Study On The Homology And Genotyping Of β-lactamases In Multiple-drug-resistant Klebsiella Pneumoniae

Klebsiella pneumoniae, especially the resistant ones, are the most prevalent gram-negative bacteria to induce nosocomial infections. In the 1970s, the strains were chiefly resistant to aminoglycoside. Since SHV-2 extend-spectrum β-lactamase (ESBL) was reported in 1983 in Germany, ESBLs-producing Klebsiella pneumoniae were becoming the prevalent strains in the world. The main resistant mechanisms of Klebsiella pneumoniae are they can produce p-lactamases, especial ESBLs. The encoding genes of these β-lactamases often locate in large plasmids that also carry other resistant genes. Furthermore, plasmids carrying these resistant genes are easily transmitted among different members of Enterobacteriaceae. It is limited to choose antibiotics against the multiple-drug-resistant Klebsiella pneumoniae.In this study, 6 strains of Klebsiella pneumoniae that had the same resistant spectrum were collected from intensive care unit (ICU) of our hospital from May 2000 to August 2000. E test, pulse field gel electrophoresis (PFGE), and conjugation experiment were performed in these strains. 6 clinical strains and their transconjugants were detected bypolymerase chain reaction (PCR). The primers of TEM-1, SHV-1, CTX-M-1, CTX-M-2, CTX-M-8, CTX-M-9, OXA-2, OXA-10 and FOX-1 that is one of plasmid mediated AmpC enzymes were designed. Cloning and sequencing were preformed for PCR products to confirm the genotypes of β-lactamases. The pIs of β-lactamases were detected by isoelectric focusing (IEF). The novel CTX-M-22 was inserted into pET28 and expressed in Escherichia coli BL21 (DE3). The transformants were detected by E test to conform if they produced ESBLs, while pIs of the expressed protein were detected by IEF.The results showed that 6 clinical Klebsiella pneumoniae were multiple resistant and had the same resistant spectrum. The resistant spectrums of transconjugants were similar to their parent strains. The clinical strains were only highly sensitive to imipenem, but had increasing MICs to piperacillin/tazobactam, cefoperazone/sulbactam, and cefepime. The MICs to ceftazidime of 6 clinical strains were still 256μg/ml after inhibited by clavulanic acid. 6 clinical strains showed the same straps by PFGE. The 6 transconjugants had isoelectric point straps that were 5.4, 7.7, 8.0, 8.2, 8.4, respectively. The pI strap of 7.7 could be inhibited by cloxacillin while other straps could be inhibited by clavulanic acid. 6 transconjugants were positive by TEM, SHV and CTX-M-1 PCR. Nucleotide sequence analysis indicated that they produced TEM-1, SHV-12 and a new CTX-M pMactamase (designed CTX-M-22, GenBank accession No AY080899), respectively. After prokaryotic expression, the transformant were confirmed to be ESBLs producing strain and the pI of the expressed protein was 8.4.In brief, 6 clinical Klebsiella pneumoniae were multiple resistance, and had the same resistant spectrum. The resistant spectrums of transconjugants were similar to their parent strains. 6 clinical strains had resistant genes of β-lactamase in plasmids including TEM-1, SHV-12, AmpC, and a novel CTX-M-22. Multiple-drug-resistant Klebsiella pneumoniae caused an epidemic outbreak in ICU of our hospital.