MICROBIAL DRUG RESISTANCE
Volume 12, Number 2, 2006
© Mary Ann Liebert, Inc.
Plasmid-Mediated Quinolone Resistance in Australia
JOSE-MANUEL RODRIGUEZ-MARTINEZ,1,2 LAURENT POIREL,1 ALVARO PASCUAL,2
and PATRICE NORDMANN1
ABSTRACT
The aim of this study was to search for plasmid-encoded quinolone resistance determinants QnrA and QnrS in
fluoroquinolone-resistant and extended-spectrum -lactamase (ESBL)-producing enterobacterial isolates recovered in Sydney, Australia, in 2002. Twenty-three fluoroquinolone-resistant, of which 16 were also ESBL-positive,
enterobacterial and nonrelated isolates were studied. PCR with primers specific for qnrA and qnrS genes and
primers specific for a series of ESBL genes were used. A qnrA gene was identified in two ESBL-positive isolates,
whereas no qnrS-positive strain was found. The QnrA1 determinant was identified in an Enterobacter cloacae isolate and in a carbapenem-resistant Klebsiella pneumoniae isolate, both of which expressed the same ESBL SHV12. Whereas no plasmid was identified in the E. cloacae isolate, K. pneumoniae K149 possessed two conjugative
plasmids, one that harbored the qnrA and blaSHV-12 genes whereas the other expressed the carbapenemase gene
blaIMP-4. The qnrA gene, was located in both cases downstream of the orf513 recombinase gene and upstream of
the qnrA1 gene, a structure identical to that found in sul1-type integron In36 and qnrA-positive strains from
Shanghai, China. However, the gene cassettes of the sul1-type integrons were different. This study identified the
first plasmid-mediated quinolone resistance determinant in Enterobacteriaceae in Australia.
INTRODUCTION
of the QnrA determinant has been identified recently as being
the water-borne environmental species Shewanella algae.11 The
aim of this study was to determine the prevalence of the qnrA
and qnrS genes in clinical isolates recovered in Sydney, Australia, to evaluate further the dissemination of these novel plasmid-mediated quinolone resistance determinants.
Q
usually results in Enterobacteriaceae
from mutations in genes coding for chromosomally encoded
type II topoisomerases or for efflux pumps and porins.2,3 However, recent reports indicate that quinolone resistance may be also
plasmid mediated.3,9,13 The first plasmid-mediated quinolone resistance protein Qnr (termed recently QnrA) was identified from
urine in a Klebsiella pneumoniae isolate from Birmingham, Alabama by L. Martinez-Martinez and G. Jacoby.7 The plasmid-mediated quinolone resistance gene qnrA encodes a 218-amino-acid
protein of the pentapeptide family that protects gyrase from
quinolone inhibition.14 Recently, another plasmid-mediated
quinolone resistance determinant, QnrS, has been reported from
Japan.1 Molecular studies showed that the qnrA gene was located
with other resistance determinants in sul1-type integrons harboring a duplication of the 3-conserved sequence of integrons and
orf513 gene,12,13 this latter gene encoding a putative recombinase
involved in site-specific acquisition of resistance genes.
Plasmid-mediated determinant QnrA has been identified
from remotely related areas, such as the United States, China,
Thailand, Korea, Turkey, Germany, and France.9,16 The origin
UINOLONE RESISTANCE
MATERIALS AND METHODS
Bacterial strains
Twenty-three ciprofloxacin-resistant nonduplicate enterobacterial strains isolated in 2002 in the metropolitan area of Sydney, Australia, and recovered during a 2-month period from urine
samples were studied, including 11 Klebsiella pneumoniae, 8 Escherichia coli, 1 Enterobacter cloacae, 1 Enterobacter agglomerans, 1 Proteus mirabilis and 1 Citrobacter youngae strain. They
were randomly taken from the strain collection of the Antibiotic
Reference Laboratory, Department of Microbiology, The Prince
of Wales Hospital, Randwick, Australia. In addition, previously
studied E. coli EC158 and K. pneumoniae K149 strains isolated
1Service de Bactériologie-Virologie, Hôpital de Bicêtre, Assistance Publique/Hôpitaux de Paris, Faculté de Médecine Paris-Sud, Université
Paris XI, K.-Bicêtre, France.
2University Hospital Virgen Macarena, University of Sevilla, Sevilla, Spain.
99
100
RODRIGUEZ-MARTINEZ ET AL.
in Melbourne in September, 2002, that both produce metallo-lactamase IMP-4 were included in this study.10 Sixteen isolates
(69.5%) produced extended-spectrum -lactamases (ESBLs) and
were detected as reported.4 E. coli NCTC50192 harboring four
plasmids of 154, 66, 38, and 7 kb was used as size marker for
plasmids. E. coli J53 AzR was used as the host in the conjugation experiments and E. coli DH10B in the transformation experiments.6 E. coli LO was used as a qnrA1-positive5 and E. coli
(pBCH2.6) as qnrS-positive control strains.1
Susceptibility testing
MICs for nalidixic acid and fluoroquinolones were determined
by an agar dilution technique and interpreted, when available, according to the guidelines of the CLST.8 MICs of -lactams, chloramphenicol, tetracycline, tobramycin, and gentamicin were determined by an agar dilution technique with Mueller-Hinton agar
(Sanofi-Diagnostic Pasteur), as described previously.5
PCR amplification
Genomic DNAs of bacterial isolates were obtained as described previously.6 PCR experiments were carried out using
TABLE 1.
specific primers for the qnrA and qnrS genes. Primers QnrAA and QnrA-B5 for the qnrA gene and QnrS-A2 (5-AGT GAT
CTC ACC TTC ACC GC-3) and QnrS-B2 (5-CAG GCT
GCA ATT TTG ATA CC-3) for the qnrS gene amplifying internal fragments of 661 and 550 bp, respectively, were used.
Once the corresponding gene was identified using a series of
primers for genes coding for ESBLs,6 primers specific for lactamase gene blaSHV (SHV-A, 5-ATG CGT TAT WTT
CGC CTG TGT-3, and SHV-B, 5-TTA GCG TTG CCA GTG
CTC G-3) were used in combination with qnrA-specific
primers to evaluate a putative colinearity between those genes.
The PCR products were sequenced with an Applied Biosystems sequencer (ABI 377).
Plasmid and Southern blot analyses
Plasmid analysis of the clinical isolates, transconjugants, and
transformants was performed by using the Kieser technique,5
followed by an agarose gel electrophoresis analysis. Southern
blot analysis was performed using whole-cell DNA restricted
with BamHI and NdeI restriction enzymes, agarose gel electrophoresis, and membrane transfer, followed by hybridization
using qnrA and blaSHV-12-specific probes.6
QUINOLONE SUSCEPTIBILITY PROFILES OF CLINICAL ISOLATES, TRANSCONJUGANT
RECOMBINANT CLONE IN E. coli REFERENCE STRAINS
FROM
K. pneumoniae K149,
AND
MICs in mg/L for strainsb
Antibioticsa
E. cloacae
Qn17
qnrA
K. pneumoniae
K149
(p149)
qnrA
NAL
CIP
OFX
NFX
MFX
SPX
AMX
AMC
PIP
TZP
CAZ
CTX
FEP
IMP
TM
GM
C
TET
256
16
32
32
32
32
512
512
64
8
256
32
0.5
0.25
128
128
128
128
32
1
4
2
2
2
512
512
512
128
512
256
64
8
128
128
128
128
E. coli J53
transconjugant
(p149)b
qnrA
32
0.25
1
1
1
1
512
8
256
4
8
4
2
0.12
4
8
128
128
E. coli J53 AzR
E. coli DH10B
(p149SN12)c
qnrA
E. coli DH10B
4
0.12
0.01
0.01
0.03
0.01
4
4
2
1
0.06
0.06
0.01
0.06
NDd
ND
ND
ND
8
0.25
0.5
1
0.5
0.5
512
128
512
128
512
128
32
0.12
ND
ND
ND
ND
2
0.002
0.01
0.01
0.002
0.005
4
4
2
2
0.06
0.06
0.06
0.06
ND
ND
ND
ND
aNAL, nalidixic acid; CIP, ciprofloxacin; OFX, ofloxacin; NFX, norfloxacin; MFX, moxifloxacin; SPX, sparfloxacin, AMX,
amoxicillin; AMC, amoxicillin-clavulanic acid; PIP, piperacillin; TZP, piperacillin-tazobactam; CAZ, ceftazidime; CTX, cefotaxime; FEP, cefepime; IMP, imipenem; TM, tobramycin; GM, gentamicin; C, chloramphenicol; TET, tetracycline. Breakpoints
used for quinolone and fluoroquinolone susceptibility testing were the following: for nalidixic acid, susceptible if MIC 16 mg/L
and resistant if MIC 32 mg/L; for ciprofloxacin, moxifloxacin, and sparfloxacin, susceptible if MIC 1 mg/L and resistant if
MIC 4 mg/L; for ofloxacin, susceptible if MIC 2 mg/L and resistant if MIC 8 mg/L; for norfloxacin, susceptible if MIC
4 mg/L and resistant if MIC 16 mg/L.
bE. coli J53 (p149) transconjugant was obtained from K. pneumoniae K149.
cE. coli DH10B (p149SN12) recombinant strain contained a SacI insert expressing the QnrA determinant.
dND, Not determined.
QNRA DETERMINANT IN AUSTRALIA
Conjugation and transformation experiments
Transfer of the qnrA1 gene into reference strain E. coli J53
AzR by conjugation was attempted by liquid and solid matingout assays. Transconjugants were selected on trypticase soy
(TS) agar plates containing sodium azide (100 mg/L) and amoxicillin (100 mg/L) or chloramphenicol (30 mg/L). In addition,
plasmid extracts of qnrA1-positive strains were used for transformation assays in E. coli DH10B, as described, giving rise to
transformants selected on plates containing amoxicillin (100
mg/L) or chloramphenicol (30 mg/L).6
Cloning experiments and sequence analysis
Cloning experiments were performed with the restriction enzymes SacI or BamHI from whole-cell DNAs extracted as previously described using the vector pBK-CMV, followed by expression of recombinant plasmids in E. coli DH10B and
selection on TS agar containing kanamicin (30 mg/L) and
nalidixic acid (4 mg/L), gentamicin (8 mg/L), or chloramphenicol (30 mg/L). Antibiograms obtained by disk diffusion
were performed with E. coli DH10B harboring recombinant
plasmids, and sizes of the plasmid inserts were determined by
restriction analysis. Both strands of each recombinant plasmid
were sequenced. The nucleotide sequences were analyzed with
software available over the internet at the National Center for
Biotechnology Information Web site (http://www.ncbi.nlm.
nih.gov).
RESULTS AND DISCUSSION
Retrospective analysis of the antibiotic resistance profile of
the SHV-12-positive E. coli transconjugant obtained from K.
101
pneumoniae isolate K149 (IMP-4 and SHV-12 positive) recovered in Australia10 indicated that the transferred plasmid
p149 conferred reduced susceptibility to several quinolones
whereas the IMP-4-positive plasmid did not. Thus, PCR experiments were performed to screen for the putative qnrA gene
that gave a positive result for the K. pneumoniae K149 isolate
but not for the IMP-4-positive E. coli EC158 strain recovered
during the same period of time. Consequently, a survey was
conducted to evaluate prevalence of QnrA and QnrS determinants among a collection of ciprofloxacin-resistant enterobacterial isolates recovered in Melbourne over a 2-month period. This
screening gave negative results for the qnrS gene (the first survey of that type), whereas one of the 23 clinical strains analyzed
was positive for the qnrA gene (8.7%). This isolate, E. cloacae
strain Qn17, was also positive for the blaSHV-12 gene. The nucleotide sequences of the two qnrA-like genes were identical to
that of the known qnrA, latter termed qnrA1.9 K. pneumoniae
K149 was resistant to ampicillin, ceftazidime, ticarcillin, ticarcillin plus clavulanic acid, imipenem, fosfomycin, tetracycline,
chloramphenicol, trimethoprim-sulfamethoxazole, tobramycin,
gentamicin, kanamycin, rifampin, and sulfonamides of intermediate susceptibility to nalidixic acid and resistant to fluoroquinolones (Table 1). E. cloacae Qn17 was resistant to amoxicillin, cefotaxime, ceftazidime, tetracycline, chloramphenicol,
trimethoprim-sulfamethoxazole, aminoglycosides, rifampin, sulfonamides, nalidixic acid, and fluoroquinolones.
Conjugation, plasmid analysis, and Southern hybridization
experiments (data not shown) did not identify any plasmid in
E. cloacae Qn17, whereas a 160-kb conjugative plasmid was
identified in K. pneumoniae K149 that possessed both the qnrA
and blaSHV-12 genes. However, PCR mapping did not reveal
any co-linearity between those two genes. In addition, PCR
combinations with primers specific for a class 1 integron did
FIG. 1. Comparison of sul1-type integrons that contain a qnrA gene. The identical structure identified in K. pneumoniae K149
and in E. cloacae Qn17 isolates is indicated in comparison with those of In36 and In37 from Shanghai.14 pMG252 is from the
QnrA-positive E. coli strain from Alabama.12
102
RODRIGUEZ-MARTINEZ ET AL.
not identify any gene cassette upstream of orf513. Another 150kb plasmid was identified in K. pneumoniae K149, which harbored the carbapenemase gene blaIMP-4 located inside a class 1
integron structure, as reported previously.16
Quinolone resistance was transferred with a conjugation frequency (the number of transconjugants divided by the number
of the donor cells) of 5 105. Transconjugant E. coli J53
(p149) containing the qnrA gene displayed resistance to ampicillin, ceftazidime, tetracycline, chloramphenicol, trimethoprim-sulfamethoxazole, tobramycin, and gentamicin. It showed
reduced susceptibility to nalidixic acid and fluoroquinolones
(Table 1), but susceptibility to sulfonamides.
Cloning experiments using DNA from K. pneumoniae K149
gave rise to an E. coli (p149SN12) recombinant strain that
showed reduced susceptibility to quinolones (Table 1). Analysis of the sequence of the 3,495-bp insert indicated that qnrA1
was located in a complex In4 family class 1 integron similar to
In36,15 with the presence of an ampR gene downstream of qnrA
(Fig. 1). The identical fragment was detected in E. cloacae
Qn17. However, further PCR mapping did not identify the overall structure of In36 or In37 in both isolates. In particular, PCR
failed to detect IS6100 downstream of qnrA, whereas it has been
detected in the qnrA-positive integrons In36 and In37.15
This study emphasizes that the QnrA determinants are also
found in enterobacterial isolates in the Australian continent.
QnrA-like determinants were identified in ESBL-producing enterobacterial isolates, as reported previously.9 It is noteworthy
that it has been detected in a carbapenem-resistant K. pneumoniae isolate, emphasizing that some strains may harbor a large
pool of resistance genes leading to panresistance. Another interesting feature observed in that study is the possible chromosomal location of the qnrA1 gene in E. cloacae Qn17, suggesting that the qnrA-structure could have integrated into the
chromosome of that isolate, possibly by a transposition process,
as already suggested.15
ACKNOWLEDGMENTS
This work was funded by a grant from the Ministère de l’Education Nationale et de la Recherche (UPRES-EA3539), Université Paris XI, France, and by a grant from the European Community (6th PCRD, LSHM-CT-2003-503335). Strains were
kindly provided by the Antibiotic Reference Laboratory, Department of Microbiology, The Prince of Wales Hospital, Randwick NSW. L.P. is a researcher from the INSERM (Paris,
France) and J.M.R.M. was a recipient of a travel grant from the
Spanish Society for Clinical Microbiology and Infectious Diseases in 2004.
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Address reprint requests to:
Dr. Patrice Nordmann
Service de Bactériologie-Virologie
Hôpital de Bicêtre
78, rue du Général Leclerc
94275 Le Kremlin-Bicêtre, France
E-mail: nordmann.patrice@bct.ap-hop-paris.fr
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