Study On The Mechanism Of The β-lactamase,integron And Novel Gene In Clinical Mult-resistant Acinetobacter Spp. Strains

Objective:To investigate the carbapenemases resistance of multi-resistant Acinetobacter species and analyze their associated resistance mechanism and guide rational use of antibiotics.To investigate the integron gene of multi-resistant Acinetobacter species and analyze their associated resistance mechanism.To study the biochemical properties of the novel OXA-146 type carbapenemase.Materials and Methods:Totally 176 clinical isolates of Acinetobacter species were collected from 35 hospitals of Anhui province in 2007. M-H agar dilution method was used to determine MICs of 14 antimicrobial agents against wild-type isolates. Multi-resistant strains of bacteria DNA extracted by boiling method,specific fragment of carbapenemases gene and extended-spectrumβ-lactamases gene,Ⅰ,ⅡandⅢintegrase gene as well as-I integrase gene-positive strains of I-type integron structure were amplified by PCR.The encoding genes of the novel OXA-typeβ-lactamase were amplified by PCR. The purified PCR products were ligated with pUC-118 vectors,expressed in Escherichia 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 EcoRΙand BamHΙ,the whole ORF amplicon was linked into the vector Escherichia coli ppET28a (+) by T4DNA lingase. And then,the recombinant plasmid was introduced into the component cell E.coli JM109, which transformed by CaCl2 method,and the transformant was selected on M-H agar plate supplemented with 2μg/ml imipenem and 60μg/ml of ampicillin. Agar dilution method was used to determine MICs against wild-type isolates, its transconjugants and transformants.The crude enzyme was extracted from transcojugant by sonication method,pI values were determined using polyacrylamide gel by isoelectric focusing. The enzymes were used for subsequentβ-lactamase assays, and checked by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Coomassie Blue staining. Southern blot was used to reveal that the gene encoding the novel enzyme was on a plasmid.ResultsAmong the 176 isolates collected, 34 resistant resistant to Piperacillin, piperacillin / tazobactam, cefotaxime, ceftriaxone, ceftazidime, cefuroxime, aztreonam, gentamicin, ciprofloxacin including imipenem and meropenem.Among the 34 mult-resistant strains, 19 were determined as OXA-23 gene, 24 TEM-1 gene,10 CTX-M-1 gene,10 CTX-M-9 gene, 23 as class 1 integrons sequence. Among the 19 isolates OXA-23 gene DNA sequence analysis conformed that Acinetobacter baumannii and A. calcoaceticus were novel OXA-23 gene (GenBank accession is FJ194460 and FJ194494, respectively). A novel enzyme had been designated as OXA-146 by G. A. Jacoby. As compared with blaOXA-23 , sequence analysis revealed that the blaOXA-146 coding gene had GGC inserted causing one insertion mutation. And 17 not only have OXA-23 gene but also have class 1 integrons. Not detected OXA-24-type enzyme, IMP, VIM, SIM-based metal enzyme. Producing-OXA-146 carbapenemases Acinetobacter baumannii AB 518 also produces TEM-1 and CTX-M-1β-lactamase. Producing-OXA-146 carbapenemases A. calcoaceticus AC1622 also produces TEM-1β-lactamase. Among 23 classⅠintegrase-positive strains had 21 class I integron detected genetic structure.A clinical strain of the novel OXA-23β-lactamase-producing and its transconjugant had the same resistance spectrum, which exhibited the same high resistant rate.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 7.7 was identified, transferred by conjugation and associated with a plasmid. Southern blot revealed that the gene encoding was on a 52-kb plasmid.ConclusionProducing OXA-23-type carbapenemase result to carbapenem antibiotic, TEM-1-type extended-spectrumβ-lactamases,CTX-M-1-type extended-spectrumβ-lactamases,CTX-M-9-type extended-spectrumβ-lactamases ancinetobacter result to resistance toβ-lactam antibiotics. ClassⅠintegrons containing aminoglycoside resistant genes related to aminoglycoside antibiotics.19 isolates OXA-23 gene DNA sequence analysis conformed that 2 isolates have novel OXA-23 gene,designated as OXA-146 by G. A. Jacoby. This is the first describes the properties of a novel molecular carbapenems-hydrolyzing oxacillinases. Meanwhile, two novels of subtypes were found on the basis of the OXA-146-type enzymes, and multiply resistance mechanisms evolved in those isolates. Therefore, it is necessary to strengthen surveillance of antimicrobial resistance in local areas and exchange data between different areas. The rational use of antimicrobial agents may improve the situation. There is extremely important epidemiology significance in this work to prevent dissemination of resistant genes.