Study On Genes Of Oxacillinase In Carbapenemresistant Acinetobacter Baumannii

Objective: To test the antibiotic resistance and OXA enzyme encoding genotype of Acinetobacter baumannii clinical isolate in this area, analyze the impact of OXA genotype and gene mutations on drug resistance. In order to providing scientific basis for clinical rational use of antimicrobial drugs and hospital infection control, and for understanding Acinetobacter bacteria resistance trend.Methods: The minimal inhibitory concentrations(MICs) of antimicrobial agents against 50 Acinetobacter baumannii were determined by agar dilution method. Using polymerase chain reaction(PCR) to detect four OXA-type carbapenemase genes, Mutation of OXA-23-like was determined by TA cloning and sequencing. Construction of E. coli Rosetta(DE3) transconjugants producing OXA-23 and OXA-423. SDSpolyacrylamide gel electrophoresis was used for detecting the producing of OXA-23 and OXA-423. The minimal inhibitory concentrations(MICs)of antimicrobial agents against transconjugants were determined by agar dilution method.Results: The resistance rates of imipenem and meropenem for 50 Acinetobacter baumannii were 80% and 78%, respectively, only Polymyxin B, Minocycline, Tigecycline has a high sensitivity. Carbapenem resistant and susceptible strains were 40 and 10, respectively. OXA-51-like gene were 100% positive, OXA-23-like gene were 80%. All isolates were nagetive for OXA-24-like and OXA-58-like. In this study, OXA-51-like gene were detected in all collected 50 strains whether carbapenem resistant ornot. While, OXA-23-like gene were detected in 39 of 40 carbapenem resistant Acinetobacter baumannii, only one was detected of 10 carbapenem non-resistant strains. Chi-square test indicated that there is a high correlation between Acinetobacter baumannii resistant to carbapenems and carrying OXA-23-like gene, but not OXA-51-like gene. DNA sequencing of nine OXA-23-lik gene showed that seven sequences were 100% homologous with the standard sequence of OXA-23 gene. For OXA-23-like gene of the others isolates(WY-1017 and WY-0713), one have a mutation site(T47C), the other have two(A336G, T392C)mutation site, shows that those two OXA-23-like gene were new subtypes and were named OXA-422 gene and OXA-423 gene, respectively. Those novel OXA gene was submitted to Gen Bank under accession number KM433671 and KM433672, respectively. The site of 392 was critical for OXA-23-like gene, made OXA-423 differed from OXA-23 by a single amino acid substitution at position 131(Val131Ala). Noticeably, the V131 A substitution was located in the second of three active sites of OXA-23-like(82-85 STFK, 129-131 SAV and 219-221 KTG). Antibiotic susceptibility testing results showed that carring the mutation of OXA-423 strain WY-0713 than the others 8 non-mutation strains was sensitive to imipenem and meropenem(meropenem and imipenem MIC values of strain WY-0713 were μg/ml and 16 μg/ml, and meropenem and imipenem MIC values of the others 8 non-mutation strains were≥32 μg/ml). By PCR, restriction enzyme identification and sequence analysis, verified the successful construction of the recombinant plasmid(p E T-OXA-23 and p ET-OXA-423). After inducing, SDS-PAGE electrophoresis confirmed OXA-23 gene and OXA-423 gene were successfully expressed. Antibiotic susceptibility testing showed that Escherichia coli transconjugant harboring p ET-OXA-423 showed 4-and 8- times lower imipenem and meropenem MICs, respectively, as compared to that with p ET-OXA-23. In addition, the amoxicillin and amoxicillin+CLA MICs for OXA-423 producing clones were also reduced 4- to 8-fold when compared with OXA-23 producing clones. But, the MICs of cefotaxime and ceftazidime for both transconjugants remained the same.Conclusion: Drug-resistance of Acinetobacter baumannii are severe, especially for carbapenems which were mostly used. Only polymyxin B, minocycline, tigecycline has a higher sensitivity. Acinetobacter baumannii resistant to carbapenems were highly correlated with carrying OXA-23 gene. Discovering tow novel OXA-23-like subtypes genes, OXA-422 and OXA-423. Moreover the novel OXA-423 gene can reduced the resistance of Acinetobacter baumannii to some antibiotics.