A Study On The Resistance Of Klebsiella Pneumoniae Isolated From Several Hospitals In Anhui Province And Molecular Biology Property Of The Novel Plasmid-Mediated AmpC-Type β-Lactamase

ObjectiveTo study the resistance of Klebsiella pneumoniae (K. pneumoniaee) strains isolated from several hospitals in Anhui Province ranging from 2007 to 2008 against 16 antimicrobial agents, and guide rational use of antimicrobial agents.To investigate the genotypes and epidemiology of blaampC and other resistant genes in K. pneumoniaee isolated from several hospitals of Anhui Province in 2008.To study the biochemical properties and the resistance of the novel AmpCβ-lactamase-producing K. pneumoniaee strain.Materials and MethodsIsolates180 K. pneumoniaee strains (non-repeated strains) in September, 2007 and 213 K. pneumoniaee strains (non-repeated strains) in September, 2008 were isolated from 34 different graded hospitals which were the members of Anhui Center for Surveillance of Bacterial Resistance. The isolates were identified by using the Microscan Walkaway-40 System. MethodsThe susceptibility test to K. pneumoniaee strains isolated from 34 different graded hospitals in Anhui Province for 16 antimicrobial agents was performed by means of Agar Dilution Method according to a standard procedure described by the Clinical and Laboratory Standards Institute (CLSI), 2008. The results of antimicrobial susceptibility testing were processed by statistical analysis.The AmpC-producing isolates collected from 34 different graded hospitals in Anhui Province, 2008 were choosed by cefoxitin and identified by the three-dimensional test. Conjugation experiments were performed with an azide sodium-resistant recipient, Escherichia coli J53 (E. coli J53, Azir) as the recipient in order to determine the resistance was transferable in the AmpC-producing isolates. Whether the novel AmpCβ-lactamase mediated by the plasmid or not and the size of plasmid were verified by Southern Blotting. The plasmid-mediated AmpCβ-lactamases were detected by Multiplex PCR. Extended-spectrumβ-lactamases (ESBLs) genes and inserted genes cassettes of class 1 integrons were amplified by PCR. The upstream and downstream sequences of the target gene fragment were used by thermal asymmetric interlaced PCR method in order to identify the integron genes in relation with coding genes.The encoding genes of the novel AmpC-type plasmid-mediatedβ-lactamase were amplified by PCR. The purified PCR products were ligated with pUC-118 vectors, expressed in E. coli JM109, and sequenced by Sanger's dideoxy chain termination composition method. Then, Blastn program was used to ascertain the genotype at GenBank. After digestion by PstΙand BamHΙ, the whole ORF amplicon was linked into the vector pHSG398 by T4 DNA lingase. And then, the recombinant plasmid was introduced into the component cell E. coli JM109, which was transformed by CaCl2 method, and the transformant was selected on M-H agar plate supplemented with 50μg/ml of chloromycetin and 2mg/L of cefotaxime. Agar dilution method was used to determine minimal inhibitory concentrations (MICs) against wild-type isolates, its transconjugants and transformants. The crudeβ-lactamase was extracted from transcojugant by sonication method,pI values were determined using polyacrylamide gel by isoelectric focusing (IEF). The enzymes were used for subsequentβ-lactamase assays, and checked by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Coomassie Blue staining. Southern blotting was used to reveal that the gene encoding the novel enzyme located on a plasmid.ResultsThe susceptibility results of K. pneumoniaee strains isolated from Anhui Province in 2007 and 2008 showed the sensitivity of some antimicrobial agents presented significant difference. It was necessary of the surveillance of antimicrobial agents periodically.Among the 213 isolates in 2008, 14 (6.57%) clinical strains produced plasmid- mediated AmpCβ-lactamases. The resistant rates of AmpCβ-lactamase-producing strains against most kinds of antimicrobial agents were higher than the non-producing strains. They were all sensitive to imipenem and meropenem.Among the 14 AmpCβ-lactamase-producing strains, 14 strains were multidrug- resistant, 14 strains were determined as different resistant genes encodingβ-lactamases, 9 strains carried class 1 integrons sequence.Among the 14 isolates, genotypes of AmpCβ-lactamases were blaDHA (8), blaEBC (4), and blaCIT (2), respectively. DNA sequence analysis conformed that 1 novel ampC gene (GenBank accession number is FJ237367) was detected in comparison with GenBank, which had been designated as MIR-5 by Jacoby GA.A clinical strain producing the novel plasmid-mediated AmpCβ-lactamase and its transconjugant had the similar resistance spectrum, which exhibited the same high resistant rate to ampicillin, cefuroxime, cefotaxime, ceftriaxone, and cefoxicin. However, compared with wild-type strains, the transconjugant had decreased resistant ability to cefuroxime, ceftazidime, cefepime, aztreonam, gentamicin, and ciprofloxacin.This novel enzyme with apparent pI of approximately 9.01 was identified, transferred by conjugation and associated with a plasmid. The matureβ-lactamase could calculate approximate molecular masses of 41 kDa. Southern blotting revealed that the gene encoding MIR-likeβ-lactamase was on a 57-kb plasmid. There was not the typical structure of integron according to the analysis of upstream and downstream target sequence.Kinetic study of this novelβ-lactamase suggested that it effectively hydrolyzed broad- spectrumβ-lactams. Affinity of thisβ-lactamase for ceftazidime was quite low and resulted in the lowest hydrolytic efficiency for substrates with measurable rates of hydrolysis.ConclusionThe resistant rates of AmpCβ-lactamase-producing K. pneumoniaee against most kinds of antimicrobial agents were the high level. The AmpC-producing isolates were multidrug-resistance and had several resistance mechanisms. Class 1 integrons and production of AmpCβ-lactamases and ESBLs were the main resistance mechanisms. Carbopenems, amikacin and fourth cephalosporins should be chosen in clinical empirical medication.CIT, EBC, and DHA were the mainly epidemic genotypes of plasmid-mediated AmpCβ-lactamase in our area. Meanwhile, one novel subtype was found on the basis of the AmpC-type enzyme (MIR-5, GenBank accession number FJ237367) in the world. Resistance could be transferred between different vclinical strains. Therefore, it was necessary to strengthen surveillance of antimicrobial resistance in local areas. The rational use of antimicrobial agents might improve the situation. There was extremely important epidemiology significance in this work to prevent dissemination of resistant genes and control resistance.