Characterization of Klebsiella pneumoniae associated with cattle infections in southwest China using multi-locus sequence typing (MLST), antibiotic resistance and virulence-associated gene profile analysis

h tt p : / / w w w . b j m i c r o b i o l . c o m . b r /

Veterinary

Microbiology

Characterization

of

Klebsiella

pneumoniae

associated

with

cattle

infections

in

southwest

China

using

multi-locus

sequence

typing

(MLST),

antibiotic

resistance

and

virulence-associated

gene

profile

analysis

Fangjun

Cheng

a,b,∗,1

_,

_Zhangcheng

_Li

a,1

_,

_Shimei

_Lan

a

_,

_Wei

_Liu

a

_,

_Xiaoyan

_Li

a

_,

Zuoyong

Zhou

a,b

_,

_Zhenhui

_Song

a,b

_,

_Juan

_Wu

a

_,

_Manli

_Zhang

a

_,

_Wenjie

_Shan

a

a_{RongchangCampusofSouthwestUniversity,DepartmentofVeterinaryMedicine,Chongqing,China} b_{ChongqingEngineeringResearchCenterofVeterinaryScience,Chongqing402460,China}

a

r

t

i

c

l

e

i

n

f

o

Articlehistory:

Received20February2018 Accepted22June2018

Availableonline16August2018 AssociateEditor:NiltonLincopan

Keywords: Cattle Antimicrobialsusceptibility Klebsiellapneumonia MLST Virulencegenes

a

b

s

t

r

a

c

t

Klebsiellapneumoniaeisimportanthumanandanimalpathogenthatcausesawide spec-trumofinfections.Inthisstudy,isolatesfromcattlenasalswabssampleswereidentified by16SrRNA,andtoevaluatetheantimicrobialsusceptibility,virulencegenecarrying lev-els,andmultilocussequencetypingofK.pneumoniaeisolates.33isolatesofK.pneumoniae

wereisolatedandidentifiedin213nasalswabssamples,ofwhich12werehypervirulentK. pneumoniaestrains.ExtendedSpectrumBeta-Lactamasesgeneswerefoundin93.4%ofthe strains.Ofwhich,TEMwasthemostprevalent(93.4%),followedbyCTX-MandSHVwere 57.6%and39.4%,respectively.Amainmutationpatternofquinoloneresistance-determining region,Thr83-IeuandAsp87-AsningyrAandSer87-IleinparC,wasdetectedin33K. pneumo-niaeisolates.Alltheisolatesharboredatleasttwovirulencefactorgenes,withureA(97.0%) andwabG(91.0%)exhibitinghighcarriageratesin33K.pneumoniaeisolates.MLSTrevealed 7sequencetypes,ofwhich3STs(2541,2581and2844)werenewlyassigned.UsingeBURST, ST2844andST2541wereassignedtonewclonalcomplex2844.Ourstudyprovidesevidence andbiologicalcharacteristicsofK.pneumoniaeisolatesfromcattleupperrespiratorytractin SouthwestChina.

©2018SociedadeBrasileiradeMicrobiologia.PublishedbyElsevierEditoraLtda.Thisis anopenaccessarticleundertheCCBY-NC-NDlicense(http://creativecommons.org/ licenses/by-nc-nd/4.0/).

∗ _{Correspondingauthor.}

E-mail:cfj-xn@163.com(F.Cheng).

1 _{Theseauthorscontributedequallytothisworkandshouldbeconsideredco-firstauthors.} https://doi.org/10.1016/j.bjm.2018.06.004

1517-8382/©2018SociedadeBrasileiradeMicrobiologia.PublishedbyElsevierEditoraLtda.ThisisanopenaccessarticleundertheCC BY-NC-NDlicense(http://creativecommons.org/licenses/by-nc-nd/4.0/).

94

brazilian journalof microbiology49S(2018)93–100

Introduction

Klebsiella pneumoniae (K. pneumoniae) widely existed in sur-facewater,sewage,soil,plantsandthemucosalsurfacesof mammals.1 _{Due toits broadspectrumofvirulencefactors,} the infection of K. pneumoniae usually plays an important role for the causing of pneumonia, bloodstream infection and pyogenic liver abscesses inmammals.1,2 _{The invasion} ofK.pneumoniaeindomesticanimals notonlycauses haz-ardinlivestockproductionbutalsoposesapotentialthreat topublic health since theseanimals can act as the reser-voirof multidrug-resistant K. pneumoniaestrains. Although antibiotictherapyisawidelytoolforthetreatmentof infec-tionscausedbyK.pneumoniae,however,antibioticresistance in pathogenic bacteria from food-producing animals and environmental sources is recognized as a global problem for public health.3 _{Isolates of K. pneumoniae often display} drug resistance phenotypes, making difficulty in choosing sensitive antibiotics for treatment.4 _{The ciprofloxacin is} widely used in the treatment of K. pneumoniae infections in recent years, and the increasing resistance to fluoro-quinolonesamongK.pneumoniaehasalsobeen reported.5,6 Fluoroquinolones act by inhibiting the action of target enzymes, DNA gyrase and topoisomerase IV, with both enzymesplayingaprincipalrole inDNAreplication.7 _Some researchersthinkthatalterationsinthe quinoloneresistance-determining region (QRDR) within DNA gyrase (gyrA and

gyrB) and topoisomerase IV (parC and parE) are the major mechanisms one for fluoroquinolone resistance in

K.pneumoniae.6

Genotypingisimportanttoidentifycasesoroutbreaksdue toK.pneumoniaeand tofurthertracksourceand spreading ofinfections.4_{MLSTisconsideredagoodmethodfortyping} bacterialpathogensandisusedtocharacterizegenetic rela-tionshipsamongbacterialisolatesandtoidentifyandtrack the global spread of drug-resistant strains.8–10 _{Ithas been} shownthattheturkeysourceepidemicclone-relatedST15K. pneumoniaewas ableto colonizehumans and cause severe infections.11_{Inaddition,bothhandlingandconsumptionof} productsoftheseanimalscolonizedbyK.pneumoniaemaybe animportantsourceofantibiotic-resistantK.pneumoniaeof possiblehumanhealthsignificance.11_{However,incontrastto} thestudiesofK.pneumoniaeinfectionsinhumanandother animals,4,12 _{lessis knownonthe genotypingofK.}

pneumo-niaeincattleortheirrelevantproducts.Cattleasanimportant food-producinganimals,itisofimportancetounderstandthe prevalenceandgenotypingcharacterizationofK.pneumoniae

colonizingthecattle.

In 2008, Chongqing municipality was incorporated into the national “Advantage of agricultural products regional planning” in China and recognized as the key areas for beef cattle breeding.13 _{However, the genetic features and} microbiologicalCharacteristicsofK.pneumoniaeisolatesfrom cattleinSouthwestChinahavebeenpoorlyinvestigated.To understand the prevalence and characteristics of K. pneu-moniaein cattle,we usedthe nasal swabsfrom sick cattle to isolate K. pneumoniae, tested their antimicrobial sus-ceptibility and virulence gene carrying levels, and defined MLST.

Materials

and

methods

Isolationandidentification

Duringtheperiodfrom2014to2015,213nasalswabssamples were collected from 1 to 2years age of sick cattle suffer-ingfromrespiratorymanifestations.Allnasalswabsamples were collected from different farms located in Rongchang, Fengdu, Zigong and Yongchuan regions, China. Each sam-plewasplatedonLBagar (Oxoid,ThermoFisherScientific, China). Allplates were incubated at37◦C for24h.Isolated strainswerefurtherinvestigatedbypolymerasechain reac-tion(PCR)using16SribosomalRNAprimers27Fand1492R14 withthefollowingamplificationconditions:initial denatura-tionfor5minat94◦C,followedby30cyclesof94◦Cfor1min, 50◦Cfor1min,and72◦Cfor1min,withafinalelongationof 72◦Cfor10min.PCRproductsweresequencedbothin for-wardandreversedirectionsbyGeneCreateCo.,Ltd.(Wuhan, China).ThesequencingresultsofPCRproductswereanalyzed byBLAST(BasicLocalAlignmentSearchTool)sequencesearch usingtheNCBI(NationalCenterforBiotechnology Informa-tion)database.Phylogenetictreeswereconstructedforthe16S rRNAsequencesoftheisolatesbyMAFFTMultipleSequence Alignment andMEGA7.0.17 CharacterizationofidentifiedK. pneumoniaeisolateswerefurtheranalyzedusingMLST, antibi-oticresistanceandvirulence-associatedgeneprofile.

Antimicrobialsusceptibilitytesting

The evaluation ofspecimens for antibiotic resistance was conducted according to the clinical and laboratory stan-dards institute (CLSI) guidelines.15 _{Antibiotic susceptibility} testing was performed by Kirby–Bauer disk diffusion test. Each sample was tested against 8 antimicrobial agents (Oxoid, ThermoFisherScientific, China):Ampicillin (10␮g), Amoxicillin (10␮g), Gentamicin (10␮g), Cefotaxime (30␮g), Ceftriaxone(30␮g),Ceftazidime(30␮g),Ciprofloxacin(10␮g) and Amikacin (30␮g).The␤-lactamasegenes(CTX-M,TEM andSHVgene)oftheisolatesweredeterminedasdescribed byNewireetal.2

AmplificationandsequencingofthegyrAandparC fragments

ToinvestigatethecharacteristicsofgyrAandparCgenes alter-ations, gyrA and parC gene fragments were amplified and sequencedin33isolates.ThegyrAandparCprimerswereused asdescribedbyBrisseetal.16_{Thenucleotidesequencesand} thededucedaminoacidwerecomparedwiththatofK. pneu-moniaestrain459(GenBankaccessionnumberCP018306)using theClustalWmultiplesequencealignmentprograminMEGA 7.0.17

Stringtestanddetectionofvirulencegenes

PCRassaywereperformedtocheckforthepresenceof5genes thathavepreviouslyassociatedwithvirulenceinK. pneumo-niae,includingmagA,rmpA,mrkA,ureAandwabGgenes.18,19 Reference strains including ATCC 25922 and ATCC 700603.

Strainswiththehypermucoviscosityphenotypeweredefined ashigh virulent.20_{Stringtestwasperformedtodistinguish} hypervirulentK.pneumoniae(hvKP)fromclassicK.pneumoniae

(cKP).hvKPphenotypestrainswasdefinedbyapositive“string test,”which isthe formation ofaviscous string>5mm in lengthwhenbacterialcoloniesonanagarplatearestretched byaninoculationloop.20

MLSTanalysis

MLSTofK.pneumoniaeisolateswereperformedaspreviously describedbyPasteurInstituteMLSTDatabase.21_PCR ampli-ficationforsevenhousekeepinggenes (gapA,infB,mdh, pgi, phoE,rpoB,andtonB)wascarriedoutusingprimersand proto-colsavailableatthePasteurInstituteandUniversityCollege Corkschemedescribed.21_{TheeBURSTv3softwarewasusedto} investigatethepopulationdiversityandrelationshipbetween MLSTsequencetypes(STs)andtoanalyzeclonalcomplexes (CC)basedonthestringentgroupdefinitionofsixoutofseven shared alleles.To analyze the phylogenetic relationship of these33 K.pneumoniaeisolatesand previouslyidentifiedK. pneumoniaeisolatesglobally.AlongwiththeSTsobtainedin thisstudy,16STsfromhuman(ST208,347,388,475,597,712, 1096and1230)andbovinemastitis(ST87,98,99,103,104,110, 116and 1864)4,22 _{were downloadedfrom the K.pneumoniae} MLSTwebsite,concatenatedandalignedusingMEGA7.0and usedtoconstructtheNJphylogenetictreeofK.pneumoniae.

Bootstrappingwasperformedwith1000replicates.

Results

MicrobiologiccharacterizationoftheK.pneumoniae strains

Inthisstudy,33(15.5%)strainsofK.pneumoniaeweredetected inthe213casesinvestigatedby16SrRNAanalysis.TheNCBI BLASTprogramshowed thatthe sequence dataofisolated strainshadhighsimilarity(>99%)tothoseofKlebsiella pneumo-niaesubsp.NTUH-K2044(AP006725.1).Basedonthehomology of16SrRNAsequence,strainswith16SrRNAsequencefrom relativespecieswereselected,andmultiplesequence align-mentcomparisonwasperformedbyMAFFT-LINSIprogram.A phylogenetictreewasconstructedusingtheNJmethodbased onMEGA7.0software.Theresultsofphylogeneticplacement of33isolateswereshowninFig.1.Theseisolatesweremainly clusteredinKlebsiellapneumoniaesubsp(Group1).Thegene sequenceof16SrRNAwasdepositedintheNCBInucleotide sequencedatabaseunderaccessionNo.MF767570-MF767596, MG859650-MG859655.

AntimicrobialsusceptibilityandQRDRsmutations

33 K. pneumoniae isolates resistance rates to Ampicillin, Amoxicillin,Ciprofloxacin,Cefotaxime,Ceftriaxone, Gentam-icin, Ceftazidime and Amikacin were 93.9%, 81.8%, 60.6%, 57.6%,33.3%,27.3%,18.2%and 9.1%,respectively.Extended Spectrum Beta-Lactamases (ESBLs) genes were found in 93.4%ofthestrains.Ofwhich,TEMwasthemostcommon (93.4%),followedbyCTX-MandSHVwere57.6%and39.4%,

respectively(Table1).Among33isolates,19wererevealedto haveaminoacidalterationsingyrAorparCQRDR(Table1). Thetotal mutationsfound intheseisolateswere classified into6distinctgroupsaccordingtothepatternofaminoacid alteration, in which group I showed the most common in 33 isolates.Itisnotablethatallisolatesingroup1showed ciprofloxacin resistancecharacteristics,whichmay indicate that this mutationpattern is asuitable modelfor K. pneu-moniaeresistancetociprofloxacin.Ofsixgroups,onlygroup III had two mutations in gyrA (Ser83-Thr; Asp87-Tyr) and

parC (Ser80-Ile; Glu84-Lys), respectively. DNA sequences of QRDRgyrAshowedadoublemutationingyrAwasdetected in 15 of 33 isolates. The sequences of the gyrA and parC

genes in clinical isolates of K. pneumoniae have been sub-mitted tothe GenBank database underaccession numbers MG758055-MG758108;MG869692-MG869703.

DistributionofvirulencegenesamongK.pneumoniae isolates

TheureAgenesweredetectedinmajority(97.0%)ofthestrains, andwabGgenewasfoundin30(91.0%),followedbymrkAand

rmpAin57.6%isolateseach,magA(42.4%).Accordingtothe stringtestresults,12hvKPstrainswereidentifiedinthe33 isolates(Table1).

SequencetypesofK.pneumoniastrains

In this experiment, a total of7 STs were found inthe 33

K.pneumoniaestrains, ofwhich 3STs werenewlyassigned. The4knownMLSTgenotypeswere identified:ST146(n=1), ST294(n=3),ST328(n=1),ST218(n=19).The3newlyMLST genotypes were identified and added to the K. pneumoniae

MLSTdatabase:ST2844(3-2-4-181-9-4-177;n=3),ST2541 (3-2-4-1-9-4-177;n=2),ST2581(10-1-1-14-26-1-12;n=4).eBURST generated 232 groups and 923 singletons (Until January 9, 2018).TheK.pneumoniaeisolatestestedinthisstudywere clus-tered intoCC11containingST294and ST218,andafurther CC2844 containingST2844and ST2541,whileST146, ST328 and ST2581wasasingleton (Fig.2).Thephylogenetic rela-tionshipof21STs,basedonconcatenatedsequencesofseven MLSTalleles,resultedintwomainclusters:clusterI(mainly frombovine)andclusterII(fromhumans).InclusterI,there weretwosub-clusters(SCs)(Fig.3).FourK.pneumoniaeisolates fromCURTwereclusteredtoSC1,andSC2mainlycontainsof theclinicalmastitisisolatesfromcow.

Discussion

ManyreportssuggestthatK.pneumoniaeisoneofthemajor pathogensofbovinemastitis.23–25_{However,thereisverylittle} informationaboutK.pneumoniaeisolatesfromtheupper res-piratorytractofcattle.Inthestudy,33(15.5%)K.pneumoniae

strainswereisolatedfromtheupperrespiratorytractof cat-tle.OurresultswerehighertothedetectionfrequencyofK. pneumoniaeinZimbabwe,23_{butlowerthanthatinTurkey.}26

Themajorityof33K.pneumoniaeisolatesshowedrelatively lowresistancetoCeftazidimeandAmikacin,whichis simi-lartotheresistanceofK.pneumoniaeisolatedfrompatients

96

brazilian journalof microbiology49S(2018)93–100 K.pneumoniae QLR5-1 K.pneumoniae YYM20-3 K.pneumoniae QLR1-3 K.pneumoniae ZB20 K.pneumoniae QLR3-1 K.pneumoniae FD26-2 K.pneumoniae QLR5-3 K.pneumoniae YYM30-1 K.pneumoniae FDI-1 K.pneumoniae FD30-1 K.pneumoniae QLR6-2 K.pneumoniae YYM28-1 K.pneumoniae WJ44-1 K.pneumoniae QLR8-2 K.pneumoniae ZG19 K.pneumoniae YYR25 K.pneumoniae YYR52-1 K.pneumoniae WJ1-1 K.pneumoniae YYR54-1 K.pneumoniae ZG26 K.pneumoniae ZG14 K.pneumoniae QLR10-2 K.pneumoniae QLR2-3 K.pneumoniae ZG16 K.pneumoniae ZG2 K.pneumoniae YYM25-3 K.pneumoniae YYM20-1 K.pneumoniae QLR2-1

Klebsiella pneumoniae subsp. pneumoniae ATCC 13883 K.pneumoniae YYM25-1

K.pneumoniae QLR18-1 K.pneumoniae FD28-2 K.pneumoniae ZB27 K.pneumoniae YYM1-1

Klebsiella pneumoniae subsp. rhinoscleromatis ATCC13884 Klebsiella pneumoniae subsp.ozaenae ATCC 11296

Klebsiella trevisanii strain ATCC 33558T 16S ribosomal RNA gene Klebsiella terrigena 16S rRNA gene strain ATCC33257T Klebsiella michiganensis JQ070300

AF129440.1 Klebsiella oxytoca 16S ribosomal RNA gene Escherichia coli ATCC43895

Escherichia coli ATCC 35218 Escherichia coli.coil 11775T 100 100 91 81 75 100 100 100 100 100 95 43 33 31 56 58 75 52 74 21 48 51 12 45 11 23 25 14 10 11 31 14 1 1 1 12 28 6 1 0 0.0050 100

Outgroup

Group 2

Group 1

Fig.1–PhylogenetictreeoftheK.pneumoniaebasedon16SrRNAsequences.Note:The33sequencesdeterminedinthis studyareindicatedbyboldfont.3Escherichiacolistrains,Z83205.1,AM980865.1andX80725.1,wereusedasoutgroups; Bootstrapvaluesof1000replicationsareindicatedatbranches.Thesequencesingroup1correspondtotheclustersin

b r a z i l i a n j o u r n a l o f m i c r o b i o l o g y 4 9 S (2 0 1 8) 93–100

97

gyrA parC

Resistanttociprofloxacin Ser83(TCC) Asp87(GAC) Ser80(AGC) Glu84(GAA)

QLR8-2 Leu(TTG) Asn(AAC) Ile(ATC) I + + + + + 218 + + + +

FD1-1 Leu(TTG) Asn(AAC) Ile(ATC) I − + − + + 218 − + -

-FD26-2 Leu(TTG) Asn(AAC) Ile(ATC) I + − + + + 2844a _{− + + +}

FD28-2 Leu(TTG) Asn(AAC) Ile(ATC) I − − − + − 294 − + +

-QLR1-3 Leu(TTG) Asn(AAC) Ile(ATC) I + + + + + 218 + + +

-YYM28-1 Leu(TTG) Asn(AAC) Ile(ATC) I + + − + + 218 − + - +

FD30-1 Leu(TTG) Asn(AAC) Ile(ATC) I − + − + + 218 + + - +

QLR2-1 Thr(ACC) Tyr(TAC) Ile(ATC) II + + − + + 218 + + +

-YYR54-1 Thr(ACC) Tyr(TAC) Ile(ATC) II − + + + + 218 + + +

-ZB20 Thr(ACC) Tyr(TAC) Ile(ATC) II + − + + + 2844a _{− + -}

-YYM30-1 Thr(ACC) Tyr(TAC) Ile(ATC) II − + + − + 218 + + -

-ZB27 Thr(ACC) Tyr(TAC) Ile(ATC) II − + + + + 218 + + +

-YYM1-1 Thr(ACC) Tyr(TAC) Ile(ATC) Lys(AAA) III − − + + + 2581a _{− + + +}

YYM20-1 Thr(ACC) Try(TAC) IV − + + + + 218 + + +

-ZG14 Thr(ACC) Tyr(TAC) IV + − + + + 2844a _{− + + +} QLR8-1 Tyr(TAC) Ile(ATC) VI − + + + + 218 + + + -QLR10-2 Tyr(TAC) Ile(ATC) VI + + + + + 218 + + + + ZG26 Tyr(TAC) Ile(ATC) VI + − − + + 2541a _{− + - +} YYR25-1 + − − + − 146 − + - -ZG2 − − − + + 2581a _{− + +} -Susceptibletociprofloxacin WJ1-1 Tyr(TAC) V − − − + + 328 − + - + QLR3-1 + − + + + 2541a _{− + + +} QLR5-1 − + + + + 218 − - + -QLR5-3 − + + + + 218 − + + -QLR6-2 − + + + + 218 − + - -WJ44-1 − − − + + 2581a _{− + -} -YYR52-1 + + + + + 218 + + + + YYM20-3 + + + + + 218 + + + -YYM25-1 − + − + + 218 − + + -ZG16 − − − + + 294 − + - + ZG19 − − − + + 294 − - - -YYM25-3 + + − + + 218 + + - + QLR2-3 − − + + − 2581a _{− + -}

-Note:+:positivereaction;−:negativereaction.

98

brazilian journalof microbiology49S(2018)93–100 ST146 ST294 ST2581 ST328 ST2844 ST2541 ST2844 ST2729 ST2541 ST218 CC11 CC2844

Fig.2–ThepopulationsnapshotoftheKlebsiellapneumoniaeisolateswasdiagramedbyeBURSTonthebasisofallelic profiles.Note:The7STs(ST146,ST294,ST328,ST218,ST2844,ST2541andST2581.)inthisstudyareindicatedbyredfont.

1230 388 208 712 347 1096 475 597 84 50 34 26 43 41 100 294 110 87 98 99 47 47 28 35 116 1864 2541 2844 218 2581 103 104 99 49 52 41 24 39 24 0.0050 100 cluster I cluster II sub-clusters I sub-clusters II

Fig.3–PhylogeneticorganizationofK.pneumoniaeisolates ofdifferentMLSTsequencetypes(STs)basedon

neighbor-joiningmethod.Note:The5STsdeterminedin thisstudyareindicatedbyblackdot.

withhospital-acquiredpneumoniainAsian.27 _Inthisstudy, itisnoticeablethatK.pneumoniaeisolatesshowedrelatively highresistancetociprofloxacin,whichishigherthanthe resis-tance ofK. pneumoniaefrom patient inIranand China.28,29 Theprolongeduseofantibioticsinthetreatmentof respira-toryillnessandasgrowthpromoterswillledtotheadditional problemofemergenceantibioticresistantstrains,for exam-pletheresistant strainsentering thefoodchain.Therefore, thehighresistancestrainofciprofloxacinmaybeapotential publichealthhazardinchina.Severalreportshaveshown5,6 thatchangesinthestructureoftheQRDRsingyrAandparCare oneofthesignificantmechanismsinconferringaresistance tofluoroquinoloneingramnegativebacilli.Themutationsof Ser83withPhe,Ile,TyrandLeuisfrequentlydisplayedinK. pneumoniaeinsomestudies.5,30_{However,theresultsfromthis} studysuggestedthemainlyexistenceofSer83-Thralteration in K.pneumoniae isolates,which may besimplydueto the investigation ofanewpopulation,sincemostreportshave focusedonhumanclinicalisolatesorbovinemastitis.Inthis study,itwasfoundthatintheCiprofloxacin-resistantK. pneu-moniae, the mutationof the 80thsite inthe bacterial parC

genewascommonlyaccompaniedbythemutationofthegyrA

gene,andwhenparCgeneandgyrAgenemutatedthesites together,the Ciprofloxacin-resistancerateincreased signifi-cantly,theresultsindicatingthatmutationsofparCgeneand

gyrA genewillmakeK.pneumoniaemorehighlyresistantto Ciprofloxacin.DNA sequencingofgyrA andparC inclinical strainshasrevealedsomemutationsintheQRDRassociated withfluoroquinoloneresistance.However,QRDRsofthetwo isolates(YYR25-1and ZG2)withresistancetociprofloxacin inthisstudydidnotpossessalterationsassociatedwith flu-oroquinolones resistance in the sequence of either genes, indicatingthatotherunknownresistancemechanisms con-tribute to K.pneumoniae became resistant toCiprofloxacin, suchaseffluxsystemsandbiofilmsshouldbeconsideredin theseisolates.

Inthisstudy,mostoftheisolatesharbored atleasttwo virulencefactorgenes, suggestingthatCURTK.pneumoniae

isolates in this region were potentially pathogenic. Major-ity of studied isolates were positive for ureA (97.0%) and

wabG(91.0%)gene,similarlyhighprevalenceofthesegenes observedinisolateofhumanclinicalandotheranimals,19,20 thisresultindicatesthattheureAandwabGgenesare preva-lentin K.pneumoniae. Thetype3 fimbrialshaft mrkAgene detectedin57.6%ofisolates, whichisless than that100% reportedearlierbyAlcantar-Curieletal.18_{Thisresultshows} thattherearedifferencesbetweenbovineisolatesandhuman clinicalisolates.ManyreportssuggestthatthermpA-carrying

strainswereassociatedwiththehypermucoviscosity pheno-type.Inthisstudy,thedetectionratesofrmpAandmagAwere 57.6%and42.4%,respectively,andstringtestresultsrevealed that36.4%K.pneumoniaeisolatesfromcattleshowed hyper-mucoviscosity.ThehvKPratewassignificantlylowerthanthat humanclinicalisolates(85%).29_{However,thestringtestresult} islowerthanthermpAgenedetectionrate,whichindicatethat stringtestmightnotbeareliablemethodtoidentifyhvKP.

Inthisstudy,ST218wasthemostcommon,whichoccurred inapproximately57.6%oftheK.pneumoniaisolates.Through MLSTdatabaseandsomeresearchreports27,31_thatST218was isolatedfromhumaninThailand,Vietnam,SouthKoreaand Russia,andLiaoetal.31_{reportedthatST218strainsmaycause} liverabscessdisease inhuman. ST218isclosely related to theST23prevailinginEuropeandtheUnitedStates,bothof thembelongstoCC11,andonlyahousekeepinggene(tonB) difference between each other. The ST23 has caused seri-ous public health events inmany parts of the world.1 _As mentioned above, the prevalenceof ST218 isolates in cat-tleisnotnegligible.ThreenewSTs(ST2541,2581and2844) weresub-groupedinthebranchofSC1containingST218and ST116.PreviousresearchrevealedthatST116cancausebovine mastitis.22_{Ourworksconfirmedtherelativecloserelationship} ofST2541,ST2581andST2844withST218andST116.Onthe otherhand,thedistributionofvirulencefactorsamongthe dif-ferentsequencetypeswasnotrandom.Inthisstudy,isolates belongingtoST218werewiththegenesmrkA+ureA+wabG,

whereas ST2844included isolateswith rmpA+wabGgenes. Thisisthefirststudytoreportthefrequency,antimicrobial resistanceprofile,virulencegenesandMLSTmolecular char-acteristicofK.pneumoniaefromcattleinSouthwestChina.This studywillhelpcontinuousmonitoringofthegeneticdiversity ofK. pneumoniaemaydisclosedifferences invirulence, epi-demiology,evolutionandgenetictraitsthatinfluencecontrol andtreatment.

Conflicts

of

interest

Theauthors declarednopotentialconflictsofinterest with respecttothe research,authorship,and publicationofthis article.

Acknowledgements

This study was supported by the Fundamental Research Funds for the Central Universities (XDJK2017D083), Inno-vation Project for the Social Undertakings and People’s

LivelihoodProtectioninChongqing(CSTC2015SHMSZX80020) and Frontiers and basic research projects in Chongqing (cstc2016jcyA0235).

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1.SiuLK,FungCP,ChangFY,etal.Moleculartypingand virulenceanalysisofserotypeK1Klebsiellapneumoniae strainsisolatedfromliverabscesspatientsandstool samplesfromnoninfectioussubjectsinHongKong

Singapore,andTaiwan.JClinMicrobiol.2011;49(11):3761–3765. 2.NewireEA,AhmedSF,HouseB,etal.Detectionofnew

SHV-12SHV-5andSHV-2avariantsofextendedspectrum beta-lactamaseinKlebsiellapneumoniaeinEgypt.AnnClin MicrobiolAntimicrob.2013;12:16.

3.WhiteDG,ZhaoSH,SimjeeS,etal.Antimicrobialresistance offoodbornepathogens.MicrobesInfect.2002;4:405–414. 4.GuoC,YangX,WuY,etal.MLST-basedinferenceofgenetic

diversityandpopulationstructureofclinicalKlebsiella pneumoniaeChina.SciRep.2015;5:7612.

5.Al-MarzooqF,YusofMYM,TayST.Molecularanalysisof ciprofloxacinresistancemechanismsinMalaysian ESBL-producingKlebsiellapneumoniaeisolatesand developmentofmismatchamplificationmutationassays (MAMA)forrapiddetectionofgyrAandparCmutations. BioMedResInt.2014;2014:601630.

6.FuY,ZhangW,WangH,etal.SpecificpatternsofgyrA mutationsdeterminetheresistancedifferenceto ciprofloxacinandlevofloxacininKlebsiellapneumoniaeand Escherichiacoli.BMCInfectDis.2013;13:8.

7.NouriR,RezaeeMA,HasaniA,etal.TheroleofgyrAand parCmutationsinfluoroquinolones-resistantPseudomonas aeruginosaisolatesfromIran.BrazJMicrobiol.

2016;47(4):925–930.

8.EnrightMC,SprattBG.Multilocussequencetyping.Trends Microbiol.1999;7(12):482–487.

9.MaidenM,BygravesJA,FeilE,etal.Multilocussequence typing:aportableapproachtotheidentificationofclones withinpopulationsofpathogenicmicroorganisms.ProcNatl AcadSciUSA.1998;95(6):3140–3145.

10.Carvalho-CastroGA,SilvaJR,PaivaLV,etal.Molecular epidemiologyofStreptococcusagalactiaeisolatedfrom mastitisinBraziliandairyherds.BrazJMicrobiol. 2017;48(3):551–559.

11.DavisGS,WaitsK,NordstromL,etal.IntermingledKlebsiella pneumoniaepopulationsbetweenretailmeatsandhuman urinarytractinfections.ClinInfectDis.2015;61(6):892–899. 12.JinY,ShaoC,LiJ,etal.Outbreakofmultidrugresistant

NDM-1-producingKlebsiellapneumoniaefromaNeonatalUnit inShandongProvince,China.PLOSONE.2015;10:3.

13.ZhouZ,ZhangM,LiH,etal.Prevalenceandmolecular characterizationofStaphylococcusaureusisolatedfromgoats inChongqing,China.BMCVetRes.2017;13(1):352.

14.LaneDJ.16S/23SrRNAsequencing.NuclAcidTechBacterial Syst.1991:125–175.

15.WattsJL.PerformanceStandardsforAntimicrobialDiskand DilutionSusceptibilityTestsforBacteriaIsolatedfromAnimals: ApprovedStandardClinicalandLaboratoryStandardsInstitute; 2013.

16.BrisseS,VerhoefJ.PhylogeneticdiversityofKlebsiella pneumoniaeandKlebsiellaoxytocaclinicalisolatesrevealedby randomlyamplifiedpolymorphicDNA,gyrAandparCgenes sequencingandautomatedribotyping.IntJSystEvolut Microbiol.2001;51(3):915–924.

100

brazilian journalof microbiology49S(2018)93–100

17.KumarS,StecherG,TamuraK.MEGA7:molecular evolutionarygeneticsanalysisversion7.0forbigger datasets.MolBiolEvol.2016;33(7):1870–1874. 18.Alcantar-CurielMD,BlackburnD,SaldanaZ,etal.

Multi-functionalanalysisofKlebsiellapneumoniaefimbrial typesinadherenceandbiofilmformation.Virulence. 2013;4(2):129–138.

19.WangJ,ShangY,GuoS,etal.Serotypeandvirulencegenes ofKlebsiellapneumoniaeisolatedfromminkandits pathogenesisinmiceandmink.SciRep.2017;7:17291. 20.ItoR,ShindoY,KobayashiD,etal.Molecularepidemiological

characteristicsofKlebsiellapneumoniaeassociatedwith bacteremiaamongpatientswithpneumonia.JClinMicrobiol. 2015;53(3):879–886.

21.DiancourtL,PassetV,VerhoefJ,etal.Multilocussequence typingofKlebsiellapneumoniaenosocomialisolates.JClin Microbiol.2005;43(8):4178–4182.

22.Paulin-CurleeGG,SingerRS,SreevatsanS,etal.Genetic diversityofmastitis-associatedKlebsiellapneumoniaeindairy cows.JDairySci.2007;90(8):3681–3689.

23.KatsandeS,MatopeG,NdenguM,etal.Prevalenceof mastitisindairycowsfromsmallholderfarmsinZimbabwe. OnderstepoortJVetRes.2013;80:523.

24.MunozMA,WelcomeFL,SchukkenYH,etal.Molecular epidemiologyoftwoKlebsiellapneumoniaemastitisoutbreaks onadairyfarminNewYorkState.JClinMicrobiol.

2007;45(12):3964–3971.

25.OsmanKM,HassanHM,OrabiA,AbdelhafezAST.

Phenotypic,antimicrobialsusceptibilityprofileandvirulence factorsofKlebsiellapneumoniaeisolatedfrombuffaloandcow mastiticmilk.PathogensGlobHealth.2014;108(4):191.

26.AslanV,MadenM,ErganisO,etal.Clinicalefficacyof florfenicolinthetreatmentofcalfrespiratorytract infections.VetQ.2002;24(1):35–39.

27.LeeMY,KoKS,KangC,etal.Highprevalenceof

CTX-M-15-producingKlebsiellapneumoniaeisolatesinAsian countries:diverseclonesandclonaldissemination.IntJ AntimicrobAgents.2011;38(2):160–163.

28.MoiniAS,SoltaniB,ArdakaniAT,etal.Multidrug-resistant EscherichiacoliandKlebsiellapneumoniaeisolatedfrom patientsinKashanIran.JundishapurJMicrobiol. 2015;8:e2751710.

29.QuT,ZhouJ,JiangY,etal.Clinicalandmicrobiological characteristicsofKlebsiellapneumoniaeliverabscessinEast China.BMCInfectDis.2015;15:161.

30.FuY,GuoL,XuY,etal.AlterationofGyrAaminoacid requiredforciprofloxacinresistanceinKlebsiellapneumoniae isolatesinChina.AntimicrobAgentsChemotherapy.

2008;52(8):2980–2983.

31.LiaoCH,HuangYT,ChangCY,etal.Capsularserotypesand multilocussequencetypesofbacteremicKlebsiella pneumoniaeisolatesassociatedwithdifferenttypesof infections.EurJClinMicrobiolInfectDis.2014;33(3):365–369.