Enterobacter cloacae plasmid-mediated quinolone resistance
mechanisms of drug
Author: Wu Jian Lu Deng Chuang Hung-wen, ZHANG
【Abstract】 Objective To understand the Shenzhen area Enterobacter cloacae in the prevalence of qnrA genes, gene
targeting and its mediated quinolone resistance mechanisms generated. Methods in clinical isolates of Enterobacter cloacae 45, using PCR method combined with screening for qnrA gene sequencing technology, large plasmid extraction, Southern
hybridization, and plasmid conjugal transfer experiments positioning, agar dilution method for drug susceptibility detection. Results 45 Enterobacter cloacae, the seven-PCR and
sequencing confirmed qnrA gene. Seven strains of bacteria carried by the 6 plasmid have been successfully extracted and carried out Southern hybridization, qnrA gene located in 80 ~ 200kb size of the natural low copy number plasmid. 4 bacteria successful conjugal transfer test, so that zygote ciprofloxacin MIC values increased 32 ~ 64-fold. Conclusion
plasmid-mediated quinolone resistance gene qnrA in
Enterobacter cloacae in the Shenzhen area has a high prevalence may be the cause of the quinolone antibacterial agents Enterobacter cloacae acquired resistance to important
Key words qnrA gene Enterobacter cloacae quinolone resistance plasmid
Plasmid mediated quinolone resistance in clinical isolates
ABSTRACT Objective To investigate the prevalence and
location of qnrA gene in clinical isolates of Enterobacter cloacae and its function in the development of quinolone resistance. Method Forty five Enterobacter cloacae were
screened for the qnrA gene by PCR and then conformed with direct sequencing. Large plasmid extraction and Southern hybridization were done to determine the location of qnrA. Conjugation experiments were done to determine the
transferability and its effects in the development of quinolone resistance. Results qnrA were detected in 7 (15.6%) of 45 strains. The qnrA sequence matched that of NCBI GenBank with 100%. Plasmid extraction and Southern hybridization were
succeed in 6 stains. The qnrA were located in plasmids in size of 80 ~ 200kb. Conjugation experiments were done in 4 strains. Transconjugants had 32 ~ 64 fold increases in the MIC of
ciprofloxacin relative that of the recipient but were still susceptible to ciprofloxacin according to CLSI stardard. Conclusion Transferable plasmid mediated quinolone
resistance associated with qnrA were high prevalent in clinical isolates of Enterobacter cloacae from Shenzhen city and may contribute to the quinolone resistance of thiese bacteria .
KEY WORDS qnrA; Enterobacter cloacae; Quinolone; Drug resistance; Plasmid
In 1998, Martinez and other first time that Klebsiella pneumoniae quinolone antibacterial agents on the existence of plasmid-mediated resistance mechanisms, and that the
resistance gene named qnr (now named qnrA) . After the Chinese scholar Wang Minggui such study found that quinolone resistance qnrA gene in E. coli antibacterial agents in the detection rate as high as 7.7%, suggesting that qnrA gene in Enterobacteriaceae-mediated resistance mechanisms may play an important role, causing widespread concern .
Currently Enterobacter cloacae nosocomial infections has become an important pathogen, and the pairs of quinolone antibacterial drug resistance increased rapidly, to the clinical anti-infective therapy pose serious challenges to
[3,4]. To understand the qnrA gene in Enterobacter cloacae resistance mediated by the role of spread, this study used PCR technology to our hospital from 2004 to 2005 clinical isolates of Enterobacter cloacae were qnrA gene carrier rate of screening, DNA sequencing confirmed that and with the reported
qnrA sequence comparison and by Southern hybridization and conjugal transfer test qnrA gene of plasmid positioning, are reported below.
1 Materials and methods
1.1 Source and identification of strains
From 2003 to 2004 in our hospital and the Shenzhen Municipal People's Hospital, a non-duplicate clinical isolates
of Enterobacter cloacae 45. Using French Bio Merieux's API
20E system for species identification. Strains of E.coli
J53/pMG252 research as qnrA gene PCR detection and Southern blotting positive quality control strain, E.coli J53 Azr (sodium azide resistant) as a receptor in conjugal transfer experiments with reference strains of bacteria and MICs by the The United States provided by Professor Jacoby.
1.2 antimicrobial drug susceptibility testing
Using agar dilution and bonding sub-clinical isolates of
ciprofloxacin MICs value, other antimicrobial drug susceptibility assay using KB. MH medium and drug susceptibility were Oxoid paper products. The results of
clinical and laboratory standards in accordance with the United States Commission (CLSI) 2005 edition of the recommended standard of interpretation. Quality control strain E. coli ATCC25922 and Enterobacter cloacae ATCC13047.
1.3 qnrA amplification and sequencing
PCR method combined with sequencing using PCR primers with reference to literature  Ying-chun commissioned by
Shanghai Bio Co., Ltd. synthesis. Upstream primer 5 '
TCAGCAAGAGGATTTCTCA 3', downstream primer 5 '
GGCAGCACTATTACTCCCA 3', by fragment was 627bp. PCR cycle
parameters: 94 ? predegeneration 5min, 95 ? 30s, 45 ? 60s,
72 ? 60s, loop 35 times, the last extension at 72 ? 5min.
PCR products sent Shanghai Ying Chun Biological Co., purified and directly sequenced in both directions. Positive control
strains of E.coli J53 pMG252.
1.4 qnrA sequence comparison
The measured using DNAstar software sequence fragment assembly, after correction, using the National Information Center (NCBI) online analysis tools Blastn
(http://www.ncbi.nlm.nih.gov/) procedures for analysis to determine the resistance gene type or genetic variation. qnrA gene reference sequence number is AY070235 and AY259086.
1.5 Plasmid analysis and Southern hybridization qnrA gene
QIAfilter Plasmid Maxi Kit (QiaGen, Germany) to extract
plasmid, 0.7% agarose gel electrophoresis, using LabImage software analysis and E.coli V517 and the plasmid carrying the E.coli J53/pMG252 results of this comparison in order to determine its molecular size. Electrophoresis of taking
pictures, using capillary transfer method will be transferred to the plasmid DNA on the nitrocellulose membrane. In order to cut plastic purified qnrA gene PCR product as template preparation of their probes by digoxigenin random primer
labeling and detection kit (Boster Company, Wuhan) instructions to operate.
1.6 conjugal transfer test
Donor strain for the clinical isolates of qnrA-positive
strains, the receptor for the bacteria E.coli J53 Azr. The
donor bacteria, the receptor bacteria were inoculated on LB plate, 35 ? overnight culture. Picked a single colony were inoculated into 4ml of LB broth, 37 ?