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Introduction
Three plasmid-mediated quinolone resistance genes,
qnrA[1],
qnrB[2], and
qnrS[3], have been discovered since 1998.
At least 6 qnrA, 6 qnrB, and 2
qnrS variants have been
described[4]. qnrA was found in most common Enterobacteriaceae,
including Escherichia coli (E coli),
Klebsiella spp, Enterobacter spp,
Citrobacter freundii, and Providencia stuartii
[4], and it was located in
complex sul1-type class 1
integrons[5]. qnrB and
qnrS were identified in clinical strains of
Klebsiella pneumoniae (K
pneumoniae), E coli, Enterobacter spp, and
Salmonella spp [4,6_9].
qnrB and qnrS were also detected in
Citrobacter koseri[2]and Serratia
marcescens[7], respectively.
Interestingly, qnrB and qnrS could be detected
simultaneously in a few clinical strains[8,
9]. Among 28 qnrA positive clinical strains of Enterobacteriaceae, 7 strains also harbored
qnrS[10]. A further study was only carried out on a single
strain of Enterobacter cloacae carrying
2 qnr genes
simultaneously[9].
In China, qnrA has been detected in 8% of 78 clinical
isolates of E coli[5], but
qnrB and qnrS have not yet been reported in clinical strains. We found a clinical strain of
K pneumoniae carrying both
qnrB4 and qnrS1. The aim of this study was to identify the location and the relationship
of the 2 genes, to analyze the genetic background of the
qnrB4 and qnrS1 genes, and to evaluate their respective
roles in the production of quinolone resistance.
Materials and methods
Bacterial strains A clinical strain of K
pneumoniae carrying qnrB4 and
qnrS1 was found during the study on extended-spectrum
β-lactamases. The strain was isolated from sputum of an inpatient with acute exacerbation of chronic
bronchitis at a teaching hospital in Shanghai in 2005. The
patient was treated with cefradine, cefotaxime and levofloxacin
prior to the isolation of K pneumoniae. Additional strains
used were E coli V517[1]; E
coli J53, containing plasmid R27[5] as standards for plasmid size;
E coli J53AzR (resistant to
azide)[5] as a recipient for conjugation; and
E coli DH5α, which was used in cloning.
PCR detection The qnr genes (qnrA, qnrB, and
qnrS), class A β-lactamase genes
(blaTEM,
blaSHV, blaPER,
blaVEB, blaSFO, and
blaCTX), and class C plasmid-mediated
ampC β-lactamase genes were detected by PCR with specific primer
sets in the clinical strain, transconjugants, and transformant.
The primers used for qnrA, qnrB, and
qnrS were 5'-GGG TAT GGA TAT TAT TGA TAA AG-3' and 5'-CTA ATC CGG CAG
CAC TAT TA-3', 5'-ATG ACG CCA TTA CTG TAT AA-3' and
5'-GAT CGC AAT GTG TGA AGT TT-3', 5'-ACG ACA TTC GTC AAC TGC AA-3' and 5'-TAA ATT GGC ACC CTG TAG
GC-3', respectively. The PCR conditions were 94°C for 45 s,
56°C for 45 s, and 72°C for 1min, and cycled 30 times for the
detection of qnr genes. The primers used for CTX-M were
5'-AGT GCA AAC GGA TGA TGT-3' and 5'-GGC TGG GTA AAA ATA GGT C-3'. The primers for the
ampC genes were previously described by Perez-Perez and
Hanson[11] . All positive results were confirmed by direct sequencing of the
PCR products on both strands.
Transfer of quinolone resistance Conjugation
experiments were carried out in Luria-Bertani broth with
azide-resistant E coli J53 as the recipient, as previously
described[5]. Transconjugants were selected on Trypticase soy agar
(TSA) plates containing sodium azide (200 mg/L) for
counterselection and ampicillin (100 mg/L) or cefotaxime (8
mg/L) to select for plasmid-encoded resistance. Four
hundred colonies were picked from the selection plates and
detected by PCR for qnrB4 and qnrS1.
Transformation was performed for the
qnrS1-bearing plasmid, which could not be transferred by conjugation.
Plasmid DNA was extracted from the parent K pneumoniae
strain using the QIAGEN plasmid midi kit (QIAGEN GmbH, Hilden,
Germany) and introduced into electrocompetent E coli
DH5α by electroporation. Colonies were selected on ciprofloxacin
(0.06 mg/L). The colony carrying only 1 plasmid and
harboring qnrS1 was confirmed with PCR. The
qnrS1-bearing plasmid DNA was extracted again from the colony and
introduced into E coli J53 by electroporation. Colonies were
selected on plates containing 50 mg/L ampicillin, and were
also confirmed carrying qnrS1 with PCR.
The plasmid size was estimated by agarose gel
electro-phoresis, as previously
described[5] ; the presence of
qnrB4 and qnrS1 were confirmed with Southern blot hybridization
using the DIG nucleic acid detection kit (Roche Applied
Science, Mannheim, Germany).
In vitro susceptibility testing Minimal inhibitory
concentrations (MIC) for the donor, recipient, transconjugant,
and transformant strains were measured by agar dilution in
accordance with the guidelines of the Clinical Laboratory
Standards Institute (CLSI)[12]
for ciprofloxacin, amikacin, ampicillin, cefepime, cefotaxime, ceftazidime, gentamicin,
nalidixic acid, levofloxacin, sulfamethoxazole, and trimethoprim.
The Etest (Biodisk AB, Solna, Sweden) was used to detect
minimal changes in ciprofloxacin and levofloxacin susceptibility.
Cloning and nucleotide sequence analysis
Plasmid DNA extracted from transconjugant or transformant strains
harboring qnrB4 or qnrS1 were digested with
HindIII, ligated to pUC18, and introduced into
E coli DH5α with selection on TSA plates containing 50 mg/L ampicillin. Sequencing was
carried out with an ABI Prism 3730 genetic analyzer (Applied
Biosystems, Foster City, CA, USA), and was continued by
primer walking on both DNA strands. For the sequence
comparisons, the NCBI BLAST program
(www.ncbi.nlm.nih.gov/blast/Blast.cgi) was utilized.
Nucleotide sequence accession numbers The nucleotide
sequences in pHS7 and pHS8 containing qnrB4 and
qnrS1, respectively, have been submitted to GenBank and have been
assigned accession numbers EF683583 and EF683584, respectively.
Results
PCR detection and MIC determination of a clinical strain
of K pneumoniae The clinical strain of
K pneumoniae was identified as containing
qnrB4, qnrS1,
blaCTX-M-14, and
blaDHA-1 genes by PCR and DNA sequencing. The clinical strain was
susceptible to quinolones according to the CLSI criteria, with
ciprofloxacin and levofloxacin MIC of 0.75 and 1.0 mg/L,
respectively; but highly resistant to ampicillin, cefotaxime,
ceftazidime, gentamicin, amikacin, sulfamethoxazole, and
trimethoprim, and intermediate to cefepime (MIC 16 mg/L;
Table 1).
Transfer of quinolone resistance and plasmid
characterization qnrB4 and
qnrS1 were located on 2 different plasmids, pHS7 and pHS8, and were 180 and 45 kb in size,
respectively, by Southern blot hybridization.
The qnrB4-bearing plasmid pHS7 could be transferred by conjugation
to produce a transconjugant E coli J53 pHS7. Another
transconjugant J53 pHS9 carrying plasmid pHS9 with a size
similar to pHS7 bearing both qnrB4 and
qnrS1, was also obtained by conjugation.
qnrB4 was detected alone from 380 of 400 (95%) colonies picked from the selection plates by
PCR; no qnrS1 alone was detected;
qnrB4 and qnrS1 in combination were detected in 9 of 400 colonies (2.25%). The
non-conjugative plasmid pHS8 bearing qnrS1 was transferred
to J53 by transformation.
The ciprofloxacin MIC for J53 transconjugants or
transformant carrying qnrB4 only (J53 pHS7),
qnrS1 only (J53 pHS8), and both
qnrB4 and qnrS1 (J53 pHS9) were 0.19, 0.25, and 0.25 mg/L, respectively. The MIC of levofloxacin
for transconjugant J53 pHS8 and transformant J53 pHS9 were
0.38 and 0.5 mg/L, respectively, which were higher than that
of J53 pHS7 (0.19 mg/L; Table 1).
Transconjugant J53 pHS7 was resistant to β-lactam
antibiotics (ampicillin, cefotaxime, and ceftazidime), and pHS7
was found harboring blaCTX-M-14 and
blaDHA-1 β-lactamase genes. Transformant J53 pHS8, also harboring
the blaCTX-M-14 gene, was resistant to ampicillin and cefotaxime, but not to
ceftazidime. Comparing the MICs of antimicrobials other
than quinolones, we found that the resistance pattern in
pHS9 was similar to that of pHS8, except that resistance to
gentamicin and amikacin in pHS9, and resistance to
sulfamethoxazole, trimethoprim, and ceftazidime in pHS7 was
lost in pHS9 (Table 1).
Analysis of plasmid structures The DNA sequences of
plasmid pHS7 HindIII fragment showed that the upstream of
qnrB4 included sapA and partial
sapB, coding for putative peptide transport system permease, and
aphA1. Notably, insertion sequence IS26 was located between
aphA1 and sapB; downstream of
qnrB4 included psp operons, coding for putative phage shock proteins, and
ampC and ampR genes located between orf1 and partial
qacEΔ1. The plasmid structure adjacent to
qnrB4 was similar to that in plasmids, pRBDHA and
pMPDHA[13] , 2 qnrB4 and
blaDHA-1-bearing plasmids (GenBank accession numbers AJ971343 and
AJ971344, respectively), but found aphA1 and IS26 in the
upstream of qnrB4 in pHS7 (Figure 1).
The sequence of the immediate region
surrounding qnrS1 in pHS8 was nearly identical to the 3 reported
qnrS1-bearing plasmids: pAH0376, pINF5, and pK245. Insertion sequence
IS26 was found both upstream and downstream of
qnrS1, and an IS2 was directly located upstream of
qnrS1 (Figure 2).
Discussion
A clinical strain of K pneumoniae was identified as
carrying both qnrB4 and qnrS1, and was isolated from a patient
with acute exacerbation of chronic bronchitis.
qnrB4 and qnrS1 were located on 2 separated plasmids, pHS7 and pHS8,
respectively. pHS7 could be transferred to E
coli J53 by conjugation. pHS8 could not be transferred alone by
conjugation, but was successfully transferred to J53 by
transformation. A recent study on 526 clinical strains
isolated in Taiwan indicated that qnr genes were highly
prevalent in E cloacae with a positive rate of 16% (86/526). Both
qnrB2 and qnrS1 were detected simultaneously in 4 of the 86
qnr-positive strains, but there was no further study on the
relationship, transferability, and location of the 2
qnr genes on these strains[8]. The only study was on an
E cloacae clinical isolate co-expressing QnrB4 and QnrS1 determinants,
isolated in France[9]. qnrB4 and
qnrS1 were also located on 2 different plasmids, 100 and 160 kb in size, respectively, and
could be transferred to E coli TOP10 by conjugation or
transformation. The ciprofloxacin MIC of transconjugant or
transformant carrying qnrB4 or qnrS1 were 0.06 and 0.12
mg/L, respectively. In our study, transformant J53 pHS8
carrying qnrS1 had higher MIC values for ciprofloxacin or
levofloxacin (0.25_0.38 mg/L) than that of transconjugant
J53 pHS7 carrying qnrB4 (0.19 mg/L).
qnrS1 conferred higher quinolone MIC than
qnrB4.
qnrA was located on complex sul1-type class 1 integrons,
according to several reports[4,5,8].
qnrB was also located on sul1-type class 1 integrons from 2
reports[6,13]. A genetic environment analysis
of qnrB2 in a Salmonella enterica Serovar Keurmassar showed that
qnrB2 was located in a complex sul1-type integron which contained 2 class 1
integrons surrounding 2 common regions separated by a
partial 3´ conserved segment[6]; the structure was similar to
pMG252, the first qnrA-bearing
plasmid[4]. During the study on the genetic organization of
the ampC and ampR genes in
Morganella morganii (M
morganii), a qnrB4 gene was found to be located upstream of
psp operons, and this was a complex
sul1-type integron (qnrB4 was not labeled in the
original figure, as it had not been reported at that
time)[13]. In our study, the genetic environment of
qnrB4 was similar to that of M
morganii, but found aphA1 and IS26 upstream of
qnrB4. Partial qacESΔ1 was found downstream of
ampR, and intl1 gene and orf513 were detected by PCR (data not shown), so
we supposed that qnrB4 was also located in a
sul1-type class 1 integron in K
pneumoniae.
Unlike qnrA and qnrB, qnrS was not located on any
integrons according to 3 reported qnrS-bearing plasmid
structure analyses[3,14,15], but qnrS
was directly downstream of IS2. The PCR amplification was negative
for intl1 and orf513 in qnrS-bearing plasmid pHS8 (data not shown), indicating
that pHS8 did not carry any class 1 integron. Like in pK245,
the fts1 and a blaLAP-1 genes were
found upstream of qnrS1 in this study, and notably, insertion sequence IS26 was found
both upstream and downstream of qnrS1.
qnrS could also be located on a mobilizable incQ-related
plasmid[16].
A transconjugant was obtained carrying plasmid pHS9,
of a similar size to pHS7, but bore both
qnrB4 and qnrS1. We supposed that there was an integration between pHS7
and pHS8 to produce pHS9 during the conjugation experiment. There was a possibility that a segment of
plasmid pHS8 containing qnrS1 was integrated to pHS7. The
blaLAP-1 gene that was located upstream of
qnrS1 in pHS8, but not harbored by pHS7, was detected in pHS9 by PCR
(data not shown). The movement of qnrS1 might be related
to the insertion sequences of IS26 located upstream and
downstream of qnrS1, and IS2 directly upstream of
qnrS1. The resistance to sulfamethoxazole, trimethoprim, and
ceftazidime in pHS7 was lost in pHS9, indicating that the
qnrS1 segment might replace part of plasmid DNA in pHS7
or interrupt the expression of some genes in pHS7. However,
the MIC of ciprofloxacin in pHS9 was not shown to be
augmented from qnrB4 to qnrS1. Further study is ongoing on
the analysis of the structure of pHS9 to understand the
mechanism of the integration between the 2 plasmids.
This is the first report of qnrB and
qnrS from China and these 2 genes are harbored by a single clinical strain of
K pneumoniae.
Acknowledgements
We thank George A JACOBY and David C HOOPER for
providing reference strains E coli
J53AzR, E coli V517, and E
coli R27. We are also grateful to George A JACOBY for
critically reviewing the manuscript.
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