产质粒介导AmpC酶地肺炎克雷伯菌耐药性与新基因、整合子研究.docxVIP

安徽医科大学硕士学位论文 sulfate-polyacrylamide gel electrophoresis (SDS), and stained with Coomassie Brilliant Blue G-250 (Sigma, USA). The pI values were determined using polyacrylamide gel by isoelectric focusing. Moreover, we amplify the conserved segment of class Ⅰ integron from the parental and exconjugant strains, and Purified PCR products were ligated with the pMD18-T easy vector (TaKaRa), expressed in E. coli JM109 on a Luria-Bertani agar plate with blue-white selection, and nucleotide sequences were determined by bidirectional sequencing of PCR production with a 3730 automatic DNA sequencer three times. Southern blot was used to reveal that the gene encoding the conserved segment of class integron was on a plasmid. Results Of the 180 strains, 21 strains were proved to hyperproduct plasmid-mediated AmpC by DNA Sequence test, with a positive rate of 11.67%. Blast results indicated that positive AmpC group was comprised of 17 strains which carried DHA type AmpC β-lactamases and 4 strains which carried EBC type AmpC β-lactamases. DNA sequence analysis revealed three novel AmpC genotypes (GenBank accession is FJ237366, FJ237367 and FJ237368). All AmpC positive isolates exhibited high resistance to the third or fourth generation cephalosporins, aminoglycosides and quinolones. But all of them were susceptible to imipenem and meropenem. Conjugation Experiment and Southern blot hydridization have demonstrated the novel AmpC beta-lactamase (K. pneumoniae 701) is mediated by the plasmid. The substitutions of the the novel AmpC-type plasmid-mediated β-lactamase could not result in the resistance pattern changes. The novel AmpC beta-lactamase (K. pneumoniae 701) 9 安徽医科大学硕士学位论文 is mediated by the plasmid and its pI is 8.4, and it hydrolyzes cefoxitin with high measurable hydrolysis rate. The inhibition profile showed that the pI 8.4 β-lactamase was not inhibited well by any of the β-lactamase inhibitors tested. In our study we amplify the class Ⅰ integron of the K. pneumoniae E701,