The Study Of High AmpC Producing Clinical Strains Of Escherichia Coli And Klebsiella Pneumoniae

[OBJECTIVE]To screen high AmpC producing clinical strains of Escherichia coli and Klebsiella pneumoniae and to investigatethe prevalence of ESBLs among them. To detect the gene of pi asmid mediated AmpC by multiplex PCR method. To detect andcompare AmpC regulator gene of chromosomal high AmpC produ cing isolates of Escherichia coli. To explore the effect of regulat or mutation on AmpC expressing of Escherichia coli. To study t he impact of expression of high-level AmpC 3 -lactamase and E SBLs on antibiotics susceptibility of clinical strains of Escherichi a coli and Klebsiella pneumoniae.[METHOD] Standard disk diffusion susceptibility tests were u sed as initial screen tests. 3 -lactamases of the isolates which i ntermediated or resist to cefoxitin were investigated by three-dim ensional extract test, isoelectric focusing overlay technique and c onjugation experiment. PCR and DNA sequencing were used to determine the genotypes of ESBLs. Multiplex PCR method was used to screen plasmid mediated AmpC gene and their genotypeswere determined by DNA sequencing. The regulator genes of 1 9 chromosomal high AmpC producing isolates of Escherichia coli were amplified, sequenced and the difference between them wereanalyzed by blast method. Then the regulator genes of above w ere cloned into pCAT3-basic vector (a promotorless reporter genevector) and two ways were used to assess the expressing level of the different regulator: one is directly to assay the concentrati on of CAT protein by Elisa method and other is indirectly to examine the MIC of chloramphenicol against the isolates which car rying different regulators. In order to make clear the impact of t he expression of AmpC beta -lactamase and ESBLs on antibiotics susceptibility of Escherichia coli and Klebsiella pneumoniae, the MICs of 24 antimicrobial agents against 25 high AmpC producin g isolates of Escherichia coli and 4 high AmpC producing isolat es of Klebsiella pneumoniae were determined by standard micro dilution susceptibility tests.[RESULTS ] Among total 719 clinical isolates of Escherichia coli, 59 isolates were intermediated or resist to cefoxitin and abo ve them, 25 (3.48%) isolates were proved as high AmpC produ cing. Among these 25 isolates, 17(2.36%) only produced high-lev el AmpC beta-lactamase, 8(1.13%) produced both high-level Amp C beta -lactamase and ESBLs. The genotype of ESBLs was foundas a new member of CTX-M. Among total 86 clinical isolates of Klebsiella pneumoniae, 1 isolates were intermediated or resist to cefoxitin and above them, 4(4.65%) produced both high-level AmpC beta -lactamase and SHV-12. 6 isolates (0.83%) of Escheri chia coli were screened as plasmid mediated AmpC producer bymultiplex PCR method and the sequence of them showed that itis a new CMY type gene. 4 isolates (4.65%) of Klebsiella pneu moniae were screened as plasmid mediated AmpC producer also and the sequence of them are identical to DHA-1. The regulatorgene of 19 chromosomal high AmpC producing isolates of Esche richia coli were contrasted to that of Escherichia coli K-12 and based on the mutation characteristic, they can be classified as fol lows groups: A: mutations in -35 region and attenuator (7 isolat es); B: mutations in attenuator (1 isolates); C: insertion between -10 and -35 region and deletion of attenuator (7 isolates); D: formation of a new pribnow box (1 isolates); F: IS10 insertion do wnstream of attenuator. Those regulator gene of different group were cloned into pCAT3-basic vector and directly and indirectly measurement indicated that regulator gene of A, B, C, D and F group could enhance the CAT expressing level as much as 19, 1 0, 12, 13 and 18 times respectively than that of Escherichia coliK-12. The Klebsiella pneumoniae strains which producing both high-level AmpC and SHV-12 are totally resistant to quinolones, a minoglycosides, penicillins and cephamycin. Their resistant rates to sulfami do, third generation cephalosporins, beta-lactams combined with beta-lactam ase inhibitor, cefepine and oxacephems were 50%, 2...