Detection of O25b-ST131 clone, CTX-M-1 and CTX-M-15 genes via real-time PCR in Escherichia coli strains in patients with UTIs obtained from a university hospital in Istanbul

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ContentslistsavailableatScienceDirect

Journal

of

Infection

and

Public

Health

jo u r n al ho me p ag e :h t t p : / /w w w . e l s e v i e r . c o m / l o c a t e / j i p h

Detection

of

O25b-ST131

clone,

CTX-M-1

and

CTX-M-15

genes

via

real-time

PCR

in

Escherichia

coli

strains

in

patients

with

UTIs

obtained

from

a

university

hospital

in

Istanbul

Mehmet

Demirci

a,∗

_,

_Özge

_Ünlü

a

_,

_Ays¸

_{e ˙Istanbullu}

_Tosun

b

a_Beykent_University_School_of_Medicine,_Department_of_Medical_{Microbiology,}_34520,_Istanbul,_Turkey b_Medipol_University_School_of_Medicine,_Department_of_Medical_{Microbiology,}_34214,_Istanbul,_Turkey

a

r

t

i

c

l

e

i

n

f

o

Articlehistory:

Received12October2018

Receivedinrevisedform6December2018 Accepted13February2019 Keywords: O25b-ST131 Escherichiacoli UTI CTX-M-15 Real-timePCR

a

b

s

t

r

a

c

t

Background:Escherichiacolisequencetype131isanimportantmultidrugresistantcloneresponsible frommorethanhalfofESBL-producingE.coliisolates.Aimofthisstudywastoinvestigatethepresence ofO25b-ST131clone,CTX-M-15andCTX-M-1genesintheE.colistrainsisolatedfrombothhospitaland communityacquiredUTIsbyreal-timePCRandtorevealmolecularepidemiologicaldata.

Methods:Non-duplicateE.coli(n=101)strainsisolatedfromUTIpatientswereincluded.Bacterial iden-tiﬁcationswereperformedwithVITEKCompact.Antimicrobialsusceptibilitytests,phenotypicESBLand E-testswereperformedconventionally.Real-timePCRwasutilizedtodetectpresenceofO25b-ST131 clone,blaCTX-M-15andblaCTX-M-1.

Results:O25b-ST131clone,CTX-M-1andCTX-M-15weredetectedin22%,73%,37%inUTIs,respectively. PresenceofO25b-ST131clonesandCTX-M-1genesamongE.colistrainsisolatedfrominpatientswere foundstatisticallyhigherthanoutpatients.Themosteffectivechoicewasfoundtobefosfomycinand nitrofurantoininoutpatientsandinpatients,respectively.TheMIC90valuesofAmikacin,Cefotaxime,

Cefepimeand Ciproﬂoxacinwerehigher ininpatientsthaninoupatients,whereasCefotaxime and CiproﬂoxacinMIC50valueswerefoundtobehigherininpatientsthaninoutpatients.Thehighestincrease

ofMIC90valueswasobservedinO25b-ST131,CTX-M-1andCTX-M-15coexistence.

Conclusion:ThepresenceofO25b-ST131clone,CTX-M-1andCTX-M-15genesinE.colistrainsinpatients withUTIhasbeenrevealed.InthepresenceoftheO25b-ST131clone,asigniﬁcantincreasewasobserved intheciproﬂoxacinMICvaluesindicatingtheimportanceofmonitorizationofthecloneusingmolecular epidemiology.

©2019TheAuthors.PublishedbyElsevierLimitedonbehalfofKingSaudBinAbdulazizUniversity forHealthSciences.ThisisanopenaccessarticleundertheCCBY-NC-NDlicense(http:// creativecommons.org/licenses/by-nc-nd/4.0/).

Introduction

Urinarytractinfections(UTIs) are amongthemostcommon

bacterial infections affecting millions of people every year [1].

Escherichia coli (E. coli) is one of the most important causes

ofurinarytractinfections (UTI),whetheritis hospital-acquired

orcommunity-acquired[2]. InfectionscausedbyGram-negative

multidrug-resistant(MDR)bacteriaareaworldwidehealth

prob-lem [3]. In recent years, the production of extended-spectrum

ˇ-lactamase(ESBL)intheEnterobacteriaceaefamily,especiallyin

E.coli,hasincreasedsigniﬁcantly[4].Amongthese,E.colisequence

type 131(ST131)-O25b:H4 is an important multidrugresistant

clonewhichisabletospreadglobally[3].E.coliST131clone,

dis-∗ Correspondingauthor.

E-mailaddress:demircimehmet@hotmail.com(M.Demirci).

coveredin2008,isamemberofhighlyvirulentphylogeneticgroup

B2,alsothestrainsinthiscloneoftenhaveserotypeO25:H4and

thespeciﬁctypeofO25iscalledO25b[5].Besidesbeingoneofthe

maincausesofhospital-acquiredandcommunity-acquiredurinary

tractinfectionsinEurope,thiscloneisthoughttoberesponsible

forapproximately20%ofallextraintestinalE.coliinfections[6,7].

Inaddition,amongthestrainsidentiﬁedinmanyinfections,the

majorityofﬂuoroquinolone-resistantisolates[8]andmorethan

halfofESBL-producingisolatesareassociatedwiththisclone[9].

CTX-M-15,themostwidelyspreadingESBLenzymeamong

CTX-Ms,isfrequentlydetectedinE.coliST131clones[10].Thus,E.coli

O25b-ST131cloneshowahighresistanceproﬁletomanydrugsand

thisleavesafeweffectiveantibioticoptionsthatcanbeusedtotreat

patients[6].Ciproﬂoxacinisthemostcommonlyprescribed

ﬂuo-roquinoloneforUTIssinceoralandintravenouspreparationsare

available[11].Increasedresistancetoﬂuoroquinolonesmayhave

seriousclinicalconsequencesastheyareoneofthemosteffective

https://doi.org/10.1016/j.jiph.2019.02.017

1876-0341/©2019TheAuthors.PublishedbyElsevierLimitedonbehalfofKingSaudBinAbdulazizUniversityforHealthSciences.Thisisanopenaccessarticleunderthe CCBY-NC-NDlicense(http://creativecommons.org/licenses/by-nc-nd/4.0/).

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therapeuticoptionsinsevereSalmonellaspp.andE.coliinfections

[12].Inthelightofallthisinformation,weaimedtoinvestigate

thepresenceofO25b-ST131cloneaswellasproducingof

CTX-M-15andCTX-M-1genesintheE.colistrainsdetectedasacausative

agentsofbothhospitalandcommunityacquiredUTIsbyreal-time

PCRandtorevealmolecularepidemiologicaldatasaboutour

coun-tryandalsopresentstatusagaintantimicrobialsconventionally.

Materialandmethods

Non-duplicateE.coli(n=101)strainsisolatedfromurine

sam-plesofpatientsadmittedtooneofa privateuniversityhospital

inTurkey,betweenAprilandAugust2018.Isolatesweredeemed

duplicatesifthesameorganismfromsamepatientswiththesame

antibiogramisgrownfromthesamesampletypewithin14days

(forinpatients)or91days(foroutpatients).Weconsideredpatients

whostayedfortheclinicaltreatmentinagivenwardofthe

hos-pitaltobeinpatients,andpatientswhowerenothospitalizedbut

whovisitedanacutedaywardorpolyclinicwereconsideredto

beoutpatients.Theinclusioncriteriafortheoutpatientswere:not

bepregnant,nothaveusedantibioticswithinonemonthpriorto

enrollmentandwasn’trecordedanyhospitalaswithin72h.Cobas

6500(RocheDiagnostics,Mannheim,Germany)automatedurine

analysersystemwasusedtodetectwhitebloodcells(WBC)count

fromurinesamplesandreportsgeneratedautomaticallythe

micro-scopicresultsascells/highpowerﬁeld(HPF).Patientspresenting

withUTI,whereCFUcountswere>1×105_mL_were_included_in_this

study.Bacterialidentiﬁcationswereconductedwithautomatized

testVITEKCompact(BioMerieux,MarcyL’Etoile,France).

Antimi-crobialsusceptibilitytestswereperformedwithKirbyBauersdisc

diffusiontestsonMueller-Hintonagar(BioMerieux,MarcyL’Etoile,

France). The following antibiotic disks were used,

amoxicillin-clavulanicacid(20/10␮g), ampicillin(30␮g),cefazolin (30␮g),

ceﬁxime(30␮g),cefotaxime (30␮g),cefepime(30␮g),

ceftriax-one (30_␮g),cefuroxime (30_␮g),fosfomycin (200_␮g), amikacin

(30␮g), gentamicin (10␮g), imipenem (10␮g), meropenem

(10␮g),nitrofurantoin(300␮g),ciproﬂoxacin(5␮g),levoﬂoxacin

(5␮g), trimethoprim-sulfamethoxazole (1.25/23.75␮g) (Oxoid,

Basingstoke, UK) interpreted according to the EUCAST clinical

breakpoints (EUCAST, 2016). To conﬁrm ESBL phenotype, ESBL

testwasperformedusingthedouble-discsynergyprocedureon

Mueller-Hinton agar (BioMerieux, Marcy L’Etoile, France) [13].

MICvalues ofciproﬂoxacin, amikacin,cefotaxime and cefepime

were also determined with E-test (AB Biodisk, Solna, Sweden)

onMueller-Hintonagar(BioMerieux,MarcyL’Etoile,France)and

interpretedaccordingtotheEUCASTclinicalbreakpoints[14].

A single colony of each strain’s overnight culture on eosin

methylene blue (EMB) agar was suspended in 50␮L of

ultra-purewater. Thesuspensionwasheatedat95◦Cfor 10minand

centrifuged at 14.000rpm for 10min. Thirty microliters of the

supernatantwasusedasaDNAtemplateforreal-timePCR[15].

AllDNAwasstored−80◦_C_until_processing._Previously_designed

primers; ST131TF(5-GGT GCTCCA GCA GGTG-3),ST131TR

(5-TGG GCGAAT GTCTGC-3),ST131AF(5-GGCAAT CCA ATATGA

CCC-3),ST131AR(5-ACCTGGCGAAATTTTTCG-3),MC-3-15F

(5-TGGGGGATAAAACCGGCAG-3),MC-3-15R(5-GCGATATCGTTG

GTGGTGC-3),blaCTX-M-1F (5-AACCGTCACGCTGTTGTT

AG-3)andblaCTX-M-1R(5-TTGAGGCTGGGTGAAGTAAG-3)were

usedtodetectpresenceofO25b-ST131clone,blaCTX-M-15and

blaCTX-M-1respectively[15,16].Real-timePCRampliﬁcationand

meltingcurveanalysiswereperformedusingaLightCycler480II

systemwithsoftwareversion1.5(RocheDiagnostics,Mannheim,

Germany). The real-time PCR mixture was prepared using the

LightCycler480SYBR GreenIMastermixkit(RocheDiagnostics,

Mannheim,Germany)followingthemanufacturer’sinstructions.

CyclingconditionsfortheO25b-ST131assayswere:initial

denatu-Table1

DemographicdataofpatientsanddistributionofUrineWBC/HPF,CTX-M-1, CTX-M-15genesandO25b-ST131clone.

Inpatient(n:42) Outpatient(n:59) Total(n:101) Age(Mean-SD) 35.40±25.18 37.24±25.23 37.23±25.18

Sex(Male/female) 13/29 11/48 24/77

UrineWBC/HPF O25b-ST131positive(n)/total(n)

≤10 1/6 2/6 3/12 11–20 2/5 0/4 2/9 20–50 2/4 0/3 2/7 >50 9/27 7/46 16/73 ESBLpositivity 28(66.67) 23(38.98) 51(50.49) blaCTX-M-1 38(90.48) 36(61.02) 74(73.27) blaCTX-M-15 16(38.10) 22(37.29) 38(37.62) O25b-ST131clone 14(33.33) 9(15.25) 23(22.27) Table2

DistributionofCTX-M-1 andCTX-M-15genesandO25b-ST131cloneby ESBL condition.

E.coli ESBLpositive(n:51) ESBLnegative(n:50)

blaCTX-M-1 46(90.20) 28(56.00)

blaCTX-M-15 18(35.30) 20(40.00)

O25b-ST131clone 16(31.37) 7(14.00)

rationfor5minat95◦Cand40cyclesof5sat95◦Cand10sat58◦C;

thosefortheblaCTX-M-15andblaCTX-M-1assaywere:5minat

95◦Cand50cyclesof5sat95◦Cand10sat70◦C.Theﬂuorescence

signalwasmeasuredattheendofeachannealingstep.Following

ampliﬁcation,ameltingcurvewasgeneratedbyheatingthePCR

productto95◦Cwitharamprateof0.05◦C/s.Statistical

analy-siswereperformedwithchi-squaretestonSPSSvers.20software

(IBM,USA).

Results

Inthisstudyweevaluated101E.colistrains,42wereisolated

frominpatients(meanage35years)and59isolatedfrom

outpa-tients(meanage37years).O25b-ST131clonewasdetectedin22%,

CTX-M-1wasdetectedin73%andCTX-M-15in37%ofallisolates

(Table1).DistributionofO25b-ST131clone,accordingtoWBC/HPF

resultsshowedinTable1.

InpatientswerefoundtohavesigniﬁcantlymoreE.coli

O25b-ST131clonescomparedtooutpatients(p<0.05).TheCTX-M-1gene

responsibleforESBLproductioninstrainsisolatedfrominpatients

wasstatisticallysigniﬁcantlyhigher thanin thestrainsisolated

fromoutpatients(p<0.05).Ontheotherhand,producingof

CTX-M-15geneinstrainsisolatedfrominpatientswasnotstatistically

higherthaninthestrainsisolatedfromoutpatients(p>0.05).When

presenceofphenotypicESBLwasexaminedamongthestrains,it

wasfoundtobepositive in66.67% of inpatientsand38.98% of

outpatients.

AmongthephenotypicallyESBLpositivestrains,itwasfound

that producing of CTX-M-1 and CTX-M-15 gene and

O25b-ST131clonepositivitywere90.2%,35.3%and31.37%,respectively

(Table2).CTX-M-1geneandO25b-ST131clonepresencewere

sig-niﬁcantlyhigherintheESBLpositivestrains(p<0.05).

Antimicrobial susceptibilities of the strains were given in

Table 3. According to susceptibility results carbapenems were

foundtobethebesttherapeuticchoiceforallpatientswhichwas

followedbynitrofurantoin,amikacinandfosfomycin.The

suscepti-bilitypercentageforallantimicrobials,detectedininpatientswere

lowerthaninoutpatients.

E.colistrainsisolatedfrominpatientswerefoundtobemore

resistant to antimicrobials than the strains isolated from

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Table3

Antimicrobialsusceptibilitesofthestrains.

Antimicrobials Inpatient(n:42) Outpatient(n:59) Total(n:101)

Amoxicillin-clavulanicacid(20/10␮g) 14(33.33) 33(55.93) 47(46.53) Ampicillin(30␮g) 7(16.67) 12(20.34) 19(18.81) Cefazolin(30␮g) 9(21.43) 18(30.51) 27(26.73) Cefixime(30␮g) 13(30.95) 33(55.93) 46(45.54) Cefotaxime(30␮g) 14(33.33) 34(57.63) 48(47.52) Ceftriaxone(30␮g) 14(33.33) 36(61.02) 50(49.50) Cefuroxime(30␮g) 7(16.67) 18(30.51) 25(24.75) Cefepime(30␮g) 22(52.38) 45(76.27) 67(66.34) Fosfomycin(200␮g) 36(85.71) 57(96.61) 93(92.08) Amikacin(30␮g) 37(88.10) 56(94.92) 93(92.08) Gentamicin(10␮g) 26(61.90) 47(79.66) 73(72.28) Imipenem(10␮g) 41(97.62) 59(100.0) 100(99.01) Meropenem(10␮g) 42(100.0) 58(98.31) 100(99.01) Nitrofurantoin(300␮g) 41(97.62) 56(94.92) 97(96.04) Ciprofloxacin(5␮g) 11(26.19) 33(55.93) 44(43.56) Levofloxacin(5␮g) 11(26.19) 34(57.63) 45(44.55) Trimethoprim-sulfamethoxazole(1.25/23.75␮g) 19(45.24) 34(57.63) 53(52.48) Table4

MICvaluesofthestrains(␮g/mL).

Inpatients(n:42) Outpatients(n:59) Total(n:101)

MICrange MIC50 MIC90 MICrange MIC50 MIC90 MICrange MIC50 MIC90

Amikasin 0.5–32 2 16 0.5–8 2 4 0.5–32 2 4

Cefotaxime 0.015–8 0.5 2 0.015–2 0.12 1 0.015–8 0.5 2

Cefepime 0.015–2 0.06 1 0.015–1 0.06 0.5 0.015–2 0.06 0.5

Ciproﬂoxacin 0.004–64 0.5 16 0.004–16 0.03 4 0.04–64 0.12 8

Table5

CiproﬂoxacinsusceptibilityaccordingtoCTX-M-1,CTX-M-15andO25b-ST131positivity.

E.coli Ciproﬂoxacin

S R MICrange MIC50 MIC90

CTX-M-1positive(n:51) 25(49.02) 26(50.98) 0.04–1 0.06 0.5

CTX-M-15positive(n:8) 5(62.5) 3(37.5) 0.004–4 0.03 4

CTX-M-1&CTX-M-15positive(n:9) 4(44.44) 5(55.56) 0.08–4 0.015 4

CTX-M-1&O25b-ST131positive(n:2) 0(0) 2(100) 0.5–2 0.5 2

CTX-M-15&O25b-ST131positive(n:9) 0(0) 9(100) 2–16 4 16

O25b-ST131&CTX-M-1&CTX-M-15positive(n:12) 0(0) 12(100) 4–64 16 32

O25b-ST131&CTX-M-1&CTX-M-15negative(n:10) 10(100) 0(0) 0.004–0.03 0.015 0.03

*_S:_Sensitive,_R:_Resistant.

fosfomycinaftercarbapenems(96.61%)foroutpatients,thisoption

wasfoundasnitrofurantoin(97.62%)ininpatients.

MICvalues ofE.colistrainsfor Cefotaxime,Cefepim

demon-stratingthirdandfourthgenerationcephalosporinsrespectively,

AmikacinandCiproﬂoxacinareshowninTable4.TheMIC90

val-uesof Amikacin, Cefotaxime, Cefepime and Ciproﬂoxacin were

higherininpatientsthaninoupatients,whereasCefotaximeand

CiproﬂoxacinMIC50valueswerefoundtobehigherininpatients

thaninoutpatients.

CTX-M-1detectedin2,CTX-M-15in9oftheO25b-ST131

pos-itive E. coli strains, also in 12 strainsCTX-M-1 and CTX-M-15

werecoexisted.AllE.coliO25b-ST131strainswereresistantto

ciproﬂoxacin(100%).Moreover,all10strainsthatarenot

carry-ingCTX-M-1orCTX-M-15genesandthatarenotST131clonewere

foundtobesensitivetociproﬂoxacin(Table5).

TheMICrange,MIC50andMIC90valuesoftheCiproﬂoxacinwere

examinedaccordingtothepresenceofCTX-M-1,CTX-M-15genes

andO25b-ST131clones.InthecaseofCTX-M-1andO25b-ST131or

CTX-M-15andO25b-ST131associations,anincreaseinMICvalues

weredetectedforthestrains,whereasinthecaseofco-occurrence

ofO25b-ST131,CTX-M-15andCTX-M-1thesevalueswerefound

attheirhighestlevels.

CefotaximeMICrange,MIC50andMIC90valueswereanalyzed

accordingtothepresenceofCTX-M-1,CTX-M-15genesand

O25b-ST131.InthecaseofpresenceofoneCTX-MgeneandO25b-ST131

cloneanincreasehasbeennoticedinMIC90values whereasthe

highestincreasewasobservedinO25b-ST131,CTX-M-1and

CTX-M-15coexistence(Table6).

Inaddition,8outof10strainsthatwerenotO25b-ST131clone

andnotcarryingeitherCTX-M-1orCTX-M-15geneswerefoundto

besusceptibletocefotaxime.Also,all12O25b-ST131clones

pos-itivestrainsthatarecarryingbothCTX-M-1andCTX-M-15genes

werefoundtoberesistanttocefotaxime.

Discussion

E.coliisoneofthemostimportantcausesofurinarytract

infec-tionsin bothinpatientsandoutpatients[17].ParticularlyE.coli

ST131 clone was considered to be an important public health

problem,duetoitsepidemicpotential,virulenceandmultidrug

resistanceabilitywhichweredramaticallyhigherthannonST131

clones[18].TheST131cloneisalsoreportedtobestrongly

asso-ciatedwithESBLssuchastheproducingofCTX-M-15.Thisleaves

limitedtherapeuticoptionsforthetreatmentofinfectionscaused

bythiscloneandincreasestheinterestinmonitorizationofthis

infectiousagent[5].

Whenthestudiesconductedonthedistributionofphenotypic

ESBL,O25b-ST131,CTX-M-15andCTX-M-1casesinE.colistrains

identiﬁedasthecausativeagentofUTIwereexamined,Namaei

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O25b-Table6

CefotaximesusceptibilityaccordingtoCTX-M-1,CTX-M-15andO25b-ST131positivity.

E.coli Cefotaxime

S R MICrange MIC50 MIC90

CTX-M-1positive(n:51) 19(37.25) 32(62.75) 0.015–2 0.5 1

CTX-M-15positive(n:8) 6(75.0) 2(25.0) 0.003–2 0.06 2

CTX-M-1&CTX-M-15positive(n:9) 8(88.89) 1(11.11) 0.015–0.5 0.06 0.5

CTX-M-1&O25b-ST131positive(n:2) 0(0) 2(100) 0.5–2 0.5 2

CTX-M-15&O25b-ST131positive(n:9) 7(77.78) 2(22.22) 0.015–1 0.06 1

O25b-ST131&CTX-M-1&CTX-M-15positive(n:12) 0(0) 12(100) 0.5–8 1 4

O25b-ST131&CTX-M-1&CTX-M-15negative(n:10) 8(80) 2(20) 0.015–0.5 0.06 0.25

*_S:_Sensitive,_R:_Resistant.

ST131as24.7%.ThepresenceofO25b-ST131cloneinthestrains

isolatedfromoutpatientandinpatients,wasreportedas22.8%and

55.6%,respectively.Also,inthesamestudy,O25b-ST131,CTX-M-1

andCTX-M-15presencewerereportedinESBLpositivepatientsas

78.5%,100%,95.5%,respectively[5].MarialouisandSanthanam,in

theirstudyconductedinIndiain2016,reportedESBLpresenceas

47%inE.colistrainsisolatedfrompatientswithUTI.Inthesame

study,theyreportedthepresenceofO25b-ST131clonein41%of

thestrains[19].Tovaletal.studiedonE.colistrainswhichwere

foundascausativeagentsin265patientswithUTIinGermanyin

2014.O25b-ST131clonewasdetectedin22ofthestrains,whereas

13wereisolatedfrominpatients,9wereisolatedfromoutpatients

[20].Talanetal.,studiedwiththestrainsobtainedfrompatients

withUTIintheUnitedStates in2016,and reportedESBL

posi-tivityas2.6%ofoutpatientsand12.2%ofinpatients[21].Inthe

studyconductedbyGuyomard-Rabenirinaetal.inFrancein2016,

CTX-M-15wasdetectedin5,CTX-M-1wasdetectedin4outof11

ESBLpositiveE.colistrains[22].Canetal.,reportedthepresence

ofESBLas24%,CTX-M-15geneas14%,andO25b-ST131cloneas

12%ofE.colistrainsisolatedfrompatientswithUTIintheirstudy

conductedinIstanbulat2015[23].Whenweexaminetheresults

obtainedfromallthesestudiesperformedindifferentcountries,the

presenceofESBL,CTX-M-1,CTX-M-15andO25b-ST131clonesare

variable.In2015,Goossensetal.reportedthatthetreatment

poli-cies[24],whichvaryfromcountrytocountry,maybeeffectivein

thisdifference.Inadditiontothis,becauseofthelackofconsensus

regardingtheuseofmoleculartechniques,webelievethatthe

dif-ferentmoleculartechniqueswithdifferentprotocolsusedinthese

studiesmaycausedifferencesintheresults.Besides,similartoour

studyresults,mostofthestudiesrevealedthatantibioticresistance,

presenceof CTX-M-1,CTX-M-15genes,and O25b-ST131clones

werefoundtobehigherinthestrainsisolatedfrominpatientsthan

outpatientssimilartoourresults.Becauseofthefactthatinpatients

strainsencountermoredrugsinteractionthanoutpatientsstrains.

According to the antimicrobial susceptibility studies

con-ducted on E. coli strains identiﬁed as causative agent of UTI;

Namaeiet al., reported susceptibilies toamikacin,

amoxycillin-clavulanicacid, cefepim,cefotaxime,gentamycin,

trimethoprim-sulfamethoxazole, ciproﬂoxacine,imipenemand meropenemas

97.2%,88.4%,79.4%,33.4%,67.7%,29.9%,23.2%,0%and0%,

respec-tively in2017 [5]. Talanet al., intheir studyconducted inthe

United States in 2016, reported antimicrobial susceptibility to

ampicillin,cefazoline,ceftriaxone,ciproﬂoxacin,levoﬂoxacin,

gen-tamycin,imipenemandmeropenemforoutpatientsandinpatients

as; 45.1%,44.9%;92.8%, 77%;98.4%,85.5%;94.7%, 80.4%;94.9%,

83.3%;93.7%,87.3%;0,0%;and0%,0%respectively[21].Whenall

ofthesestudiesareexamined,itcanbeseenthatthereisachange

inantimicrobialsusceptibilitiesbytheeffectofdifferent

antibi-oticsusedfromcountrytocountry, evenfromregiontoregion.

However,itisseenthatthereisahigherlevelofresistance

pro-ﬁleininpatientsthanoutpatients,asinourstudy,andantibiotic

optionsthatcanbeusedinthesepatientsaremorelimitedthanin

outpatients.

Sedighietal.,identiﬁed97%sensitivityforamikacininE.coli

strainsidentiﬁed asthecausativeagentof UTIin Iranin 2014.

TheMIC50,MIC90andMICrangesofthesestrainswerereported

as0.75,1.6and0.125–32␮g/mL,respectively[25].Inourstudy,

theMICrangewasfoundas0.5–32␮g/mLwhichissimilartothe

Sedighietal.[25],however,ourMIC50andMIC90rateswerehigher

whichare2␮g/mLand 4␮g/mL,respectively.Cubaetal,

exam-ined theMICvalues of someantibioticscommonly usedin the

treatmentintheirstudyconductedwithE.colistrainsisolatedas

UTIagentfromoutpatientsandinpatientsinBrazilin2014.They

reported75.7%sensitivityforceftriaxoneininpatientswithaMIC

range 0.016–256␮g/mL whereastheyobtained%97.5sensitivity

and0.008–256␮g/mLMICrangeforoutpatients.Inthesamestudy,

sensitivityandMICrangeforciproﬂoxacinwerereportedas64.3%,

0.008–32␮g/mLand83.3%,0.008–0.032␮g/mLininpatientsand

outpatients,respectively[26].Cerquettietal.reported9.8%

CTX-M-15positivityintheirstudyconductedwithstrainsisolatedfrom

patientswithUTIin Italy in 2010,also 90%of CTX-M-15

posi-tivestrainsdetectedas O25b-ST131clone[27]. Suzikiet al.,in

2009,reported91.5%CTX-M-15positivityandamongthesestrains

21%identiﬁedasO25b-ST131cloneinJapan[28].In2015,Drawz

etal.detectedO25b-ST131clonein13 outof15CTX-M-1

posi-tiveand6in10ofCTX-M-15positivestrains[29].Inourstudy,

we detectedCTX-M-15 in 9,CTX-M-1 in 2and CTX-M-1,

CTX-M-15 coexistence in 12 O25b-ST131 E.coli strains. The results

ofourstudyaresimilartootherstudiesindicating O25b-ST131

cloneiscloselyrelatedtotheCTX-M-1andCTX-M-15genes.The

factthatthisclonecarriesthesegenesclearlyexplainsthe

poten-tialofthis clonetodevelopmultidrugresistance. In2011,Ruiz

etal. reportedtheMICvalueforcefotaxime >32␮g/mL andfor

ciprofloxacine>8␮g/mL, whentheyidentifiedthefirst

commu-nityacquired CTX-M-15-producing O25b-ST131E.coli strainin

SouthAmerica.Theyemphasizedthattherewasanincreaseddrug

resistanceespecially in these strainsand monitorization of the

prevalenceofthesestrainswasimportantfortheclinicians[30].

Satoetal.,2017,intheirstudyconductedwithO25b-ST131clones,

bothcephalosporinsensitiveandresistantstrainswerereported.

However,whenthesestrainsexaminedintermsofciproﬂoxacin,

itwasseenthatalloftheO25b-ST131cloneswerestrainswere

resistant.Moreover,alloftheseciproﬂoxacinresistantstrainshad

MICvalues>128␮g/mL[31].Röderovaetal.,in2016,reported69%

O25b-ST131positivityandhighlevelsofciproﬂoxacinresistance(>

32␮g/mL)[32].Theresultsofourstudyaresimilartothese

stud-ies,accordingtoourresults,incaseofO25b-ST131presence,there

isadramaticincreaseinMICrange,MIC50andMIC90valuesfor

ciproﬂoxacin.

Conclusions

Asa result ofour study,thepresence of O25b-ST131clone,

CTX-M-1 and CTX-M-15 genes in E. coli strains identiﬁed as

causative agents in patients withUTI in our countryhas been

(5)

ciproﬂoxacin,whichiscommonlyusedinthetreatmentregimens,

wasinvestigatedamongthesestrains.Thereisadramaticincrease

inMICrange,MIC50andMIC90valuesforciproﬂoxacin,presence

of O25b-ST131clone in strains isolated fromUTIs. We believe

thatE.colistrainsproducingCTX-M-15andbelongingO25b-ST131

cloneshouldbefollowedbyreliablemethodssuchasmolecular

techniques,andthesemolecularepidemiologicaldatashouldbe

monitorizedtosupportclinicians.

Funding

NofundingSources.

Competinginterests

Nonedeclared.

Ethicalapproval

Thestudywasapprovedbythenon-invasiveclinicalresearch

ethicscommitteeoftheMedipolUniversitySchoolofMedicine.

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