThisisanopenaccessarticleundertheCC BY-NC-NDlicense(http://creativecommons.org/licenses/by-nc-nd/4.0/).

ContentslistsavailableatScienceDirect

Journal

of

Global

Antimicrobial

Resistance

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isolatedfromTBpatients[6–8].Accurateandearlydetermination of M. tuberculosis complex separation and drug resistance in patients suspected of TB is crucial to establish an effective treatmentprotocoland forpromptimplementationofinfection preventionandcontrolmeasures[9].

The emergence of MDR phenotypes in M. tuberculosis is a seriousprobleminthetreatmentofTB.Investigationofresistance mechanism using molecular techniques provides important information regarding the level of resistance to antitubercular drugsaswellasbasicknowledgeoftheteststobedevelopedfor rapiddiagnosispurposes.Molecularstraintypingtechniquesused toenhanceourunderstandingofclonalandphylogenetic relation-ships of M. tuberculosis strains provide information on the epidemiology of TB such as disease surveillance, outbreak investigations, transmission patterns and risk factors among apparentlydisparate cases in a population, among populations andthroughouttheworld[10–13].Clusteringisconsideredtobe anindicatorofrecentorongoingtransmission[14,15].Usingrecent TBtransmissioninastudypopulation,theeffectivenessofaTB controlprogrammeinthisenvironmentcanbeestimated.Boththe highincidenceofTBdrugresistanceandhighTBtransmissionrates in Turkey are an indication of the shortcomings of TB control programmes[10,15–17].

Molecularepidemiological studies have been very useful in outbreak investigation, determining re-infection,understanding virulenceand resistancemechanismsin differentstrains, deter-miningthespreadofinfectiousdiseases,determiningriskfactors causedbythespreadofthedisease,anddevelopingtreatmentand prevention strategies [4]. Molecular typing methods including spoligotyping and mycobacterial interspersed repetitive unit– variable-numbertandemrepeat(MIRU-VNTR)providean under-standingofthetransmissiondynamicsofTBwithinacommunity and offer the opportunity to develop more effective control measuresagainstthedisease[4,7].Despitetherapidincreasein thestudyof themolecularepidemiology ofTB in theworldin recentyears,researchconductedtoidentifytheepidemiological characteristicsofM.tuberculosisinTurkeyislimited.Theaimof thisstudywastodeterminetheclonalrelationshipbetweenM. tuberculosisstrainsbyspoligotypingandMIRU-VNTR.

2.Methods

2.1.Clinicalisolatesandidentification

Atotal of 200 M. tuberculosis strains that wereresistantto rifampicin (RIF) and/or isoniazid (INH), isolated from patients (males53.0%,females47.0%)withTBbetween2008and2019in SivasProvince (n=80)andKonyaProvince(n=120)ofTurkey were investigated in the present study. Regarding patient age, 70.0%werebetweentheageof20–50yearsand30.0%wereaged >50 years. Informed consent and ethical approval were not requiredsincepatientdatawereanonymised.Isolatesthatwere resistanttoat leastINHand RIFweredefined asMDR.Isolates were identified as M. tuberculosis using routine cultural, biochemicalandphenotypictests.Drugresistanceto streptomy-cin, INH, RIF and ethambutol was determined using the 1.0% proportionmethod.Afterdecontaminationand homogenisation ofclinicalsampleswith4.0%NaOH-NALC,microscopic examina-tionwithacid-faststainingandcultureonLöwenstein–Jensen(LJ) medium (Salubris, Istanbul, Turkey) was performed. Positive cultures were submitted to M. tuberculosis identification and differentialtestingincludingniacinaccumulation,nitrate reduc-tionandheat-stable catalasereaction.Thestandardstrainused for control of the drug susceptibilitytests was M. tuberculosis H37Rv(ATCC 27,294).

2.2.Antibioticsusceptibilitytesting

Susceptibilityof the strainsto INHand RIF was examined using the 1.0% proportion methods as described by Ceyahan et al.[18]. Foreach isolate,three 50mL polypropylene tubes with a conical bottom were used. To prepare the inoculum suspension,alargenumberofbacteriafromthecoloniesgrown on LJ mediumwere taken into a tubecontaining 10–12glass beads and 6 mL of sterile distilled water. The bacterial suspensionwas homogenised withmixing by vortexandwas allowedtosettlefor30mintoprecipitatethelargepartsinthe suspension. The turbidity of the inoculum suspension was archivedtobeequivalentto107CFU/mL.Dilutionsof102CFU/ mLand104CFU/mLwerepreparedfromthissuspension.After cultivation,themediumwasincubatedat37Cfor4weeksina 5–10%CO2 incubator(Nüve,Ankara, Turkey).Antibiotic tubes withgrowthwerecomparedwiththereproductiverateinthe controlmediumandtherateofresistance(numberofcolonies >1.0%,resistant)wasevaluated.

2.3.ExtractionofgenomicDNA

TodeterminetheclonalrelationshipofM.tuberculosisstrains via spoligotyping, DNAextraction was performed fromLJ solid mediumbytheCTAB(cetyltrimethylammoniumbromide) meth-od.AfterDNAextraction,theconcentrationofDNAwasestimated by spectrophotometric measurement. Spoligotyping was per-formedonDNAextractfromeachM.tuberculosisisolate[19]. 2.4.Spoligotyping

Spoligotyping was performed on extracted DNA using the method described by Kamerbeek et al. [20]. Briefly, primers targeting the direct repeat (DR) regions were synthesised (Fermentas,Vilnius,Lithuania).AtthePCRstageofthe spoligotyp-ing method, amplification of the spacers was performed using primersspecifictotheDRregions.Ineachstudy,M.tuberculosis H37Rvstrain(ATCC27,294) wasused asapositive control,and distilledwaterwasusedasanegativecontrol.Thebiotin-labelled amplifiedPCRproductwashybridisedwithdifferent membrane-coupled spacer probes specific for M. tuberculosis. After the membranes were exposed to X-ray film, hybridised spacers produced black dots on the membrane. The presence of a hybridised spacer was detected as a positive signal, and the absenceofspacerhybridisationwasconsideredanegativesignal. Thebinaryoroctalvaluesofthespoligotypingpatternsobtained foreach strainwereenteredintotheSpolDB4databaseandthe spoligotypeinternationaltype(SIT)number,clustersandfamilies ofthestrainweredetermined.

2.5.MIRU-VNTR

24-LocusMIRU-VNTRgenotypingofM.tuberculosiswascarried outbythemethoddescribedbySupplyetal.[21].Theprimersfor MIRU loci were synthesised (Table 1). MIRU-VNTR loci were amplified with eight different multiplex PCR mixtures using fluorescently-labelled oligonucleotide primers. Amplification products weresubjected toelectrophoresis and the number of MIRUalleleswasdeterminedaccordingtothesizeofPCRproducts ateachlocus(Table2).ThepositivePCRcontrolwasDNAfromM. tuberculosisH37Rvstrain,andthenegativecontrolwasdistilled water. ThisDNAwas usedforqualitycontrol.Whentheresults were incompatible, device control was performed using the fragment analysiscontrolsample (PN608105; Beckman Coulter, USA).

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2.6.Statisticalanalysis

Molecular typing results were analysed using BioNumerics v.3.1(AppliedMaths,Sint-Martens-Latem,Belgium).Thepairwise distance between clinical isolates was computed using the 1-Jaccardindex[22].TheUPGMA (unweightedpair-groupmethod usingarithmeticaverage)wasusedforclusteringoftheisolates

[23]. 3.Results

MycobacteriumtuberculosisisolatesconfirmedtobeMDRusing the proportion method were studied by molecular methods. Dependingonthehybridisationsignalofthe43spacersonthe spoligotypingmembrane(blackcolour formationorcolourless),

octalcodesspecificforeachstrainweredetermined.Clustering,SIT numbersandmajorfamiliesweredeterminedusingthesecodes.A totalof188strains(94.0%)wereincludedinthecluster.Thirteen different SITs were detected from 200 M. tuberculosis isolates (Table3).SIT53formedthebiggestclusterconstituting72isolates (36.0%),followedbySIT41andSIT1261with26isolates(13.0%)and 20isolates(10.0%),respectively.SIT1105,SIT91,SIT35,SIT1073and SIT1 each comprised 12 isolates (6.0%). SIT608 and SIT363 comprised 8 isolates (4.0%) each. And SIT20, SIT50, SIT34 comprised2isolates(1.0%)each.

Table1

Mycobacterialinterspersedrepetitiveunit(MIRU)lociandprimersequencesused.

Multiplex Alias PCRprimerpair(5ʹ→3ʹ)

Mix1 MIRU4 GCGCGAGAGCCCGAACTGC(5.DYD2)GCGCAGCAGAAACGCCAGC MIRU26 TAGGTCTACCGTCGAAATCTGTGACCATAGGCGACCAGGCGAATAG(5.CY5.5) MIRU40 GGGTTGCTGGATGACAACGTGT(5.CY5.5)GGGTGATCTCGGCGAAATCAGATA Mix2 MIRU10 GTTCTTGACCAACTGCAGTCGTCCGCCACCTTGGTGATCAGCTACCT(5.DYD2)

MIRU16 TCGGTGATCGGGTCCAGTCCAAGTACCCGTCGTGCAGCCCTGGTAC(5.CY5.5) MIRU31 ACTGATTGGCTTCATACGGCTTTAGTGCCGACGTGGTCTTGAT(CY5)

Mix3 VNTR42/MTUB04 CTTGGCCGGCATCAAGCGCATTATTGGCAGCAGAGCCCGGGATTCTTC(5.DYD2) VNTR43/ETR-C CGAGAGTGGCAGTGGCGGTTATCT(5.CY5.5)AATGACTTGAACGCGCAAATTGTGA ETRA AAATCGGTCCCATCACCTTCTTAT(CY5)CGAAGCCTGGGGTGCCCGCGATTT Mix4 VNTR47/MTUB30 CTTGAAGCCCCGGTCTCATCTGT(5.DYD2)ACTTGAACCCCCACGCCCATTAGTA

VNTR52/MTUB39 CGGTGGAGGCGATGAACGTCTTC(5.CY5.5)TAGAGCGGCACGGGGGAAAGCTTAG VNTR53/QUB-4156C TGACCACGGATTGCTCTAGTGCCGGCGTCCATGTT(CY5)

Mix5 QUB-11b CGTAAGGGGGATGCGGGAAATAGGCGAAGTGAATGGTGGCAT(5.DYD2) MTUB21/VNTR1995 AGATCCCAGTTGTCGTCGTC(5.CY5.5)CAACATCGCCTGGTTCTGTA QUB-26 AACGCTCAGCTGTCGGAT(CY5)CGGCCGTGCCGGCCAGGTCCTTCCCGAT Mix6 MIRU2 TGGACTTGCAGCAATGGACCAACTTACTCGGACGCCGGCTCAAAAT(5.DYD2)

MIRU23 CTGTCGATGGCCGCAACAAAACG(5.CY5.5)AGCTCAACGGGTTCGCCCTTTTGTC MIRU39 CGCATCGACAAACTGGAGCCAAACCGGAAACGTCTACGCCCCACACAT(CY5) Mix7 MIRU20 TCGGAGAGATGCCCTTCGAGTTAG(5.DYD2)GGAGACCGCGACCAGGTACTTGTA

MIRU24 CGACCAAGATGTGCAGGAATACATGGGCGAGTTGAGCTCACAGAA(5.CY5.5) MIRU27/QUB5 TCGAAAGCCTCTGCGTGCCAGTAAGCGATGTGAGCGTGCCACTCAA(CY5) Mix8 MTUB29/VNTR46 GCCAGCCGCCGTGCATAAACCT(5.DYD2)AGCCACCCGGTGTGCCTTGTATGAC

ETR-B/VNTR48 ATGGCCACCCGATACCGCTTCAGT(5.CY5.5)CGACGGGCCATCTTGGATCAGCTAC MTUB34/VNTR49 GGTGCGCACCTGCTCCAGATAA(CY5)GGCTCTCATTGCTGGAGGGTTGTAC

Table2

Mycobacterialinterspersedrepetitiveunit–variable-numbertandemrepeat (MIRU-VNTR)repeatnumbersandampliconlengthofMIRUloci.

Multiplex Alias Ampliconlength(bp) Repeatunitlength(bp) Mix3 MTUB04 537 51 ETR-C 171 58 ETRA 195 75 Mix5 QUB-11b 67 69 MTUB21 116 57 QUB-26 153 111 Mix7 MIRU20 437 77 MIRU24 395 54 MIRU27 498 53 Mix2 MIRU10 482 53 MIRU16 565 53 MIRU31 492 53 Mix8 MTUB29 335 57 ETR-B 347 57 MTUB34 326 54 Mix6 MIRU2 402 53 MIRU23 150 53 MIRU39 540 53 Mix1 MIRU4 122 77 MIRU26 285 51 MIRU40 354 54 Mix4 Mtub30 252 58 MTUB39 540 53 QUB-4156 563 59 Table3

Distribution of spoligotypesidentified among 200Mycobacterium tuberculosis isolates.

Spoligotype(SIT) No.(%)ofisolates

53 72(36.0) 1261 20(10.0) 41 26(13.0) 1105 12(6.0) 91 12(6.0) 35 12(6.0) 1073 12(6.0) 608 8(4.0) 363 8(4.0) 1 12(6.0) 20 2(1.0) 50 2(1.0) 34 2(1.0)

SIT,spoligotypeinternationaltype.

Table4

Distributionofmajorspoligofamilies.

Spoligofamily No.(%)ofisolates

T 86(43.0) LAM 52(26.0) H 16(8.0) X 12(6.0) S 12(6.0) Beijing 12(6.0) U 10(5.0)

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Ofthe200M.tuberculosisisolates,86(43.0%)and52(26.0%) werefromtheTand theLatinAmerican–Mediterranean(LAM) family,respectively.Inaddition,16isolateswerefromtheHaarlem (H) family (8.0%), 24 (12.0%) isolates were from the X and S families,12(6.0%)isolateswerefromBeijingfamilyand10(5.0%) isolateswerefromtheUfamily.Majorfamilygroupsaregivenin

Table4.

Usingthemolecularweightstandard,thebandsizesoftheloci ineachmultiplexPCRmixturewerecalculated.TheMIRU-VNTR profile,consistingof24digits,wasdeterminedfromlocussizes.A dendrogram was created using GelCompar software (Applied Maths,Inc.,AustinTX,USA)usingMIRU-VNTRdataforeachisolate (notshown).Clusteringbetweenisolateswasdetermined.There were176differentgenotypesamong200M.tuberculosisisolates typedbytheMIRU-VNTRmethod.Atotal of24isolates(12.0%) wereincludedinthecluster.

4.Discussion

Zozioet al.reported thatamong 245M.tuberculosis clinical isolatesobtainedfromAnkaraandMalatyaregion,206formed33 spoligotype clusters(clusteringrate 84.1%),whereas 39isolates wereincludedinsingle-memberedspecialclusters[13].Theyalso reportedthat thelargest clusterconsistedof LAM7-TUR family (21.0%),followedbyclustersofT1with16.3%andHaarlemwith 5.3%,whereasBeijinggenotypedeterminedtobeassociatedwith multidrug resistance was not encountered. Another study in Gaziantep Province of Turkey determined the distribution and epidemiologicalfeaturesofMDRM.tuberculosisisolatesobtained from Medical Faculty Hospital of Gaziantep University with molecular methods including PCR–restriction enzyme length polymorphism(PCR-RFLP)and spoligotyping.Theresultsofthis studydemonstratedthatspoligotypingof55isolatesdetected13 different genetic profiles. It was reported that T1 (64.0%) and LAM7-TUR(18.0%)familieswerethemostprevalentamongthe55 M. tuberculosis isolates, whereas the Beijing genotype was not detected [17]. In a study by Durmaz et al., drug-resistant M. tuberculosisisolatesobtainedinfourdifferentregionsofTurkey (Marmara,n=81;Mediterranean,n =39;Aegean,n =42;East Anatolia,n=38)weretypedbymethodsincludingspoligotyping andIS6110-RFLP[10].ST1(22.5%),knownasLAM7-TUR,wasthe predominantsharedtype(ST)detectedintheisolates,followedby ST53(19.5%, ill-defined Tsuperfamily)with39 of strains, ST50 (6.5%,Haarlem3)with13strains,ST1261(4.5%)whichiscloseto theLAM7-TURlineagewith9strains,andST47(Haarlem1,3.5%) with7 strains. The global distributionof major M. tuberculosis lineagesamongdrug-resistantstrainswasasfollows:T superfam-ily(29.0%),LAM(33.5%),Haarlem(14.0%),andtheSlineage(3.0%). Also,Beijinggenotypewasreportedtobenotencounteredintheir study. The results of an another study with a total of 467 M. tuberculosisstrainsisolated fromJanuary2007–June2010 from patientswithpulmonaryTBintheEasternMediterraneanpartof Turkeyshowedthat443(94.9%)ofthestrainswereclusteredin21 groupsandmostcommonfamilieswereT1genotypeseenin239 (51.9%)strainsandLAM7-TURgenotypeseenin54(11.5%)strains. Sixstrains(1.3%)isolatedfromSanliurfaProvinceofTurkeywere MDRstrainsandbelongedtotheBeijingfamily[24].Inanother study,theIS6110andpTBN12RFLPmethodswereusedfortyping ofM.tuberculosisstrains,suggestingthatRIF-resistantisolateshad arelativelyhighclusteringrateandwereclonallyrelated.These findingssuggestthatRIF-resistantisolatesaretransmittedamong patients [25]. In the present study conducted to elucidate the degreeofcross-contaminationbetweenMDRisolatesandtoreveal phylogeneticanalysisoftheseisolates,SIT53,SIT1261andSIT41 weremuchmoreprevalentspoligotypesintheseisolates.Ofthe isolates, 69.0% were in the T and LAM major families. It was

observedthatthedegreeofcross-contaminationwashighamong theMDRisolates.Theclusteringratewas foundtobeveryhigh (94.0%). Whilst the majority of the isolates belonging to three majorfamiliesinourstudywerealsoobservedinsimilarstudiesin otherregionsofTurkey[10,24,26],majorfamiliesvariedinsome regionsofTurkey[27].

AstudyperformedbyNiemanetal.intheRepublicofGeorgia showedthatthemostprominentfamilywith26.0%ofthestrains was Beijing,followedbyLAM(18.0%),Ural(12.0%)andHaarlem (5.0%)strains[28].In Bulgaria,themajorlineagewasT1(ST53; 25.7%)followedbytheLAM7-TUR(ST41)[29].InIran,Rohanietal. reportedthatthelargestclusterconsistsoforphanpatternswith 13 strains(11.5%), and theBeijing genotype was 7.1% [30].For Europeancountries,isolatesoftheTandLAMfamiliesareslightly higherthaninnorthernEuropeancountries[31].Thedistribution offamilies inourstudyissimilartothatreportedforEuropean countriesandsomeAsiancountries.Thesedataindicatethatthere is no dominant M. tuberculosis family in Turkey. According to spoligotyping data that are prominent in phylogenetic studies, thereisaheterogeneousTBbacilluspopulationinTurkey.Whilst SIT53 is widely observed all over the world, SIT41 is widely observedinTurkeyanditsneighbours.Morethan5.0%ofstrains exhibitspecificprofilesthatarenotavailableintheworlddatabase. TheprevalenceofSIT41inthespoligotypingmethodinTurkeyand neighbouringcountriesisabovetheworldaverageshowingthe importanceofmigrationandhumanmovementsinthespreadof M.tuberculosisgenotypes.ReasonsforthehighprevalenceofSIT41 inneighbouringcountriesmaybeduetothehistoricalwavesof migrationandthepossibleTurkishphylogeographicspecificityof theST41clonecomplex.

TheBeijingsublineageisagroupofgloballyemergingstrains thatareresponsiblefor>25%ofallcasesofTBinvariousareasof theworld.GlobalemergenceofBeijingandBeijing-likestrainsis oftenassociatedwithitshypervirulenceanddrugresistance.The BeijingfamilyhasexpandedandemergedworldwidefromEast Asia and the former Soviet Union [32]. Turkey is a country bridgingthetwocontinents oftheoldworld,AsiaandEurope. Among the countries in our region, studies monitoring M. tuberculosiscomplex membersusing molecularepidemiological methodsarequitelimited.Theresultsobtainedinthesestudies showthattheBeijinggenotypeisbecomingincreasinglycommon ineasternneighboursofTurkey[28,30].Similarly,morerecently, anincreaseintheincidenceofBeijingfamilyhasbeenobservedin various regions of Turkey [10,24,26,27,33]. This maybe due to uncontrolledmigrationfromAsia(especiallySyriaandIraq)and AfricatoTurkey.TheoccurrenceofthissubgroupinTurkeymay causeseriousproblemsinTB controlprogrammesowingtothe prevalenceofmultidrugresistancecharacterisedbyRIFresistance amongtheisolates.

Inthepresentstudy,theclusteringratewasfoundtobevery high (94.0%) by spoligotyping. The isolates, which have low discriminatorypowerbyspoligotyping,fromTurkeyhavehada highclusteringraterangingfrom38.5%to94.0%[10,13,24,26,34]

and, toimprovediscriminationonthestrains, additionaluseof anothermoleculartypingmethodsuchasMIRU-VNTRisneeded. Inthis study,therewere176differentgenotypesamong200M. tuberculosisisolatestypedbyMIRU-VNTR.Atotal of24 isolates (12.0%) were included in the cluster. Similar to other studies

[24,34],thehighdiscriminatorypoweroftheMIRU-VNTRmethod was also observed in this study. Although the spoligotyping methodrevealed188isolatesthatweregroupedintotheclusters, MIRU-VNTR method revealed 24 isolates were included in the cluster.Thespoligotypingmethodaloneisinsufficienttodetect cross-contamination and for epidemiological links among the isolates.ThesefindingsshowthatacombinationofMIRU-VNTR and spoligotypingprovides betterdiscrimination and therefore

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maybe bestgenotypingtool tounderstandtransmission ofM. tuberculosisincommunities.

5.Conclusions

Thefindings reportedfromdifferentregionsof Turkey were similarintermsofSITsandmajorspoligofamiliescomparedwith ourstudy.Inaddition,thefindingsfromthepresentstudyindicate that the high clustering rate determined by spoligotyping was insufficienttorevealtheactualcross-contamination,suggesting thatdatashouldbeconfirmedbyothertechniqueswithhigher discriminatory power such as MIRU-VNTR or IS6110-RFLP. The MIRU-VNTR results show that cross-contamination (12.0%) ob-servedbetweenMDR-TBstrainsinTurkeyiscontrollable. Funding

None.

Conflictofinterests Nonedeclared. Ethicalapproval

Notrequired. References

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