A Study On Resistance Of AmpC β-lactamase Producing Klebsiella Pneumoniae And Analysis Of The Novel Gene And The Class Ⅰ Integron.

ObjectiveTo detect of AmpC beta-lactamase in 180 clinical isolates of Klebsiella pneumoniae in several hospitals of Anhui province in 2007 and study the resistance of AmpC-producing isolates against 17 antibiotics.To investigate the genotypes distribution of plasmid-mediated beta-lactamase of the AmpC-producing isolates.To study the characterization of the novel AmpC-type plasmid-mediatedβ-lactamase and the location of the classⅠintegron.Materials and MethodsThe 180 clinical isolates of Klebsiella pneumoniae were collected from 33 hospitals of Anhui province in 2007.A total of 180 clinical isolates of Klebsiella pneumoniae were consecutively collected from different clinical significance specimens in 33 hospitals of Anhui province in September 2007 (from 1 September, 2007 to 30 September, 2007), and were identified by the standard methods in each cooperative hospitals in the entire month (from 1 September, 2007 to 30 September, 2007). All isolates were identified with the API identification System, and excluded the repeated isolates from the same patients. The AmpC-producing isolates were chose by cefoxitin firstly, and identified by the three-dimensional test, extracting plasmid and multiple PCR. Tow pairs of entire coding gene primers were designed and performed in the consequence experiments and the sequence was determined by direct sequencing of PCR products carried out by the dideoxy chain termination procedure of Sanger on an ABI377 automatic sequencer (Invotrigen Biocompany, Shanghai, China). The susceptibility to antibiotics of AmpC-producing isolates of Klebsiella pneumoniae were studied by agar dilution method.E. coli J53, Azir, resistant to sodium azide, was used as the recipient strain.The transconjugant was selected on Tryptone soya agar supplemented with sodium azide (100μg/mL) to inhibit growth of the donor strain and cefoxitin (2μg/mL) to inhibit growth of the recipient strain. Southern blot was used to reveal that the gene encoding the novel enzyme was on a plasmid.The whole ORF amplicon of the novel AmpC-type plasmid-mediatedβ-lactamase was linked into the vector Escherichia coli pHSG398 by T4DNA lingase. And then, the recombinant plasmid was introduced into the competent cells E.coli JM109, which transformed by CaCl2 method, and the transformant was selected on M-H agar plate supplemented with 50μg/ml of chloromycetin and 2μg/ml of cefoxitin. Agar dilution method was used to determine MICs against wild-type isolates, its transconjugants and transformants.The crude enzyme was extracted from transformants by sonication method. Purified enzymes were used for subsequentβ-lactamase assays, checked by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), 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 classⅠintegron 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 classⅠintegron was on a plasmid.ResultsOf 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 AmpCβ-lactamases and 4 strains which carried EBC type AmpCβ-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-mediatedβ-lactamase could not result in the resistance pattern changes. The novel AmpC beta-lactamase (K. pneumoniae 701) 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 8.4β-lactamase was not inhibited well by any of theβ-lactamase inhibitors tested.In our study we amplify the classⅠintegron of the K. pneumoniae E701, the composition of it explained for the aminoglycoside resistance. Moreover, the classⅠintegron from K. pneumoniae E701 was detectable in the same size plasmid.Conclusions1. The plasmid-mediated AmpCβ-lactamases were found in Klebsiella pneumoniae strains isolated from Anhui Province and DHA type were the mainly epidemic genotypes in our area. Moreover, three novel EBC genotypes were first found.2. The carbapenems is recommended to treat the AmpC-producing isolates in clinic.3. The substitutions of the the novel AmpC-type plasmid-mediatedβ-lactamase are neutral mutations. The integrons possibly is the main mechanism of the resistance of the plasmid-mediated AmpC-typeβ-lactamase.