Molecular Analysis of Ciprofloxacin Resistance Mechanisms...

Research ArticleMolecular Analysis of Ciprofloxacin Resistance Mechanisms inMalaysian ESBL-Producing Klebsiella pneumoniae Isolates andDevelopment of Mismatch Amplification Mutation Assays(MAMA) for Rapid Detection of gyrA and parC Mutations

Farah Al-Marzooq Mohd Yasim Mohd Yusof and Sun Tee Tay

Department of Medical Microbiology Faculty of Medicine University of Malaya 50603 Kuala Lumpur Malaysia

Correspondence should be addressed to Sun Tee Tay taystumgmailcom

Received 28 November 2013 Accepted 6 February 2014 Published 10 April 2014

Academic Editor Paul M Tulkens

Copyright copy 2014 Farah Al-Marzooq et al This is an open access article distributed under the Creative Commons AttributionLicense which permits unrestricted use distribution and reproduction in any medium provided the original work is properlycited

Ninety-three Malaysian extended-spectrum 120573-lactamase (ESBL)-producing Klebsiella pneumoniae isolates were investigated forciprofloxacin resistance Two mismatch amplification mutation (MAMA) assays were developed and used to facilitate rapiddetection of gyrA and parC mutations The isolates were also screened for plasmid-mediated quinolone resistance (PMQR) genesincluding aac(61015840)-Ib-cr qepA and qnr Ciprofloxacin resistance (MICs 4ndashge32 120583gmL) was noted in 34 (37) isolates of which 33isolates had multiple mutations either in gyrA alone (119899 = 1) or in both gyrA and parC regions (119899 = 32) aac(61015840)-Ib-cr was the mostcommon PMQR gene detected in this study (119899 = 61) followed by qnrB and qnrS (119899 = 55 and 1 resp) Low-level ciprofloxacinresistance (MICs 1-2 120583gmL) was noted in 40 (43) isolates carrying qnrB accompanied by either aac(61015840)-Ib-cr (119899 = 34) or a singlegyrA 83 mutation (119899 = 6) Ciprofloxacin resistance was significantly associated with the presence of multiple mutations in gyrAand parC regions While the isolates harbouring gyrA andor parC alteration were distributed into 11 PFGE clusters no specificclusters were associated with isolates carrying PMQR genesThe high prevalence of ciprofloxacin resistance amongst theMalaysianESBL-producing K pneumoniae isolates suggests the need for more effective infection control measures to limit the spread of theseresistant organisms in the hospital

1 Introduction

The emergence and spread of extended-spectrum 120573-lactamase (ESBL)-producing organisms have posed a greatchallenge to clinicians worldwide As ESBL-producingorganisms are usually resistant to many other antimicrobialagents limited therapeutic options are available for treatmentof infections caused by these organisms [1] Ciprofloxacinis one of the therapeutic choices for infections caused bybacteria belonging to the family Enterobacteriaceae Thisantibiotic acts by inhibiting bacterial DNA gyrase andtopoisomerase IV which are required for replication [2]

Resistance to fluoroquinolones (FQ) is now commonin many ESBL-producing Gram-negative bacteria includingKlebsiella pneumoniae [3 4] FQ resistance has been linked

to specific amino acid substitutions in the chromosomalquinolone resistance determining regions (QRDRs) in GyrAand B subunits of DNA gyrase and ParC and E subunits oftopoisomerase IV [5] Mutations at Ser83 and Asp87 codonsofGyrA subunit and Ser80 andGlu84 codons of ParC subunithave been commonly reported in FQ resistantK pneumoniaeisolates worldwide [6ndash8] DNA sequencing is the gold stan-dard method for the detection of these mutations howeverthis method is expensive laborious and time consumingHence cheaper simpler and rapid methods are requiredto facilitate mutation detection A few assays have beendeveloped for rapid detection of mutations in gyrA andorparC genes of Campylobacter jejuni [9] Escherichia coli [10]and Neisseria gonorrhoeae [11] using mismatch amplificationmutation assay (MAMA) a modified polymerase chain

Hindawi Publishing CorporationBioMed Research InternationalVolume 2014 Article ID 601630 10 pageshttpdxdoiorg1011552014601630

2 BioMed Research International

reaction that permits discriminatory amplification of specificallele sequences at QRDRs [12]

Plasmid-mediated quinolone resistance (PMQR) genesincluding qnr aac(61015840)-Ib-cr and efflux pumps are knownto confer low-level FQ resistance [13] Quinolone targetprotection by Qnr proteins are widely distributed in Enter-obacteriaceae worldwide [14] Until now six Qnr familiesnamely Qnr A B C D S and VC have been identified(httpwwwlaheyorgqnrStudies) While qnrA B and 119878genes are commonly detected at variable rates in K pneumo-niae worldwide [3 13 15] qnrC and 119863 have been reportedat low rates amongst K pneumoniae isolates in China[16] Moreover a variant of aminoglycoside acetyltransferase(AAC(61015840)-Ib-cr) with the ability to modify and inactivateciprofloxacin has been widely spread in K pneumoniaeisolates from Asia [17 18] and worldwide [3 14]

FQ resistance may also arise as a result of reducedintracellular drug accumulation caused by porin loss or activeefflux pump [5] QepA a quinolone-specific efflux pump hasbeen identified in Escherichia coli isolates from several Asiancountries such as Japan Korea and China [19ndash21] but wasrarely detected in K pneumoniae [22 23]

There is a paucity of data on the prevalence and thegenetic determinants associated with ciprofloxacin resistancein Malaysian K pneumoniae isolates Hence this study wasconducted to identify chromosomal as well as plasmid-mediated mechanisms of ciprofloxacin resistance in a groupof Malaysian ESBL-producing K pneumoniae isolates Tofacilitate rapid detection of gyrA and parC mutations twomismatch amplificationmutation assays (MAMA-PCR) weredeveloped and validated in this study

2 Materials and Methods

21 Bacterial Isolates A group of 93 nonduplicate ESBL-producing K pneumoniae isolates from patients attending toUniversity of Malaya Medical Centre and a private hospitalin Kuala Lumpur Malaysia in 2010ndash2012 were investigatedin this study The isolates were confirmed as K pneumoniaeusing a PCR assay targeting the internal transcribed spacerunit of the bacteria [24] Confirmation of ESBL productionwas performed using Cefpodoxime Combination Disc Kit(Oxoid UK)

22 Antibiotic Susceptibility Testing Minimum inhibitoryconcentration (MIC) of ciprofloxacin was determined by 119864-test strips (bioMereiux Marcy LrsquoEtoile France) on Mueller-Hinton agar (Oxoid UK) in accordance with the Clini-cal and Laboratory Standards Institute (CLSI) guidelines[25] MIC values ge4 2 and le1 120583gmL were used to defineresistance intermediate susceptibility and susceptibility tociprofloxacin respectively

23 Development of MAMA-PCR for gyrA and parC Muta-tions Detection Two duplex PCR assays (gyrA83 + parC80assay and gyrA87 + parC84 assay) were developed for thesimultaneous detection of mutations in Ser83 codon ofGyrA subunit and Ser80 codon of ParC subunit and Asp87

codon of GyrA subunit and Glu84 codon of ParC subunitrespectively

Universal gyrA and parC forward primers [16] were usedtogether with the reverse primers (MAMAprimers) designedin this study for the amplification of gyrA (Ser83 and Asp87)and parC (Ser80 and Glu84) genetic regions (Figure 1)MAMA primer design was performed using NCBIPrimer-BLAST tool (httpwwwncbinlmnihgovtoolsprimer-bl-ast) The reverse primers were complementary to the wild-type alleles of gyrA and parC sequences of K pneumoniaestrain ATCC 13883 (GenBank accession numbers DQ673325and AF303641 resp) except for a mismatch at the ante-penultimate (minus3) nucleotide of the 31015840 end of each MAMAprimer which was included to improve allele discriminationThe MAMA primertemplate mismatches included in thisstudy were CC (in gyrA83 and 87) AG (in parC80) GA(in parC84) The selection of the mismatches was based onprevious observations of their effects on the overall PCR yield[26] The presence of a single primertemplate mismatch hasminimal effect on the PCR yield thus the wild-type gene canbe amplified efficiently In case ofmutation(s) PCR efficiencywill be extremely reduced due to the presence of additionalmismatch(es) at the 31015840 end of the MAMA primer which willnot bind to the template thus amplification of the target geneis failed [27]

The performance of the primers was first evaluated usingmonoplex PCR prior to use in the duplex PCR assays whichwere finally optimized for the simultaneous detection ofmutations in Ser83 codon of GyrA subunit with Ser80 codonof ParC subunit and in Asp87 codon of GyrA subunit withGlu84 codon of ParC subunit

For the first duplex PCR assay the concentrations ofprimers were optimized to 04120583M for each parC80 MAMAprimer and parC universal forward primer in addition to025 120583M for each gyrA83 MAMA primer and gyrA universalforward primer For the second assay the concentrations ofprimers were optimized to 045120583M for each parC84 MAMAprimer and parC universal forward primer in addition to02 120583M for each of gyrA87MAMAprimer and gyrA universalforward primer The primer mixtures were added to a finalPCR reaction volume of 20120583L containing 4 120583L of 5x HOTFIREPol Blend Master Mix (Solis BioDyne Estonia) whichwas comprised of 200120583M of each dNTP 005U120583L of DNApolymerase and 15mM MgCl

2 Finally 1 120583L (lt100 ng) of

boiled bacterial extract [28] was added to each reactionAmplification was carried out on a Veriti 96-well thermalcycler (Applied Biosystems USA) programmed as followsinitial denaturation at 95∘C for 10min followed by 30 cyclesof denaturation at 95∘C for 30 s annealing at 56∘C for 40 sand extension at 72∘C for 50 s and a final extension step at72∘C for 7min PCR products were analysed on a 2 agarosegel prestained with 05 120583gmL ethidium bromide in 05x TBEbuffer

K pneumoniae strains with knownmutations in gyrA (45isolates) and parC (10 isolates) were used as quality controlstrains for the assay optimization and validation [29]

24 Detection of gyrA and parC Mutations by MAMA-PCR Following validation of theMAMA-PCR assays the 93

BioMed Research International 3

Possible mutations

ATCC 13883

(Tyr)(Ile) (Phe)

(Gly)(Ala)(Asn)

3998400-AGC CGC CAT ATG CTG TGG T-5998400MAMA gyrA-83

MAMA gyrA-87

NucleotidesK pneumoniae

gyrA geneAmino acids

235 CCG CAC GGC GAC TCC GCG GTA TAC GAC ACC ATC GTG CGT ATG GCG CAG 79

83 87P H G D S D T I V R M A QA V Y

TAC GGC

GCC

AAC

ATC

TTC

3998400-CTC TGG TAG CAC GCA TAC CG-5998400

(a)

80 84

Possible mutations

MAMA parC-84

MAMA parC-80

NucleotidesAmino acids

223 CAC CCG CAC GGC GAC AGC GCC TGC TAT GAA GCG ATG GTG CTG ATG GCG

H P H G D S A C Y E A M V L M A75

ATC (Ile) GGA

AAA (Lys)AGA (Arg)(Gly)lowast

ATCC 13883 K pneumoniae

parC gene

3998400-CG AGG ACG ATA CTT CGC TAC CA-5998400

3998400-CTG CGC TAC CAC GAC TAC-5998400

(b)

Figure 1 MAMA-PCR primers for gyrA (a) and parC (b) mutation detection Red highlighted nucleotides are the mismatched nucleotidesat the 31015840 end of eachMAMA primer Mismatches were positioned at the conserved nucleotides of each codon (highlighted by yellow) locatedat the 3rd nucleotide from the 31015840 end of each primer except for parC80 primer where the conserved nucleotide (1st nucleotide in the parC80codon) was excluded from the MAMA primer and the alteration was situated at a nucleotide outside the coding region (pink highlightednucleotide) Quality control strains with the expected mutations shown in the figure were used for the assay development and optimizationexcept the mutation with lowast which was not available

clinical isolates investigated in this study were tested foralterations in gyrA andor parC regions For confirmationpurpose amplification and sequence analysis of the entirecoding regions of gyrA and parC for 25 randomly selectedisolates were performed as described previously [16] Thenucleotide sequences and deduced proteins were analyzedby NCBI tools and BioEdit software (version 7) and werecompared with those of GyrA and ParC subunits of K pneu-moniae strain ATCC 13883 (GenBank accession numbersDQ673325 and AF303641 resp)

25 Detection of Plasmid-Mediated Quinolone Resistance(PMQR)Genes All isolateswere subjected to screening usinga multiplex PCR assay for the detection of qnr types (A BC and S) [13] and a monoplex PCR assay for the detectionof qnrD type [30] Detection of efflux pump (qepA) and theaminoglycoside acetyltransferase (aac(61015840)-Ib) was performedusing a multiplex PCR assay [13] Allele-specific PCR assaywas used to identify the cr mutation in aac(61015840)-Ib [31] Toconfirm the PCR results representative amplicons of eachPMQR gene were sequenced Additionally nine isolates fromdifferent susceptibility categories (resistant intermediatelysusceptible and susceptible to ciprofloxacin) were selected

for sequence determination of the entire qnrB gene asdescribed previously [32] The nucleotide sequences anddeduced proteins were compared to the reference sequencesin Lahey website (httpwwwlaheyorgqnrStudies) andGenBank database using BLAST search engine (httpblastncbinlmnihgovBlastcgi)

26 Pulsed-Field Gel Electrophoresis (PFGE) PFGE was usedto determine the genetic relationship of the isolates asdescribed previously [33] Fragments generated by restric-tion with XbaI enzyme (New England Biolabs USA) wereseparated by the CHEF-DR II system (Bio-Rad LaboratoriesUSA) The resultant banding patterns were analysed withBioNumerics software version 71 (Applied Maths Belgium)by the unweighted pair group method with arithmetic mean(UPGMA) algorithm Cluster designation was based onisolates showing ge80 relatedness

27 Statistical Analyses Categorical variables were comparedby the Chi-square or Fisherrsquos exact test and continuousvariables were compared by Mann-Whitney 119880 test Therelationship between ciprofloxacin MIC values with thenumber of gyrA andor parC mutations and with the total


number of quinolone resistance determinants was assessedby calculating Spearmanrsquos correlation coefficient The totalnumber of quinolone resistance determinants was calculatedby adding the number of PMQR genes and the number ofmutations in Ser83 andorAsp87 codons ofGyrA subunit andin Ser80 andor Glu84 codons of ParC subunit All tests weretwo-tailed and a 119875 value lt 005 was considered statisticallysignificant All statistical analyses were performed by PASWsoftware version 18 (SPSS Chicago IL USA)

3 Results

Reduced susceptibility to ciprofloxacin (resistance and inter-mediate susceptibility) was observed in 66 (71) isolatesinvestigated in this study Ciprofloxacin MIC values of theisolates ranged from 0032 to ge32 120583gmL with MIC

90and

MIC50

equal to ge32 and 2 120583gmL respectively Based onciprofloxacin MICs the 93 ESBL-producing K pneumoniaeisolates were grouped into three susceptibility categoriesTheMIC values with quinolone resistance determinants in eachcategory are shown in Table 1

31 Development and Validation of MAMA-PCR The uni-versal forward and MAMA reverse primers generated aPCR product from the wild-type gene in the absence ofmutation(s) On the other hand PCR was inhibited in thepresence of mutation(s) (two or more mismatches at the 31015840end of the MAMA primer) therefore negative PCR resultwas an indication of mutation in the corresponding geneticregion MAMA primers were able to distinguish wild typesfrom mutations for all of the quality control strains (45isolates for gyrA and 10 isolates for parC) The four MAMAmonoplex PCR assays were then combined into two duplexassays (gyrA83 + parC80 assay and gyrA87 + parC84 assay)Figure 2 shows the results of MAMA duplex assays for someof the isolates investigated in this study

32 Detection of gyrA and parC Mutations by MAMA-PCRAssays Alterations in gyrA andor parC genetic regions weredetected in 41 of the 93 K pneumoniae clinical isolates byMAMA method as shown in Table 2 Mutations in gyrAwere detected in 44 (119899 = 41) of the isolates of which 17isolates had both Ser83 and Asp87 alterations and 24 isolateshad Ser83 mutation detected either alone in ciprofloxacinsusceptible (119899 = 5) or intermediately susceptible isolates (119899 =3) or coupled with parC mutations in ciprofloxacin resistantisolates (119899 = 16)

Alterations in ParC subunit of DNA topoisomerase IVwere detected in 344 (119899 = 32) of the isolates of which30 isolates had Ser80 mutation and two isolates had Glu84alteration Mutations in parC were detected in ciprofloxacinresistant isolates which had single or multiple gyrA muta-tions

MAMA findings were confirmed by sequence analysisof the entire coding regions of gyrA and parC for selectedisolates (seven isolates with the wild type of gyrA and parCand 18 isolates with MAMA results indicative of gyrA andorparC alterations) Detected amino acid substitutions in GyrA

were Ser83Ile (119899 = 10) Ser83Tyr (119899 = 4) and Ser83Phe +Asp87Ala (119899 = 4) whilst substitutions in ParC were Ser80Ile(119899 = 15) Ser80Arg (119899 = 1) and Glu84Lys (119899 = 2) (Table 2)

33 PMQRGenes aac(61015840)-Ib gene was detected in 74 (796)of the isolates of which 61 (656) carried the cr variant qnrgenes were detected from 56 isolates (602) of which 55 car-ried qnrB (591) and one carried qnrS (11) Sequence anal-ysis of qnrB gene in nine randomly selected isolates revealed100 nucleotide sequence identity with qnrB1 (119899 = 4) qnrB6(119899 = 3) and qnrB7 (119899 = 2) (GenBank accession numbersDQ351241 GQ914054 and EU043311 resp) The deducedproteins (223 amino acids) also exhibited 100 amino acididentity with QnrB1 QnrB6 and QnrB7 (GenBank accessionnumbers DQ351241 ADH03417 and ABW03156 resp)

aac(61015840)-Ib-cr and qnr genes were detected from isolateswhich were susceptible (17 and 11 isolates resp) interme-diately susceptible (29 and 32 isolates resp) and resistantto ciprofloxacin (15 and 13 isolates resp) Interestingly 47isolates (502) harbored both qnrB and aac(61015840)-Ib-cr genesthus the association between both genes was statisticallysignificant (119875 lt 0001) Neither qepA efflux pump nor qnrAC and119863 genes were detected in this study

34 The Relationship between Ciprofloxacin MIC and FQResistance Determinants Table 1 shows the increase in theciprofloxacin MICs of our isolates which was accompaniedby a stepwise accumulation of FQ resistance determinantsThe increase in ciprofloxacin MICs is correlated stronglywith the increase in the total number of FQ resistancedeterminants (both gyrA andor parC mutations and PMQRgenes) (Spearmanrsquos correlation coefficient = 0918119875 lt 0001)

The lowest MIC values were noted in the isolates lackingany FQ resistance determinants (0032ndash0047 120583gmL) Forthe isolates with one FQ resistance determinant MICsof isolates expressing aac(61015840)-Ib-cr alone were significantlylower (0094ndash038 120583gmL) than those of isolates expressingqnr gene alone or having a single gyrA83 mutation (05ndash075 120583gmL) (119875 = 0001) MICs of isolates expressing twoFQ resistance determinants (qnr gene accompanied by eitheraac(61015840)-Ib-cr or a single gyrA83 mutation) were significantlyhigher (1-2 120583gmL) compared to those of isolates expressingone of the above mentioned genes alone (119875 lt 0001)

Of note 34 out of 40 isolateswhich demonstrated reducedsusceptibility to ciprofloxacin (MIC = 1-2120583gmL) harboredboth qnrB and aac(61015840)-Ib-cr genes thus both genes were sig-nificantly associated with low-level ciprofloxacin resistance(119875 lt 0001)

There is significant association between the resistancephenotype and the presence of more than one mutationin gyrA andor parC (119875 lt 0001) as most (33 out of34) ciprofloxacin resistant isolates harbored 2-3 mutationsin gyrA andor parC codons These isolates demonstratedsignificantly higher MIC values (4ndashge32120583gmL) compared tothose of isolates harboring single gyrA83 mutation with andwithout qnrB gene (MICs 05ndash2 120583gmL 119875 lt 0001) andthose harboring qnr andor aac(61015840)-Ib-cr genes without anyalterations in QRDRs (MICs 0094ndash2120583gmL 119875 lt 0001)


Table1Ciprofl

oxacin

susceptib

ilitypatte

rnsa

ndflu

oroq

uino

lone

(FQ)resistance

determ

inantsdetected

inthe9

3Kpneum

oniaeisolatesinvestig

ated

inthisstu

dy

Ciprofl

oxacin

susceptib

ility

MIC

(120583gmL)

Num

bero

fiso

lates

FQresistanced

eterminants

Totaln

umbero

fgyrA

andor

parC

alteratio

nsTo

taln

umbero

fFQ

resistanced

eterminants

PMQRlowast

genes(119899)

gyrA

andor

parC

alteratio

ns(119899)

Susceptib

le(119899=2729

)

0032ndash0047

2Non

e(2)

Non

e(2)

Non

eNon

e0094ndash

038

12aac(61015840)-Ib-cr(12)

Non

e(12)

Non

e1

05ndash075

5qnrB

(2)qnrS

(1)

Non

e(2)

Non

e(3)

gyrA

83(2)

Non

e1

1 11

8qnrB

+aac(61015840)-Ib-cr(5)

qnrB

(3)

Non

e(5)

gyrA

83(3)

Non

e1

2 2Interm

ediately

susceptib

le(119899=3234

)2

32qnrB

+aac(61015840)-Ib-cr

(29)

qnrB

(3)

Non

e(29)

gyrA

83(3)

Non

e1

2 2

Resistant

(119899=3437

)

4ndash6

4Non

e(4)

gyrA

83+parC

80(4)

22

ge32

30

qnrB

+aac(61015840)-Ib-cr

(8)

qnrB

+aac(61015840)-Ib-cr

(2)

qnrB

+aac(61015840)-Ib-cr(1)

qnrB

+aac(61015840)-Ib-cr

(1)aac(61015840)-Ib-cr

(2)

qnrB

+aac(61015840)-Ib-cr(1)

gyrA

83+parC

80(10)

gyrA

83+parC

84(2)

gyrA

83+gyrA

87(1)

gyrA

83+gyrA

87+parC

80(16)

Non

e(1)

2 2 2 3Non

e

2or

44 4 3ndash5 2

lowastPM

QR

Plasmid-m

ediatedqu

inolon

eresistance


Table 2 Alterations in gyrA and parC genes detected by MAMA-PCR and confirmed by sequence analysis for selected isolates

Total number ofmutations

Number ofisolates

Alterations detected by MAMA-PCR Confirmation by sequencing (119899)gyrA parC

83 87 80 84 gyrA parCNone 52 None None None None Wild type (7) Wild type (7)

1 8 Mutation None None None Ser83Tyr (4) ND

21 Mutation Mutation None None ND ND14 Mutation None Mutation None Ser83Ile (8) Ser80Ile (9) Ser80Arg (1)2 Mutation None None Mutation Ser83Ile (2) Glu84Lys (2)

3 16 Mutation Mutation Mutation None Ser83Phe + Asp87Ala (4) Ser80Ile (6)ND not done

3000

1000

500

300

200

100

(bp)MM1 2 3 4 5 6

gyrA83 = 259bp

parC80 = 190bp

(a) gyrA83 + parC80 duplex PCR

3000

1000

500

300

200

100

(bp)MM1 2 3 4 5 6

gyrA87 = 272bp

parC84 = 197bp

(b) gyrA87 + parC84 duplex PCR

Figure 2 Agarose gel electrophoresis image of PCR products generated from duplex MAMA-PCR assays (a) gyrA83 + parC80 and (b)gyrA87 + parC84 Identification of each target was based on the expected product size Lanes 1-2 represent PCR products generated in thepresence of the wild-type alleles Lanes 3ndash6 are examples of products generated in case of mutations in one gene or in both target genes MDNA molecular size marker (100 bp DNA Ladder Solis BioDyne Estonia)

Thus the increase in ciprofloxacinMICs is correlated stronglywith the increase in the total number of mutations in gyrAandor parC subunits (Spearmanrsquos correlation coefficient =078 119875 lt 0001)

Notably aac(61015840)-Ib-cr and qnrB were detected in someof the ciprofloxacin resistant isolates (15 and 13 isolatesresp) The real contribution of PMQR on ciprofloxacin MICis not clear in the ciprofloxacin resistant isolates as thereis nonsignificant difference in ciprofloxacin MICs of theresistant isolates with and without aac(61015840)-Ib-cr and qnrBgenes (119875 gt 005)

35 PFGE The 93 isolates investigated in this study weredifferentiated into 41 PFGE clusters (Figure 3) The isolatesharboring gyrA andor parC alteration were distributed into11 clusters of which six clusters (X1ndashX6) were composed of 2ndash14 genetically related isolates whereas the remaining five clus-ters were comprised of only one isolate each Identical gyrAandor parC mutations were found amongst isolates withinthe same cluster with the only exception of two ciprofloxacinresistant isolates in cluster X4 In this cluster two highlyrelated isolates (923) harboring gyrA83 and parC84 muta-tions were genetically related (less than 89) to another

two isolates without any gyrA and parC mutations (one wassensitive and the other was intermediately susceptible tociprofloxacin) While gyrA andor parC alteration were lim-ited to isolates within 11 clusters PMQR genes were detectedin isolates distributed into 38 clusters The FQ resistancedeterminants of the isolates in different clusters were pre-sented in the supplementary data file (Supplementary Mate-rial available online at httpdxdoiorg1011552014601630)

4 Discussion

A big proportion of our isolates (71) were nonsusceptible(resistant and intermediately susceptible) to ciprofloxacinwhich is a common finding in ESBL-producing isolates asreported in several countries such as Taiwan (591) [34]France (603) [3] and UK (623) [35] According to thelatest study of antimicrobial resistance trends (SMART) inthe Asia-Pacific region ciprofloxacin nonsusceptibility in Kpneumoniaewasmuch higher in the ESBL-producing isolates(658) compared to the non-ESBL-producing isolates (74)[36] This may explain the reason why our ciprofloxacinnonsusceptibility rate (71)was higher than the rate reportedin a previous Malaysian study (18) because the isolates


M63

M56K5

2M

27

M44

M71

M74

M99

M82

M35

M21

M91

M58M

5M

60M

104

M92

M69

M37

M96

M73

M66

M70

M75

M80

M93

M46

M33

M67

M50

M78

M30

M98

M10

3M

81K92

K22

K106

M10

5

X4 X5 X6X1 X2 X3

K129K5

M38

M40

M85

M90

M95

M32

M51

M10

7M

100

M65

M52

M77

M11

M89

M79

M61

M87

M59

M86M

2M

57M

109

M11

1

M11

0

M36

M28

M13

M72M

1M

68

M47

K112K1

2

K103 K5

0

ND

M

M10

6M

48 K27

K66

K24

K40

M84 M

4K1

26

K114 K1

4

M49

K109

M15

M10

2K1

30

gyrA83 gyrA83 gyrA83 gyrA83 gyrA83+ parC80 + parC80 + parC80 + parC84 gyrA83 and 87

+ parC80

60

65

70

75

80

85

90

95

100

976964958943525

977

976

966

963

897871

865

840

947

976

973

937

893

852

761

722

718

790

780

751

664

590676

739

743

820

910

947

919

732

760

752

750

882

938925

842

971

760

711

709

818

914

970

904

699

608596577

625

617

648

871

743

750

649

669

626

666

690

886

930

646

794

813

587

797

906

950

886

886

822

923 907

927

930

940

974

927

960

973

971

651

911

774

675

Study ID

Sim

ilarit

y (

)

Figure 3 PFGE dendrogram of the 93 K pneumoniae isolates investigated in this study Alterations in GyrA andor ParC subunits weredetected in the isolates which belong to 11 clusters Black triangles represent clusters with multiple isolates possessing the same gyrA andorparCmutations Black circles represent monoisolate clusters with gyrA andor parCmutationsThe dashed line represents the similarity level(80) used in the clusters definition

investigated in that study were a mixture of ESBL and non-ESBL-producing K pneumoniae isolates [37]

In this study two multiplex MAMA-PCR assays havebeen successfully developed for the detection of mutationsin gyrA83 + parC80 and in gyrA87 + parC84 codons ofquinolone resistance determining regions in K pneumoniaeTo the best of our knowledge no specific assay has beendeveloped previously for the detection of alterations in theQRDRs ofK pneumoniaeA fewmonoplex PCR assays usingMAMAmethod have been developed to detect alterations inbacterial QRDRs For C jejuni and N gonorrhoeae MAMAprimers were designed to amplify particular mutations in thegyrA codon and not the wild type [9 11] On the contrarythe primers used in the E coli MAMA-PCR method weredesigned in a way that gyrA or parCwild types were amplifiedand no PCR product would be obtained if mutations werepresent [10] A similar approach was used in designing theMAMA primers in this study Additionally instead of usingfour monoplex MAMA-PCR assays as described in the Ecoli study two duplex assays were designed for simultaneousamplification of the gyrA and parC genetic regions Theduplex assay strategy is expected to facilitate rapid detectionof mutations in the gyrA and parC since it shortens the timeand the steps involvedTheMAMAmethod developed in thisstudy is rapid and cost effective compared toDNAsequencingapproach and is useful for screening of a large number ofK pneumoniae isolates Isolates with results suggestive ofmutations can be selected for sequence analysis in order todefine the amino acid at the mutation site

DNA sequence analysis for selected isolates revealedseveral types of amino acid substitutions in GyrA (Ser83IleSer83Tyr Ser83Phe and Asp87Ala) and ParC (Ser80IleSer80Arg and Glu84Lys) Similar amino acid substitutionswere detected in GyrA regions of K pneumoniae isolatesfrom Malaysia [29] and other Asian countries [16 38] Nodata is available on ParC alterations in the Malaysian Kpneumoniae isolates however the amino acid substitutions

detected in our study (Ser80Ile Ser80Arg and Glu84Lys)have been reported previously inK pneumoniae isolates fromother Asian countries such as Singapore [39] Japan [6] andTaiwan [38]

In agreement with previous reports [6 40] multiplealterations in GyrA andor ParC have been associated withciprofloxacin resistance Isolates with single alterations ingyrA83 exhibited reduced susceptibility to ciprofloxacin(MIC = 05ndash2120583gmL) This observation has been reportedpreviously [5] and is considered as the first step for thedevelopment of full resistance to ciprofloxacin CiprofloxacinMICs of our isolates increased with the acquisition ofadditional mutations in gyrA andor parC genetic regionsThis was expected as previous studies have shown that theprogression fromFQ susceptible towards resistant phenotypeis a gradual process starting from mutations in gyrA theprimary target of FQ and followed by parC alterations whichhas a complementary role in the development of higherresistance [2 41]

Surprisingly we had one ciprofloxacin resistant isolate(MIC ge 32 120583gmL) which lacked any mutation in gyrAand parC genes but harbored both qnrB and aac(61015840)-Ib-crgenes Similar observations have been reported in isolatesfrom other geographical regions [39 42 43] The possibleinvolvement of resistance mechanisms such as the reductionof bacterial drug uptake due to active efflux system (forinstance OqxAB) andor membrane impermeability due toporin loss or mutation in other genetic regions such as gyrBor parE is yet to be explored

The high prevalence of PMQR genes (656 for aac(61015840)-Ib-cr and 602 for qnr) in our ESBL-producing K pneu-moniae isolates is in agreement with previous findings fromother parts of the world [34 44] This high prevalence canbe attributed to the coexistence of ESBL and PMQR genes onthe same plasmid as reported previously [34 45] Similarlythe simultaneous detection of both qnrB and aac(61015840)-Ib-crgenes in 502 of the isolates is an indication that they are


most likely located on the same plasmid as shown in previousstudies [17 45]

aac(61015840)-Ib-crwas themost commonPMQRgene detectedin this study No information is available on the prevalenceof this gene in the Malaysian Enterobacteriaceae isolateshowever studies from other Asian countries such as China[18] Korea [17] andThailand [44] confirmed the emergenceand spread of this gene amongst K pneumoniae isolates

qnrB was the predominant qnr gene identified in thisstudy in agreement with a previous report from our hospital[29] and reports from other Asian countries [15 46] qnrB1qnrB6 and qnrB7 were detected by sequence analysis in someof our isolates Both qnrB1 and qnrB6 have been previouslydetected in K pneumoniae isolates from Malaysia [29] andother parts of Southeast Asia [38 44] whereas qnrB7 has onlybeen reported in two K pneumoniae isolates from Norwayand Sweden [47]

qnrS was detected at a very low frequency in our isolatesin contrast to a recent Thai report whereby this gene wasthe dominant qnr type in K pneumoniae isolates [44] Otherqnr types including qnrA C and 119863 were not detected inthis study The prevalence of qnr genes is variable from timeto time and in different geographical locations [13] Thisphenomenon has been attributed to the variations in the qnr-carrying plasmids which can also possess multiple antibioticresistance genes As a result of antibiotic selective pressuresome plasmids may dominate in certain clinical settings [48]qepA efflux pump gene was not detected in our isolates Thiswas expected as the prevalence of this gene is generally lowworldwide [22 23]

In this study the progressive increase in ciprofloxacinMICs of our isolates is correlated with the stepwise accu-mulation of FQ resistance determinants Isolates with singleFQ resistance determinant (a single PMQR gene (qnr oraac(61015840)-Ib-cr) or a single gyrA83 mutation) demonstratedciprofloxacin MICs (0094ndash075120583gmL) lower than thoseof isolates expressing two FQ resistance determinants (1-2 120583gmL) including two PMQR genes (qnrB and aac(61015840)-Ib-cr) or qnrB with a single gyrA83 mutation (Table 1) Ourresults indicate that the effect of different FQ resistancedeterminants on ciprofloxacinMIC is cumulative as reportedpreviously [14]

Although the expression of qnr and aac(61015840)-Ib-cr con-fers low-level ciprofloxacin resistance it may have a nega-tive impact on the therapeutic efficacy of ciprofloxacin asobserved in rat animal models of experimental infectionwith qnr producing K pneumoniae [49 50] Moreover theexpression of these genes can increase the mutant preventionconcentration which is the lowest antimicrobial concentra-tion required to prevent the emergence of resistant mutantsthus resistant mutants can be selected under ciprofloxacintherapeutic levels [51 52]

The PFGE results in this study show the evidence ofthe spread of ciprofloxacin resistant isolates harboring GyrAandor ParC alterations by clonal expansion as identicalmutations in gyrA andor parC were detected from geneti-cally related isolates Similar findings have also been reportedpreviously by other investigators [35] Interestingly bothgyrA83 and parC84 mutations were detected in two highly

related isolates (923) which were genetically related toanother two isolates without any gyrA and parC mutationsAll the four isolates were hospital-associated (data notshown) therefore it is possible that they have originatedfrom a common ancestor in the hospital environmentCiprofloxacin resistance in two of the four isolates wasprobably caused by de novomutations in gyrA83 and parC84induced by ciprofloxacin selective pressure as previouslyobserved in E coli [53] This assumption was supportedby the finding of both qnrB and aac(61015840)-Ib-cr in the twociprofloxacin resistant isolates as both genes have the abilityto enhance the selection of chromosomal mutations [2]

PMQR genes were widely distributed into isolates withindifferent PFGE clusters Horizontal dissemination of theplasmids carrying PMQR genes is probably responsible forthe high prevalence of PMQR genes in our isolates due totheir wide distribution amongst genetically unrelated isolates[44]

5 Conclusions

A high prevalence of ciprofloxacin resistance was reportedamongst the Malaysian ESBL-producing K pneumoniae iso-lates investigated in this study The current scenario cancomplicate the clinical management of patients because veryfew antimicrobial agents are active against these bacteriaThisstudy also identified chromosomal and plasmid-mediatedgenetic determinants associated with ciprofloxacin resis-tance TheMAMAmethod developed in this study is simplecost effective and rapid for detection of gyrA and parCmutations It is important for epidemiologic investigationsparticularly when a large number of bacterial isolates needto be screened The high prevalence of PMQR genes in ourisolates is alarming as these genes can be widely spreadvia plasmids confer low-level ciprofloxacin resistance andfacilitate the selection of chromosomal mutations implicatedin higher level ciprofloxacin resistance Regular surveillanceof ciprofloxacin resistance determinants and molecular epi-demiologic investigations are essential in order to follow upthe progress of resistance development and spread in ourhospital settings

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Acknowledgments

This study was supported by the University of MalayaPostgraduate ResearchGrant (PV037-2012A) and theUniver-sity of Malaya High Impact Research Grant (HIRE000013-20001) PFGE analysis was performed using a fully functionaltemporary evaluation license of BioNumerics software (ver-sion 71) Permission to publish the PFGE results was obtainedfrom Applied Maths Belgium The authors thank Ms AmalS Saiful Anuar for her kind assistance in this study


References

[1] E Lautenbach J B Patel W B Bilker P H Edelstein andN O Fishman ldquoExtended-spectrum 120573-lactamase-producingEscherichia coli andKlebsiella pneumoniae risk factors for infec-tion and impact of resistance on outcomesrdquo Clinical InfectiousDiseases vol 32 no 8 pp 1162ndash1171 2001

[2] K Drlica X Zhao M Malik T Salz and R Kerns ldquoFluoro-quinolone resistance mechanisms restrictive dosing and anti-mutant screening strategies for new compoundsrdquo in AntibioticDiscovery and Development T J Dougherty and M J PucciEds pp 485ndash514 Springer New York NY USA 2012

[3] L Cremet N Caroff S Dauvergne A Reynaud D Lepelletierand S Corvec ldquoPrevalence of plasmid-mediated quinoloneresistance determinants in ESBL Enterobacteriaceae clinicalisolates over a 1-year period in a French hospitalrdquo PathologieBiologie vol 59 no 3 pp 151ndash156 2011

[4] D L Paterson LMulazimoglu JM Casellas et al ldquoEpidemiol-ogy of ciprofloxacin resistance and its relationship to extended-spectrum 120573-lactamase production in Klebsiella pneumoniaeisolates causing bacteremiardquoClinical Infectious Diseases vol 30no 3 pp 473ndash478 2000

[5] G A Jacoby ldquoMechanisms of resistance to quinolonesrdquoClinicalInfectious Diseases vol 41 no 2 pp S120ndashS126 2005

[6] T Deguchi A Fukuoka M Yasuda et al ldquoAlterations in thegyrA subunit of DNA gyrase and the parC subunit of topoiso-merase IV in quinolone-resistant clinical isolates of Klebsiellapneumoniaerdquo Antimicrobial Agents and Chemotherapy vol 41no 3 pp 699ndash701 1997

[7] Y Fu L Guo Y Xu et al ldquoAlteration of GyrA amino acidrequired for ciprofloxacin resistance in Klebsiella pneumoniaeisolates in Chinardquo Antimicrobial Agents and Chemotherapy vol52 no 8 pp 2980ndash2983 2008

[8] Y Fu W Zhang H Wang et al ldquoSpecific patterns of gyrAmutations determine the resistance difference to ciprofloxacinand levofloxacin in Klebsiella pneumoniae and Escherichia colirdquoBMC Infectious Diseases vol 13 no 1 article 8 2013

[9] G Zirnstein Y Li B Swaminathan and F Angulo ldquoCipro-floxacin resistance inCampylobacter jejuni isolates detection ofgyrA resistance mutations by mismatch amplification mutationassay PCR and DNA sequence analysisrdquo Journal of ClinicalMicrobiology vol 37 no 10 pp 3276ndash3280 1999

[10] Y Z Qiang T Qin W Fu W P Cheng Y S Li and G YildquoUse of a rapid mismatch PCR method to detect gyrA andparC mutations in ciprofloxacin-resistant clinical isolates ofEscherichia colirdquo Journal of Antimicrobial Chemotherapy vol 49no 3 pp 549ndash552 2002

[11] Z Sultan S Nahar B Wretlind E Lindback and M RahmanldquoComparison of mismatch amplification mutation assay withDNA sequencing for characterization of fluoroquinolone resis-tance inNeisseria gonorrhoeaerdquo Journal of ClinicalMicrobiologyvol 42 no 2 pp 591ndash594 2004

[12] D N Birdsell T Pearson E P Price et al ldquoMelt analysisof mismatch amplification mutation assays (Melt-MAMA) afunctional study of a cost-effective SNP genotyping assay inbacterial modelsrdquo PLoS ONE vol 7 no 3 Article ID e328662012

[13] B K Hong H P Chi J K Chung E C Kim G A Jacobyand D C Hooper ldquoPrevalence of plasmid-mediated quinoloneresistance determinants over a 9-year periodrdquo AntimicrobialAgents and Chemotherapy vol 53 no 2 pp 639ndash645 2009

[14] J Ruiz M J Pons and C Gomes ldquoTransferable mechanismsof quinolone resistancerdquo International Journal of AntimicrobialAgents vol 40 no 3 pp 196ndash203 2012

[15] A Wang Y Yang Q Lu et al ldquoPresence of qnr genein Escherichia coli and Klebsiella pneumoniae resistant tociprofloxacin isolated from pediatric patients in Chinardquo BMCInfectious Diseases vol 8 no 1 article 68 2008

[16] B Li Y Yi Q Wang et al ldquoAnalysis of drug resistancedeterminants in Klebsiella pneumoniae isolates from a tertiary-care hospital in Beijing Chinardquo PLOS ONE vol 7 no 7 ArticleID e42280 2012

[17] S Y Shin K C Kwon J W Park et al ldquoCharacteris-tics of aac(61015840)-Ib-cr gene in extended-spectrum 120573-lactamase-producing Escherichia coli and Klebsiella pneumoniae isolatedfrom Chungnam areardquo Korean Journal of Laboratory Medicinevol 29 no 6 pp 541ndash550 2009

[18] H Yang H Chen Q Yang M Chen and H Wang ldquoHighprevalence of plasmid-mediated quinolone resistance genes qnrand aac(61015840)-Ib-cr in clinical isolates of Enterobacteriaceae fromnine teaching hospitals in Chinardquo Antimicrobial Agents andChemotherapy vol 52 no 12 pp 4268ndash4273 2008

[19] K Yamane J I Wachino S Suzuki et al ldquoNew plasmid-mediated fluoroquinolone efflux pump qepA found in anEscherichia coli clinical isolaterdquo Antimicrobial Agents andChemotherapy vol 51 no 9 pp 3354ndash3360 2007

[20] E S Kim J Y Jeong S H Choi et al ldquoPlasmid-mediatedfluoroquinolone efflux pump gene qepA in Escherichia coliclinical isolates in Koreardquo Diagnostic Microbiology and Infec-tious Disease vol 65 no 3 pp 335ndash338 2009

[21] J H Liu Y T Deng Z L Zeng et al ldquoCoprevalence of plasmid-mediated quinolone resistance determinants qepA Qnr andAAC(61015840)-Ib-cr among 16S rRNA methylase RmtB-producingEscherichia coli isolates from pigsrdquo Antimicrobial Agents andChemotherapy vol 52 no 8 pp 2992ndash2993 2008

[22] J Ma Z Zeng Z Chen et al ldquoHigh prevalence of plasmid-mediated quinolone resistance determinants qnr aac(61015840)-Ib-crand qepA among ceftiofur-resistant Enterobacteriaceae isolatesfrom companion and food-producing animalsrdquo AntimicrobialAgents and Chemotherapy vol 53 no 2 pp 519ndash524 2009

[23] Y Luo J Yang Y Zhang L Ye LWang andLGuo ldquoPrevalenceof 120573-lactamases and 16S rRNA methylase genes amongst clin-ical Klebsiella pneumoniae isolates carrying plasmid-mediatedquinolone resistance determinantsrdquo International Journal ofAntimicrobial Agents vol 37 no 4 pp 352ndash355 2011

[24] Y Liu C LiuW Zheng et al ldquoPCRdetection ofKlebsiella pneu-moniae in infant formula based on 16S-23S internal transcribedspacerrdquo International Journal of Food Microbiology vol 125 no3 pp 230ndash235 2008

[25] Performance Standards for Antimicrobial Susceptibility TestingClinical and Laboratory Standards Institute document M100-S23 Wayne Pa USA 2013

[26] S Kwok D E Kellogg N McKinney et al ldquoEffects of primer-template mismatches on the polymerase chain reaction humanimmunodeficiency virus type 1 model studiesrdquo Nucleic AcidsResearch vol 18 no 4 pp 999ndash1005 1990

[27] B L Parsons and R H Heflich ldquoGenotypic selection methodsfor the direct analysis of point mutationsrdquo Mutation Researchvol 387 no 2 pp 97ndash121 1997

[28] T M Huang Y F Chang and C F Chang ldquoDetection ofmutations in the gyrA gene and class I integron fromquinolone-resistant Salmonella enterica serovar Choleraesuis isolates in


Taiwanrdquo Veterinary Microbiology vol 100 no 3-4 pp 247ndash2542004

[29] A S Saiful Anuar M Y Mohd Yusof and S T Tay ldquoPrevalenceof plasmid-mediated qnr determinants and gyrase alterationin Klebsiella pneumoniae isolated from a university teachinghospital in Malaysiardquo European Review for Medical and Phar-macological Sciences vol 17 no 13 pp 1744ndash1747 2013

[30] L M Cavaco H Hasman S Xia and F M Aarestrup ldquoqnrDa novel gene conferring transferable quinolone resistance inSalmonella enterica serovar Kentucky and Bovismorbificansstrains of human originrdquo Antimicrobial Agents and Chemother-apy vol 53 no 2 pp 603ndash608 2009

[31] D W Wareham I Umoren P Khanna and N C GordonldquoAllele-specific polymerase chain reaction (PCR) for rapiddetection of the aac(61015840)-Ib-cr quinolone resistance generdquo Inter-national Journal of Antimicrobial Agents vol 36 no 5 pp 476ndash477 2010

[32] M D Tamang S Y Seol J Y Oh et al ldquoPlasmid-mediatedquinolone resistance determinants qnrA qnrB and qnrS amongclinical isolates of Enterobacteriaceae in a Korean hospitalrdquoAntimicrobial Agents and Chemotherapy vol 52 no 11 pp4159ndash4162 2008

[33] R K Gautom ldquoRapid pulsed-field gel electrophoresis protocolfor typing of Escherichia coli O157H7 and other gram-negativeorganisms in 1 dayrdquo Journal of Clinical Microbiology vol 35 no11 pp 2977ndash2980 1997

[34] C J Lin L K Siu L Ma Y T Chang and P L Lu ldquoMolecularepidemiology of ciprofloxacin-resistant extended-spectrum 120573-lactamase-producing Klebsiella pneumoniae in TaiwanrdquoMicro-bial Drug Resistance vol 18 no 1 pp 52ndash58 2012

[35] A A Dashti R Paton and S G B Amyes ldquoLinkage ofciprofloxacin resistance with a single genotypic cluster ofKlebsiella pneumoniaerdquo International Journal of AntimicrobialAgents vol 27 no 1 pp 73ndash76 2006

[36] P R Hsueh ldquoStudy for Monitoring Antimicrobial ResistanceTrends (SMART) in the Asia-Pacific region 2002ndash2010rdquo Inter-national Journal of Antimicrobial Agents vol 40 supplement 1pp S1ndashS3 2012

[37] K T Lim C C Yeo R M Yasin G Balan and K LThong ldquoCharacterization of multidrug-resistant and extended-spectrum120573-lactamase-producingKlebsiella pneumoniae strainsfromMalaysian hospitalsrdquo Journal of Medical Microbiology vol58 no 11 pp 1463ndash1469 2009

[38] C H Liao P R Hsueh G A Jacoby and D C Hooper ldquoRiskfactors and clinical characteristics of patients with qnr-positiveKlebsiella pneumoniae bacteraemiardquo Journal of AntimicrobialChemotherapy vol 68 no 12 pp 2907ndash2914 2013

[39] T Schneiders S G B Amyes and S B Levy ldquoRole of AcrRand RamA in fluoroquinolone resistance in clinical Klebsiellapneumoniae isolates from Singaporerdquo Antimicrobial Agents andChemotherapy vol 47 no 9 pp 2831ndash2837 2003

[40] S Bansal and V Tandon ldquoContribution of mutations in DNAgyrase and topoisomerase IV genes to ciprofloxacin resistancein Escherichia coli clinical isolatesrdquo International Journal ofAntimicrobial Agents vol 37 no 3 pp 253ndash255 2011

[41] L Drago L Nicola R Mattina and E de Vecchi ldquoIn vitroselection of resistance in Escherichia coli andKlebsiella spp at invivo fluoroquinolone concentrationsrdquo BMC Microbiology vol10 no 1 article 119 2010

[42] A Mazzariol J Zuliani G Cornaglia G M Rossolini andR Fontana ldquoAcrAB efflux system expression and contribution

to fluoroquinolone resistance in Klebsiella spprdquo AntimicrobialAgents and Chemotherapy vol 46 no 12 pp 3984ndash3986 2002

[43] J Ruiz ldquoMechanisms of resistance to quinolones target alter-ations decreased accumulation and DNA gyrase protectionrdquoJournal of Antimicrobial Chemotherapy vol 51 no 5 pp 1109ndash1117 2003

[44] W Pasom A Chanawong A Lulitanond et al ldquoPlasmid-mediated quinolone resistance genes aac(6rsquo)-Ib-cr qnrS qnrBand qnrA in urinary isolates of Escherichia coli and Klebsiellapneumoniae at a Teaching Hospital Thailandrdquo Japanese Journalof Infectious Diseases vol 66 no 5 pp 428ndash432 2013

[45] Y Jiang Z Zhou Y Qian et al ldquoPlasmid-mediated quinoloneresistance determinants qnr and aac(61015840)-Ib-cr in extended-spectrum120573-lactamase-producing Escherichia coli andKlebsiellapneumoniae in Chinardquo Journal of Antimicrobial Chemotherapyvol 61 no 5 pp 1003ndash1006 2008

[46] J W P Teo K Y Ng and R T P Lin ldquoDetection and geneticcharacterisation of qnrB in hospital isolates of Klebsiella pneu-moniae in Singaporerdquo International Journal of AntimicrobialAgents vol 33 no 2 pp 177ndash180 2009

[47] N Karah L Poirel S Bengtsson et al ldquoPlasmid-mediatedquinolone resistance determinants qnr and aac(61015840)-Ib-cr inEscherichia coli and Klebsiella spp from Norway and SwedenrdquoDiagnosticMicrobiology and InfectiousDisease vol 66 no 4 pp425ndash431 2010

[48] H Pai M R Seo and T Y Choi ldquoAssociation of QnrB deter-minants and production of extended-spectrum 120573-lactamasesor plasmid-mediated AmpC 120573-lactamases in clinical isolates ofKlebsiella pneumoniaerdquo Antimicrobial Agents and Chemother-apy vol 51 no 1 pp 366ndash368 2007

[49] K Fuursted and H Schumacher ldquoSignificance of low-levelresistance to ciprofloxacin in Klebsiella pneumoniae and theeffect of increased dosage of ciprofloxacin in vivo using the ratgranuloma pouch modelrdquo Journal of Antimicrobial Chemother-apy vol 50 no 3 pp 421ndash424 2002

[50] J M Rodrıguez-Martınez C Pichardo I Garcıa et al ldquoActivityof ciprofloxacin and levofloxacin in experimental pneumoniacaused by Klebsiella pneumoniae deficient in porins expressingactive efflux and producing QnrA1rdquo Clinical Microbiology andInfection vol 14 no 7 pp 691ndash697 2008

[51] A Briales J M Rodrıguez-Martınez C Velasco et al ldquoIn vitroeffect of qnrA1 qnrB1 and qnrS1 genes on fluoroquinoloneactivity against isogenic Escherichia coli isolates with mutationsin gyrA and parCrdquoAntimicrobial Agents and Chemotherapy vol55 no 3 pp 1266ndash1269 2011

[52] J M Rodrıguez-Martınez C Velasco I Garcıa M E Cano LMartınez-Martınez and A Pascual ldquoMutant prevention con-centrations of fluoroquinolones for Enterobacteriaceae express-ing the plasmid-carried quinolone resistance determinantqnrA1rdquo Antimicrobial Agents and Chemotherapy vol 51 no 6pp 2236ndash2239 2007

[53] B C van Hees M Tersmette R J L Willems B de JongD Biesma and E J van Hannen ldquoMolecular analysis ofciprofloxacin resistance and clonal relatedness of clinical Es-cherichia coli isolates from haematology patients receivingciprofloxacin prophylaxisrdquo Journal of Antimicrobial Chemother-apy vol 66 no 8 pp 1739ndash1744 2011

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

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PeptidesInternational Journal of


Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology


Molecular Biology International

GenomicsInternational Journal of


The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014


BioinformaticsAdvances in

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Signal TransductionJournal of


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Evolutionary BiologyInternational Journal of



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ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014


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Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational



Enzyme Research



Microbiology


reaction that permits discriminatory amplification of specificallele sequences at QRDRs [12]

Plasmid-mediated quinolone resistance (PMQR) genesincluding qnr aac(61015840)-Ib-cr and efflux pumps are knownto confer low-level FQ resistance [13] Quinolone targetprotection by Qnr proteins are widely distributed in Enter-obacteriaceae worldwide [14] Until now six Qnr familiesnamely Qnr A B C D S and VC have been identified(httpwwwlaheyorgqnrStudies) While qnrA B and 119878genes are commonly detected at variable rates in K pneumo-niae worldwide [3 13 15] qnrC and 119863 have been reportedat low rates amongst K pneumoniae isolates in China[16] Moreover a variant of aminoglycoside acetyltransferase(AAC(61015840)-Ib-cr) with the ability to modify and inactivateciprofloxacin has been widely spread in K pneumoniaeisolates from Asia [17 18] and worldwide [3 14]

FQ resistance may also arise as a result of reducedintracellular drug accumulation caused by porin loss or activeefflux pump [5] QepA a quinolone-specific efflux pump hasbeen identified in Escherichia coli isolates from several Asiancountries such as Japan Korea and China [19ndash21] but wasrarely detected in K pneumoniae [22 23]

There is a paucity of data on the prevalence and thegenetic determinants associated with ciprofloxacin resistancein Malaysian K pneumoniae isolates Hence this study wasconducted to identify chromosomal as well as plasmid-mediated mechanisms of ciprofloxacin resistance in a groupof Malaysian ESBL-producing K pneumoniae isolates Tofacilitate rapid detection of gyrA and parC mutations twomismatch amplificationmutation assays (MAMA-PCR) weredeveloped and validated in this study

2 Materials and Methods

21 Bacterial Isolates A group of 93 nonduplicate ESBL-producing K pneumoniae isolates from patients attending toUniversity of Malaya Medical Centre and a private hospitalin Kuala Lumpur Malaysia in 2010ndash2012 were investigatedin this study The isolates were confirmed as K pneumoniaeusing a PCR assay targeting the internal transcribed spacerunit of the bacteria [24] Confirmation of ESBL productionwas performed using Cefpodoxime Combination Disc Kit(Oxoid UK)

22 Antibiotic Susceptibility Testing Minimum inhibitoryconcentration (MIC) of ciprofloxacin was determined by 119864-test strips (bioMereiux Marcy LrsquoEtoile France) on Mueller-Hinton agar (Oxoid UK) in accordance with the Clini-cal and Laboratory Standards Institute (CLSI) guidelines[25] MIC values ge4 2 and le1 120583gmL were used to defineresistance intermediate susceptibility and susceptibility tociprofloxacin respectively

23 Development of MAMA-PCR for gyrA and parC Muta-tions Detection Two duplex PCR assays (gyrA83 + parC80assay and gyrA87 + parC84 assay) were developed for thesimultaneous detection of mutations in Ser83 codon ofGyrA subunit and Ser80 codon of ParC subunit and Asp87

codon of GyrA subunit and Glu84 codon of ParC subunitrespectively

Universal gyrA and parC forward primers [16] were usedtogether with the reverse primers (MAMAprimers) designedin this study for the amplification of gyrA (Ser83 and Asp87)and parC (Ser80 and Glu84) genetic regions (Figure 1)MAMA primer design was performed using NCBIPrimer-BLAST tool (httpwwwncbinlmnihgovtoolsprimer-bl-ast) The reverse primers were complementary to the wild-type alleles of gyrA and parC sequences of K pneumoniaestrain ATCC 13883 (GenBank accession numbers DQ673325and AF303641 resp) except for a mismatch at the ante-penultimate (minus3) nucleotide of the 31015840 end of each MAMAprimer which was included to improve allele discriminationThe MAMA primertemplate mismatches included in thisstudy were CC (in gyrA83 and 87) AG (in parC80) GA(in parC84) The selection of the mismatches was based onprevious observations of their effects on the overall PCR yield[26] The presence of a single primertemplate mismatch hasminimal effect on the PCR yield thus the wild-type gene canbe amplified efficiently In case ofmutation(s) PCR efficiencywill be extremely reduced due to the presence of additionalmismatch(es) at the 31015840 end of the MAMA primer which willnot bind to the template thus amplification of the target geneis failed [27]

The performance of the primers was first evaluated usingmonoplex PCR prior to use in the duplex PCR assays whichwere finally optimized for the simultaneous detection ofmutations in Ser83 codon of GyrA subunit with Ser80 codonof ParC subunit and in Asp87 codon of GyrA subunit withGlu84 codon of ParC subunit

For the first duplex PCR assay the concentrations ofprimers were optimized to 04120583M for each parC80 MAMAprimer and parC universal forward primer in addition to025 120583M for each gyrA83 MAMA primer and gyrA universalforward primer For the second assay the concentrations ofprimers were optimized to 045120583M for each parC84 MAMAprimer and parC universal forward primer in addition to02 120583M for each of gyrA87MAMAprimer and gyrA universalforward primer The primer mixtures were added to a finalPCR reaction volume of 20120583L containing 4 120583L of 5x HOTFIREPol Blend Master Mix (Solis BioDyne Estonia) whichwas comprised of 200120583M of each dNTP 005U120583L of DNApolymerase and 15mM MgCl

2 Finally 1 120583L (lt100 ng) of

boiled bacterial extract [28] was added to each reactionAmplification was carried out on a Veriti 96-well thermalcycler (Applied Biosystems USA) programmed as followsinitial denaturation at 95∘C for 10min followed by 30 cyclesof denaturation at 95∘C for 30 s annealing at 56∘C for 40 sand extension at 72∘C for 50 s and a final extension step at72∘C for 7min PCR products were analysed on a 2 agarosegel prestained with 05 120583gmL ethidium bromide in 05x TBEbuffer

K pneumoniae strains with knownmutations in gyrA (45isolates) and parC (10 isolates) were used as quality controlstrains for the assay optimization and validation [29]

24 Detection of gyrA and parC Mutations by MAMA-PCR Following validation of theMAMA-PCR assays the 93


Possible mutations

ATCC 13883

(Tyr)(Ile) (Phe)

(Gly)(Ala)(Asn)


MAMA gyrA-87





TAC GGC

GCC

AAC

ATC

TTC


(a)

80 84

Possible mutations

MAMA parC-84

MAMA parC-80




ATC (Ile) GGA



parC gene



(b)









3 Results


90and

MIC50














Table1Ciprofl

oxacin

susceptib

ilitypatte

rnsa

ndflu

oroq

uino

lone

(FQ)resistance

determ

inantsdetected

inthe9

3Kpneum


ated

inthisstu

dy

Ciprofl

oxacin

susceptib

ility

MIC

(120583gmL)

Num

bero

fiso

lates

FQresistanced

eterminants

Totaln

umbero

fgyrA

andor

parC

alteratio

nsTo

taln

umbero

fFQ

resistanced

eterminants

PMQRlowast

genes(119899)

gyrA

andor

parC

alteratio

ns(119899)

Susceptib

le(119899=2729

)

0032ndash0047

2Non

e(2)

Non

e(2)

Non

eNon

e0094ndash

038

12aac(61015840)-Ib-cr(12)

Non

e(12)

Non

e1

05ndash075

5qnrB

(2)qnrS

(1)

Non

e(2)

Non

e(3)

gyrA

83(2)

Non

e1

1 11

8qnrB

+aac(61015840)-Ib-cr(5)

qnrB

(3)

Non

e(5)

gyrA

83(3)

Non

e1

2 2Interm

ediately

susceptib

le(119899=3234

)2

32qnrB

+aac(61015840)-Ib-cr

(29)

qnrB

(3)

Non

e(29)

gyrA

83(3)

Non

e1

2 2

Resistant

(119899=3437

)

4ndash6

4Non

e(4)

gyrA

83+parC

80(4)

22

ge32

30

qnrB

+aac(61015840)-Ib-cr

(8)

qnrB

+aac(61015840)-Ib-cr

(2)

qnrB

+aac(61015840)-Ib-cr(1)

qnrB

+aac(61015840)-Ib-cr

(1)aac(61015840)-Ib-cr

(2)

qnrB

+aac(61015840)-Ib-cr(1)

gyrA

83+parC

80(10)

gyrA

83+parC

84(2)

gyrA

83+gyrA

87(1)

gyrA

83+gyrA

87+parC

80(16)

Non

e(1)

2 2 2 3Non

e

2or

44 4 3ndash5 2

lowastPM

QR

Plasmid-m

ediatedqu

inolon

eresistance




Number ofisolates






3000

1000

500

300

200

100

(bp)MM1 2 3 4 5 6

gyrA83 = 259bp

parC80 = 190bp


3000

1000

500

300

200

100

(bp)MM1 2 3 4 5 6

gyrA87 = 272bp

parC84 = 197bp







4 Discussion



M63

M56K5

2M

27

M44

M71

M74

M99

M82

M35

M21

M91

M58M

5M

60M

104

M92

M69

M37

M96

M73

M66

M70

M75

M80

M93

M46

M33

M67

M50

M78

M30

M98

M10

3M

81K92

K22

K106

M10

5

X4 X5 X6X1 X2 X3

K129K5

M38

M40

M85

M90

M95

M32

M51

M10

7M

100

M65

M52

M77

M11

M89

M79

M61

M87

M59

M86M

2M

57M

109

M11

1

M11

0

M36

M28

M13

M72M

1M

68

M47

K112K1

2

K103 K5

0

ND

M

M10

6M

48 K27

K66

K24

K40

M84 M

4K1

26

K114 K1

4

M49

K109

M15

M10

2K1

30


+ parC80

60

65

70

75

80

85

90

95

100

976964958943525

977

976

966

963

897871

865

840

947

976

973

937

893

852

761

722

718

790

780

751

664

590676

739

743

820

910

947

919

732

760

752

750

882

938925

842

971

760

711

709

818

914

970

904

699

608596577

625

617

648

871

743

750

649

669

626

666

690

886

930

646

794

813

587

797

906

950

886

886

822

923 907

927

930

940

974

927

960

973

971

651

911

774

675

Study ID

Sim

ilarit

y (

)



















5 Conclusions




Acknowledgments



References
































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


Possible mutations

ATCC 13883

(Tyr)(Ile) (Phe)

(Gly)(Ala)(Asn)


MAMA gyrA-87





TAC GGC

GCC

AAC

ATC

TTC


(a)

80 84

Possible mutations

MAMA parC-84

MAMA parC-80




ATC (Ile) GGA



parC gene



(b)









3 Results


90and

MIC50














Table1Ciprofl

oxacin

susceptib

ilitypatte

rnsa

ndflu

oroq

uino

lone

(FQ)resistance

determ

inantsdetected

inthe9

3Kpneum


ated

inthisstu

dy

Ciprofl

oxacin

susceptib

ility

MIC

(120583gmL)

Num

bero

fiso

lates

FQresistanced

eterminants

Totaln

umbero

fgyrA

andor

parC

alteratio

nsTo

taln

umbero

fFQ

resistanced

eterminants

PMQRlowast

genes(119899)

gyrA

andor

parC

alteratio

ns(119899)

Susceptib

le(119899=2729

)

0032ndash0047

2Non

e(2)

Non

e(2)

Non

eNon

e0094ndash

038

12aac(61015840)-Ib-cr(12)

Non

e(12)

Non

e1

05ndash075

5qnrB

(2)qnrS

(1)

Non

e(2)

Non

e(3)

gyrA

83(2)

Non

e1

1 11

8qnrB

+aac(61015840)-Ib-cr(5)

qnrB

(3)

Non

e(5)

gyrA

83(3)

Non

e1

2 2Interm

ediately

susceptib

le(119899=3234

)2

32qnrB

+aac(61015840)-Ib-cr

(29)

qnrB

(3)

Non

e(29)

gyrA

83(3)

Non

e1

2 2

Resistant

(119899=3437

)

4ndash6

4Non

e(4)

gyrA

83+parC

80(4)

22

ge32

30

qnrB

+aac(61015840)-Ib-cr

(8)

qnrB

+aac(61015840)-Ib-cr

(2)

qnrB

+aac(61015840)-Ib-cr(1)

qnrB

+aac(61015840)-Ib-cr

(1)aac(61015840)-Ib-cr

(2)

qnrB

+aac(61015840)-Ib-cr(1)

gyrA

83+parC

80(10)

gyrA

83+parC

84(2)

gyrA

83+gyrA

87(1)

gyrA

83+gyrA

87+parC

80(16)

Non

e(1)

2 2 2 3Non

e

2or

44 4 3ndash5 2

lowastPM

QR

Plasmid-m

ediatedqu

inolon

eresistance




Number ofisolates






3000

1000

500

300

200

100

(bp)MM1 2 3 4 5 6

gyrA83 = 259bp

parC80 = 190bp


3000

1000

500

300

200

100

(bp)MM1 2 3 4 5 6

gyrA87 = 272bp

parC84 = 197bp







4 Discussion



M63

M56K5

2M

27

M44

M71

M74

M99

M82

M35

M21

M91

M58M

5M

60M

104

M92

M69

M37

M96

M73

M66

M70

M75

M80

M93

M46

M33

M67

M50

M78

M30

M98

M10

3M

81K92

K22

K106

M10

5

X4 X5 X6X1 X2 X3

K129K5

M38

M40

M85

M90

M95

M32

M51

M10

7M

100

M65

M52

M77

M11

M89

M79

M61

M87

M59

M86M

2M

57M

109

M11

1

M11

0

M36

M28

M13

M72M

1M

68

M47

K112K1

2

K103 K5

0

ND

M

M10

6M

48 K27

K66

K24

K40

M84 M

4K1

26

K114 K1

4

M49

K109

M15

M10

2K1

30


+ parC80

60

65

70

75

80

85

90

95

100

976964958943525

977

976

966

963

897871

865

840

947

976

973

937

893

852

761

722

718

790

780

751

664

590676

739

743

820

910

947

919

732

760

752

750

882

938925

842

971

760

711

709

818

914

970

904

699

608596577

625

617

648

871

743

750

649

669

626

666

690

886

930

646

794

813

587

797

906

950

886

886

822

923 907

927

930

940

974

927

960

973

971

651

911

774

675

Study ID

Sim

ilarit

y (

)



















5 Conclusions




Acknowledgments



References
































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology



3 Results


90and

MIC50














Table1Ciprofl

oxacin

susceptib

ilitypatte

rnsa

ndflu

oroq

uino

lone

(FQ)resistance

determ

inantsdetected

inthe9

3Kpneum


ated

inthisstu

dy

Ciprofl

oxacin

susceptib

ility

MIC

(120583gmL)

Num

bero

fiso

lates

FQresistanced

eterminants

Totaln

umbero

fgyrA

andor

parC

alteratio

nsTo

taln

umbero

fFQ

resistanced

eterminants

PMQRlowast

genes(119899)

gyrA

andor

parC

alteratio

ns(119899)

Susceptib

le(119899=2729

)

0032ndash0047

2Non

e(2)

Non

e(2)

Non

eNon

e0094ndash

038

12aac(61015840)-Ib-cr(12)

Non

e(12)

Non

e1

05ndash075

5qnrB

(2)qnrS

(1)

Non

e(2)

Non

e(3)

gyrA

83(2)

Non

e1

1 11

8qnrB

+aac(61015840)-Ib-cr(5)

qnrB

(3)

Non

e(5)

gyrA

83(3)

Non

e1

2 2Interm

ediately

susceptib

le(119899=3234

)2

32qnrB

+aac(61015840)-Ib-cr

(29)

qnrB

(3)

Non

e(29)

gyrA

83(3)

Non

e1

2 2

Resistant

(119899=3437

)

4ndash6

4Non

e(4)

gyrA

83+parC

80(4)

22

ge32

30

qnrB

+aac(61015840)-Ib-cr

(8)

qnrB

+aac(61015840)-Ib-cr

(2)

qnrB

+aac(61015840)-Ib-cr(1)

qnrB

+aac(61015840)-Ib-cr

(1)aac(61015840)-Ib-cr

(2)

qnrB

+aac(61015840)-Ib-cr(1)

gyrA

83+parC

80(10)

gyrA

83+parC

84(2)

gyrA

83+gyrA

87(1)

gyrA

83+gyrA

87+parC

80(16)

Non

e(1)

2 2 2 3Non

e

2or

44 4 3ndash5 2

lowastPM

QR

Plasmid-m

ediatedqu

inolon

eresistance




Number ofisolates






3000

1000

500

300

200

100

(bp)MM1 2 3 4 5 6

gyrA83 = 259bp

parC80 = 190bp


3000

1000

500

300

200

100

(bp)MM1 2 3 4 5 6

gyrA87 = 272bp

parC84 = 197bp







4 Discussion



M63

M56K5

2M

27

M44

M71

M74

M99

M82

M35

M21

M91

M58M

5M

60M

104

M92

M69

M37

M96

M73

M66

M70

M75

M80

M93

M46

M33

M67

M50

M78

M30

M98

M10

3M

81K92

K22

K106

M10

5

X4 X5 X6X1 X2 X3

K129K5

M38

M40

M85

M90

M95

M32

M51

M10

7M

100

M65

M52

M77

M11

M89

M79

M61

M87

M59

M86M

2M

57M

109

M11

1

M11

0

M36

M28

M13

M72M

1M

68

M47

K112K1

2

K103 K5

0

ND

M

M10

6M

48 K27

K66

K24

K40

M84 M

4K1

26

K114 K1

4

M49

K109

M15

M10

2K1

30


+ parC80

60

65

70

75

80

85

90

95

100

976964958943525

977

976

966

963

897871

865

840

947

976

973

937

893

852

761

722

718

790

780

751

664

590676

739

743

820

910

947

919

732

760

752

750

882

938925

842

971

760

711

709

818

914

970

904

699

608596577

625

617

648

871

743

750

649

669

626

666

690

886

930

646

794

813

587

797

906

950

886

886

822

923 907

927

930

940

974

927

960

973

971

651

911

774

675

Study ID

Sim

ilarit

y (

)



















5 Conclusions




Acknowledgments



References
































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


Table1Ciprofl

oxacin

susceptib

ilitypatte

rnsa

ndflu

oroq

uino

lone

(FQ)resistance

determ

inantsdetected

inthe9

3Kpneum


ated

inthisstu

dy

Ciprofl

oxacin

susceptib

ility

MIC

(120583gmL)

Num

bero

fiso

lates

FQresistanced

eterminants

Totaln

umbero

fgyrA

andor

parC

alteratio

nsTo

taln

umbero

fFQ

resistanced

eterminants

PMQRlowast

genes(119899)

gyrA

andor

parC

alteratio

ns(119899)

Susceptib

le(119899=2729

)

0032ndash0047

2Non

e(2)

Non

e(2)

Non

eNon

e0094ndash

038

12aac(61015840)-Ib-cr(12)

Non

e(12)

Non

e1

05ndash075

5qnrB

(2)qnrS

(1)

Non

e(2)

Non

e(3)

gyrA

83(2)

Non

e1

1 11

8qnrB

+aac(61015840)-Ib-cr(5)

qnrB

(3)

Non

e(5)

gyrA

83(3)

Non

e1

2 2Interm

ediately

susceptib

le(119899=3234

)2

32qnrB

+aac(61015840)-Ib-cr

(29)

qnrB

(3)

Non

e(29)

gyrA

83(3)

Non

e1

2 2

Resistant

(119899=3437

)

4ndash6

4Non

e(4)

gyrA

83+parC

80(4)

22

ge32

30

qnrB

+aac(61015840)-Ib-cr

(8)

qnrB

+aac(61015840)-Ib-cr

(2)

qnrB

+aac(61015840)-Ib-cr(1)

qnrB

+aac(61015840)-Ib-cr

(1)aac(61015840)-Ib-cr

(2)

qnrB

+aac(61015840)-Ib-cr(1)

gyrA

83+parC

80(10)

gyrA

83+parC

84(2)

gyrA

83+gyrA

87(1)

gyrA

83+gyrA

87+parC

80(16)

Non

e(1)

2 2 2 3Non

e

2or

44 4 3ndash5 2

lowastPM

QR

Plasmid-m

ediatedqu

inolon

eresistance




Number ofisolates






3000

1000

500

300

200

100

(bp)MM1 2 3 4 5 6

gyrA83 = 259bp

parC80 = 190bp


3000

1000

500

300

200

100

(bp)MM1 2 3 4 5 6

gyrA87 = 272bp

parC84 = 197bp







4 Discussion



M63

M56K5

2M

27

M44

M71

M74

M99

M82

M35

M21

M91

M58M

5M

60M

104

M92

M69

M37

M96

M73

M66

M70

M75

M80

M93

M46

M33

M67

M50

M78

M30

M98

M10

3M

81K92

K22

K106

M10

5

X4 X5 X6X1 X2 X3

K129K5

M38

M40

M85

M90

M95

M32

M51

M10

7M

100

M65

M52

M77

M11

M89

M79

M61

M87

M59

M86M

2M

57M

109

M11

1

M11

0

M36

M28

M13

M72M

1M

68

M47

K112K1

2

K103 K5

0

ND

M

M10

6M

48 K27

K66

K24

K40

M84 M

4K1

26

K114 K1

4

M49

K109

M15

M10

2K1

30


+ parC80

60

65

70

75

80

85

90

95

100

976964958943525

977

976

966

963

897871

865

840

947

976

973

937

893

852

761

722

718

790

780

751

664

590676

739

743

820

910

947

919

732

760

752

750

882

938925

842

971

760

711

709

818

914

970

904

699

608596577

625

617

648

871

743

750

649

669

626

666

690

886

930

646

794

813

587

797

906

950

886

886

822

923 907

927

930

940

974

927

960

973

971

651

911

774

675

Study ID

Sim

ilarit

y (

)



















5 Conclusions




Acknowledgments



References
































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology




Number ofisolates






3000

1000

500

300

200

100

(bp)MM1 2 3 4 5 6

gyrA83 = 259bp

parC80 = 190bp


3000

1000

500

300

200

100

(bp)MM1 2 3 4 5 6

gyrA87 = 272bp

parC84 = 197bp







4 Discussion



M63

M56K5

2M

27

M44

M71

M74

M99

M82

M35

M21

M91

M58M

5M

60M

104

M92

M69

M37

M96

M73

M66

M70

M75

M80

M93

M46

M33

M67

M50

M78

M30

M98

M10

3M

81K92

K22

K106

M10

5

X4 X5 X6X1 X2 X3

K129K5

M38

M40

M85

M90

M95

M32

M51

M10

7M

100

M65

M52

M77

M11

M89

M79

M61

M87

M59

M86M

2M

57M

109

M11

1

M11

0

M36

M28

M13

M72M

1M

68

M47

K112K1

2

K103 K5

0

ND

M

M10

6M

48 K27

K66

K24

K40

M84 M

4K1

26

K114 K1

4

M49

K109

M15

M10

2K1

30


+ parC80

60

65

70

75

80

85

90

95

100

976964958943525

977

976

966

963

897871

865

840

947

976

973

937

893

852

761

722

718

790

780

751

664

590676

739

743

820

910

947

919

732

760

752

750

882

938925

842

971

760

711

709

818

914

970

904

699

608596577

625

617

648

871

743

750

649

669

626

666

690

886

930

646

794

813

587

797

906

950

886

886

822

923 907

927

930

940

974

927

960

973

971

651

911

774

675

Study ID

Sim

ilarit

y (

)



















5 Conclusions




Acknowledgments



References
































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


M63

M56K5

2M

27

M44

M71

M74

M99

M82

M35

M21

M91

M58M

5M

60M

104

M92

M69

M37

M96

M73

M66

M70

M75

M80

M93

M46

M33

M67

M50

M78

M30

M98

M10

3M

81K92

K22

K106

M10

5

X4 X5 X6X1 X2 X3

K129K5

M38

M40

M85

M90

M95

M32

M51

M10

7M

100

M65

M52

M77

M11

M89

M79

M61

M87

M59

M86M

2M

57M

109

M11

1

M11

0

M36

M28

M13

M72M

1M

68

M47

K112K1

2

K103 K5

0

ND

M

M10

6M

48 K27

K66

K24

K40

M84 M

4K1

26

K114 K1

4

M49

K109

M15

M10

2K1

30


+ parC80

60

65

70

75

80

85

90

95

100

976964958943525

977

976

966

963

897871

865

840

947

976

973

937

893

852

761

722

718

790

780

751

664

590676

739

743

820

910

947

919

732

760

752

750

882

938925

842

971

760

711

709

818

914

970

904

699

608596577

625

617

648

871

743

750

649

669

626

666

690

886

930

646

794

813

587

797

906

950

886

886

822

923 907

927

930

940

974

927

960

973

971

651

911

774

675

Study ID

Sim

ilarit

y (

)



















5 Conclusions




Acknowledgments



References
































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology











5 Conclusions




Acknowledgments



References
































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


References
































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology




































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology

Molecular Analysis of Ciprofloxacin Resistance Mechanisms...

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