[Study on the genetic structure and transmission mechanism of a plasmid-mediated AmpC beta-lactamase].
OBJECTIVE
To clone the gene of plasmid-mediated AmpC beta-lactamase from the plasmid of multiple-drug resistance Klebsiella pneumoniae producing plasmid-mediated AmpC beta-lactamase and demonstrate its mechanism of transmission.


METHODS
Plasmids of the transconjugant were extracted and digested with restriction endonuclease HindIII. Taq DNA polymerase was applied to fill the recessed 3' termini, and a single deoxyadenosine was added to the 3' termimi of fragments. Then these fragments were ligated with pGEM-T Easy vector. E. coli DH5alpha containing recombinant plasmid was selected on MacConkey agar plates containing ampicillin and cefoxitin. Insert fragments were sequenced by primer walking. MIC determinations and isoelectric focusing electrophoresis (IFE) were utilized to analyze recombinant.


RESULTS
The recombinant plasmid pT948 containing a 5.2-kb insert was obtained. The inserted fragment contained a bla(DHA-1) and a regulatory gene ampR. The insertion sequence (IS26), qacEDelta1 and sulI genes of the I type integron were obtained near the bla(DHA-1) gene. Recombinant expressed a beta-lactamase with pI of 7.7. MIC determinations showed that recombinant was resistant to cefoxitin and the resistance to ceftazidime could be induced by the cefoxitin.


CONCLUSIONS
The plasmid-mediated ampC gene cloned was identified as bla(DHA-1). IS26 observed on the flanks of the bla(DHA-1) maybe relate to the translocation of bla(DHA-1) gene region from the chromosome to plasmid.
Lu
Wei-xin
WX
Infectious Disease Dept, 1st Affiliated Hospital, Medical School, Zhejiang University, Key Lab of Infectious Diseases of Public Health Ministry, Zhejiang Hangzhou 310003, China.
Wei
Ze-qing
ZQ
Yu
Yun-song
YS
Chen
Ya-gang
YG
Li
Lan-juan
LJ
D000900:Anti-Bacterial Agents / Q000494:pharmacology; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D002440:Cefoxitin / Q000494:pharmacology; D002442:Ceftazidime / Q000494:pharmacology; D003001:Cloning, Molecular; D004269:DNA, Bacterial / Q000235:genetics; D004926:Escherichia coli / Q000187:drug effects / Q000235:genetics; D007711:Klebsiella pneumoniae / Q000201:enzymology* / Q000235:genetics; D008826:Microbial Sensitivity Tests; D010957:Plasmids / Q000235:genetics*; D014169:Transformation, Bacterial; D018440:beta-Lactam Resistance / Q000235:genetics; D001618:beta-Lactamases / Q000235:genetics / Q000378:metabolism*
D000900:Anti-Bacterial Agents; D001426:Bacterial Proteins; D004269:DNA, Bacterial; D002440:Cefoxitin; D002442:Ceftazidime; C048795:AmpC beta-lactamases; D001618:beta-Lactamases