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Objeetive To explore the mechanism of drug resistance of New Delhi metallo-β-lactamase-1 (NDM-1) producing Enterobacteriaceae,and to investigate the characteristics of blaNDM-1 carrying plasmid and its gene environment.Methods A total of 48 strains of carbapenem-resistant Enterobacteriaceae were successively collected from six general hospitals in south China during August 2011 and January 2013.Escherichia coli J53 was used for plasmid conjugation.Modified Hodge test was performed,and PCR method was used for the detection of carbarpenase-related genes.The relative molecular mass of the blaNDM-1 carrying plasmid was determined using pulsed field gel electrophoresis (PFGE)assay,and enzyme digestion was performed to investigate the homology and incompatibility group of the plasmid.Clinical feature of blaNDM-1 producing Enterobacteriaceae infection was also investigated.Results Among 48 strains of carbapenem-resistant Enterobacteriaceae,43 were positive in modified Hodge tests.blaVIM,blaGIM and blaSPM genes were negative in all strains,while blaNDM-1 was positive in 19 strains including 3 strains of Escherichia coli,5 strains of Klebsiella pneumoniae,6 strains of Enterobacter cloacae,3 strains of Citrobacterfreundii,1 strain of Klebsiella oxytoca and 1 strain of Providencia rettgeri.All the 19 strains were resistant to imipenem,cefotaxime,ceftazidime,cefepime,aztreonam and piperacillin/tazobactam,47.3％ strains were resistant to ciprofloxacin and levofloxacin,but 68.4％ strains were sensitive to amikacin.Conjugation experiment showed that,blaNDM-1 carrying plasmids in 13 strains were transmitted to the Escherichia coli J53.The conjugants were resistant to imipenem,ceftazidime,cefotaxime and piperacillin/tazobactam,but were sensitive to amikacin,ciprofloxacin and levofloxacin.All genes in conjugant J-FR90 (Providencia rettgeri) were negative,while the remaining 12 conjugants carried blaNDM-1,blaSHV and aac-(6')-Ib genes.PFGE showed that,the sizes of all blaNDM-1 carrying plasmids were about 50 kb,and more than 80％ of their macrorestriction maps were similar.The plasmid belonged to incompatibility group IncX3,and exhibited 100％ passage stability after 500 generations of propagation.Among 19 patients infected with NDM-1 producing Enterobacteriaceae,6 died and 13 survived.Conclusions NDM-1 producing Enterobacteriaceae is emerging in south China,and blaNDM-1 is transmitted to Enterobacteriaceae through IncX3.Patients infected with NDM-1 producing Enterobacteriaceae usually have good prognosis.
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Objective To study the molecular characteristic of the epidemic plasmids carrying blaCTX-M-15 in Guangzhou. Methods A total of 38 strains of E. coli and 47 strains of K. pneumoniae both producing CTX-M-15 ESBLs were collected from nine hospitals in Guangzhou from 2007 to 2008. The clonal relationship of isolates carrying blaCTX-M-15 was determined by PFGE and MLST. Antimicrobial susceptibility was determined by microdilution test for all isolates. Conjugative plasmids carrying blaCTX-M-15 were obtained by mating and were subject to restriction analysis. PCR was used to determine phylogenetic groups of E. coli,and to study replicon type and the genetic contexts of the plasmids harboring blaCTX-M-15. Serum agglutination test was used to detect the serotype of E. coli. Results The 37 strains of E. coli were classified into 28 genotypes, while the 47 strains of K. pneumoniae were divided into 30 genotypes. ST131 was found in E. coli but not O25 serotype. Two novel-alleles of tonB and new ST were determined in K. pneumoniae. Forty out of 58 isolates represented independent genotypes have been succeeded to transfer the plasmid carrying blaCTX-M-15 to the E. coli C600(Rif) by conjugation. The sizes of plasmids carrying blaCTX-M-15 are 65 kb in 57.9% isolates of E. coli and 92 kb in 87.5% isolates of K. pneumoniae. Two epidemic plasmids were detected in E.coli and K. pneumoniae by restriction enzyme, designated p15-e and p15-k respectively. The blaCTX-M-15 and ISEcpI were identified on p15-e, and the blaCTX-M-15 ,ISEcpI,aac(6')- Ⅰ b,aac(3')-Ⅲ ,blaOXA-1 ,qnrB,qnrS,blaDHA-1 , blaTEM-1 were determined on p15-k. The p15-k also was identified to belong to the incompatible group FⅡ. Conclusion The local dissemination of blaCTX-M-15 appears to be due to the spread of epidemic plasmids harboring blaCTX-M-15. No evidence supports the dissemination of clone strains which carried blaCTX-M-15.
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Objective To evaluate the in vitro antimicrobial activity of piperacillin-sulbactam against clinical isolates of non-fermentative bacilli isolated from common infections. Methods Microdilution was employed to study the antimicrobial resistance. Results A total of 770 strains were collected from 6 hospitals in Guangzhou, including Pseudomonas aeruginosa, Acinetobacter baumannii, Stenotrophomonas maltophilia , Burkholderia cepacia , Flavobacterium , and Alcaligenes. Compared with other β-lactams,piperacillin-sulbactam displayed the highest activity against all the isolates of P. aeruginosa, especially for imipenem non-sensitive isolates, with the susceptibility of 71.9% and 55.8%, respectively. Piperacillinsulbactam and cefoperazone-sulbactam kept good activity against imipenem sensitive isolates of A. baumannii,with the susceptibility of 71.0% and 73. 0%, respectively. For the strains of Burkholderia cepacia, 69% strains exhibited minimal inhibitory concentration (MIC) of ≤ 16 mg/L for piperacillin-sulbactam. For the strains of Flavobacterium, and Alcaligenes, piperacillin-sulbactam also had excellent activity, with the susceptibility of 70. 2% and 94. 4%, respectively. Conclusion Piperacillin-sulbactam exhibits good activity again non-fermentative bacilli, especial for imipenem non-sensitive isolates of P. aeruginosa.
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Objective To investigate the impact on the resistance of carbapenem with the expression of OprD2 or OprD2 mutation in Pseudomonas aeruginosa. Methods One hundred and one clinical strains of Pseudomonas aeruginosa with MIC for imipenem ≥8 μg/ml were studied. MIC were determined by the broth microdilution method, and the antibiotics tested were imipenem(IPM ), biapenem( BPM), meropenem(MEM) and panipenem(PEM). The expression of the oprD2 gene in Pseudomonas aeruginosa were analyzed by real-time reverse transcriptase PCR(RT-PCR). For the Pseudomonas aeruginosa with normal expression of OprD2 and resistance to imipenem, full-length oprD2 gene was amplified by PCR and the products were sequenced. Results According to the result of the expression of oprD2 gene, 101 strains of Pseudomonas aeruginosa were divided into two groups: group1 with diminished expression of OprD2, and group2 with normal expression of OprD2. Comparing isolates with MIC of 4 kinds of carbepenem agents ≥ 16 μg/ml in two groups. Data showed the amount of OprD2 expression were different between two groups(P ＜0.01 or P ＜ 0.05). In group1, there are 28 isolates with MIC ≥ 16 μg/ml of all the 4 kinds of carbapenems, among which 25 isolates have obviously diminished expression of OprD2 ( ＜ 0.4). Negative correlations tendency appeared between the level of OprD2 transcription and MICs of 4 kinds of carbepenem agents in Pseudomonas aeruginosa. In group2, 16 strains with OprD2 mutation divided into 4 types according to the pattern of alteration. Compared with PAO1, these strains have increased MIC with different degree to IPM,BPM, MEM and PEM. Conclusion The deletion or diminished expression of OprD2 resulted in resistance to imipenem in Pseudomonas aeruginosa. The level of OprD2 transcription and antimicrobial activities for carbapenem agents proved to be highly correlated in Pseudomonas aeruginosa. The mutation of OprD2 in Pseudomonas aeruginosa probably decreased the sensitivity of carbapenem agents against Pseudomonas aeruginosa.