Study On The Gene Clone And Dissemination Of The DHA-1 Type Plasmid-mediated AmpC β-lactamase

The primary resistant mechanism is that bacteria can produce p-lactamases. One of them is AmpC p-lactamases. AmpC P-lactamases belong to the Bush group 1 P-lactamases. They can hydrolyze the third generation cephalosporins and can not be inhibited by p-lactamases inhibitors. Many Enterobacteriaceae and other gram-negative bacteria produce chromosomal-encoded AmpC P-lactamases. Within the last decade, chromosomal ampC genes have been transferred to plasmids and the incidence of these plasmid-encoded AmpC p-lactamases has increased. Plasmids carrying ampC genes are easily transmitted among different members of bacteria. This may have major public health implications, as the ampC genes provide the bacteria with additional resistance phenotypes.In this study, 1 strain of multiple-drug resistance Klebsiella pneumoniae carrying ampC gene was collected from intensive care unit of our hospital in July 2000. Conjugation experiment was performed in the strain. Plasmids of 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 tothe 3'termimi of fragments. Then these fragments were ligated with pGEM-T Easy vector. E. coli DH5a containing cloning vectors were selected on Mac-Conkey agar plates with ampicillin and cefoxitin. Cloned fragments were sequenced by primer walking. The software of DNAssist was used to investigate the characteristic of ampC gene and its surrounding sequence. MIC determinations and isoelectric focusing electrophoresis (IFE) were utilized to analyze recombinant.The results showed that the plasmid was successfully transferred by transconjugation to the recipient bacteria. Recombinant plasmid pT948 containing a 5.2-kb insert was obtained. The insert fragment contained a structural gene bladha-1 and a regulatory gene ampR. Insertion sequence (IS26) element was obtained near the bladha-i gene. MIC determinations showed that recombinant was resistant to cefoxitin and the resistance to ceftazidime could be induced by the cefoxitin.In brief, the ampC gene was located on the transferable plasmid. The plasmid-mediated ampC gene cloned was identified as bladha-1. IS26 observed on the flanks of the blaDHA-1 may be concerned with the translocation of blaDHA-1 gene region from the chromosome to plasmid.