Genetic Study In Beta-lactamase-producing Klebsiella Pneumoniae

Objective:To study theβ-lactamases genes of Klebsiella pneumoniae strains isolated from patients. Methods:ESBLs producing-strains were detected by the CLSI phenotypic confirmatory test, AmpC producing-strains were screened by cefoxitin-test and were confirmed by improved three dimensional test; carbapenemases were screened by Modified Hodge Test,three dimensional test and EDTA-disk synergy test. PCR and DNA sequencing were used to detect the genotype of ESBLs,AmpC and carbapenemase (3-lactamases. Results:67 strains of Cefoxitin-resistant Klebsiella pneumoniae were collected. ESBLs genes of CTX-M,SHV and TEM were identified by PCR. The incident rate of CTX-M,SHV and TEM were 64.06%,67.19%and 59.38%,respectively. The incident rate of producing more than 2 genes was 68.73%. genes of partial positive products were sequenced. By comparing their DNA sequences with the known genes exisitng in GenBank,3 CTX products,3 SHV products and 1 TEM product were identified as CTX-M3,SHV-11,SHV-1 and TEM-1.The above 64 non-duplicate strains of k. pneumoniae were detected both the producing AmpC enzyme strains by improved three dimensional test and 3 genes encoding (3-lactamases including blaDHA blaACT blaFox by Polymerase Chain Reaction(PCR),the subtypes were determined by DNA sequencing.18 positive strains were detected by improved three dimensional test, and all of these showed positive 405 bp bands by PCR,1 positive product was sequenced. By comparing its DNA sequence with the known gene existing in GenBank,1 DHA product was identified as DHA-1 type ampC gene. Carbapenemases of 814 Klebsiella pneumoniae stains were screened by Modified Hodge Test,EDTA-disk synergy test and three dimensional test. Four genes encoding (3-lactamases, including blaKPC blaIMP blaVIM-blasME were detected by Polymerase Chain Reaction(PCR). And genes of partial positive products were sequenced. Three strains of carbapenem-resistant Klebsiella pneumoniae were screened out by disk diffusion in our hospital.4 genes encodingβ-lactamases including blaKpc blaIMP blaVIM were detected by PCR, genes of partial positive products were sequenced. The was identified in 2 isolates by PCR.1 blaKPC positive product was sequenced. By comparing its DNA sequence with the known gene existing in GenBank, the isolate carried KPC-2 gene. Another carbapenem-resistant isolate carried IMP-4 gene. Conclusions:The mechanism of resistance to antibiotics in Klebsiella pneumoniae is more and more complicated. The isolates are usually multidrug resistant. The most prevalent mechanism of antibiotics resistance in Klebsiella pneumoniae is producing ESBLs and AmpC P-lactamases. The genetyping of ESBLs are major SHV and CTX-M types, following with TEM-type. The percentage of co-existance of more than two ESBLs gene is high. The gene of AmpC is all DHA type. Resistance to carbapenems in the population of Klebsiella pneumoniae strains is low. This is the first report of Klebsiella pneumoniae producing KPC-2 P-lactamase and IMP-4 MBL in Tianjin. The study provides evidence for the clinical drug usage and nosocomial infection control.