Phenotypic and molecular characterization of 5 novel CTX-M enzymes carried by Klebsiella pneumoniae and Escherichia coli.
OBJECTIVE
The aim of the present study was to study the phenotypic and molecular characterization of 5 novel CTX-M-beta-1actamases carried by 5 Klebsiella pneumoniae isolates and 3 Escherichia coli isolates collected from 4 hospitals in Hefei, China.


METHODS
The purified PCR products were ligated with pGEM-Teasy vectors, expressed, and sequenced. The complete genes of the CTX-M-beta-lactamases were ligated with the pHSG398 vector to express prokaryotic recombinant proteins. Plasmids were extracted by rapid alkaline lysis protocol, and the PCR method was performed to determine whether the prokaryotic expression was successful or not. Antimicrobial susceptibility was tested and the phenotypes of transformants were determined according to criteria recommended by the Clinical and Laboratory Standards Institute. The kinetic parameters of enzymes were confirmed. The isoelectric points (pI) were determined by isoelectric focusing assay. Pulsed-field gel electrophoresis and plasmid profiling were performed.


RESULTS
The PCR products had 1101 nucleotides and were determined as CTX-M-46, CTX-M-47, CTX-M-48, CTX-M-49, and CTX-M-50. All strains were resistant to cefotaxime, but most of them were susceptible or intermediate to ceftazidime. The phenotypes of novel enzymes were determined as extended-spectrum-beta-lactamases (ESBL). Penicillin G, cephalothin, cefuroxime, and cefotaxime were determined to good substrates, whereas ceftazidime hydrolysis was not detected. The pI of the 5 novel CTX-M-beta-lactamases were 8.0. CTX-M-derivatives could be the multiplex genesis in our area.


CONCLUSIONS
This is the first report of these 5 novel plasmid-mediated CTX-M ESBL produced from China in the world. Molecular typing reveals notably different origin in genes encoding different CTX-M variants of 8 strains.
Cheng
Jun
J
Department of Infectious Diseases, the First Affiliated Hospital of Anhui Medical University, Hefei 230022, China.
Ye
Ying
Y
Wang
Ying-ying
YY
Li
Hui
H
Li
Xu
X
Li
Jia-bin
JB
D000900:Anti-Bacterial Agents / Q000378:metabolism / Q000494:pharmacology; D003227:Conjugation, Genetic; D004926:Escherichia coli / Q000187:drug effects / Q000201:enzymology* / Q000235:genetics*; D004927:Escherichia coli Infections / Q000453:epidemiology / Q000382:microbiology; D007700:Kinetics; D007710:Klebsiella Infections / Q000453:epidemiology / Q000382:microbiology; D007711:Klebsiella pneumoniae / Q000187:drug effects / Q000201:enzymology* / Q000235:genetics*; D008826:Microbial Sensitivity Tests; D010641:Phenotype; D020133:Reverse Transcriptase Polymerase Chain Reaction; D001618:beta-Lactamases / Q000235:genetics* / Q000378:metabolism
D000900:Anti-Bacterial Agents; D001618:beta-Lactamases