The Mechanism Of Carbapenem-Resistant Clinical Isolates Of Acinetobacter Baumannii

Objective: In recent years, multiresistant Acinetobacter baumannii strains arerecognized as serious nosocomial pathogens, and carbapenem-resistant strains arebeing reported increasingly. This situation emerged serious challenge which clinicalfaced. The mechanism of Acinetobacter baumannii to carbapenem has become amajou issue. Nevertheless, OXA-type carbapenemase production is one of the mainmechanism, meanwhile outer membrane protein deficiencies may also be involved in.In this study, we explore the mechanisms of resistance to imipenem and meropenemin Acinetobacter baumannii clinical strains using molecular biology method. Thesignificance of this research is not only reduced the transmission or the emergence ofbacterial resistance by proper antibacterial agents, but also developed a new drug toovercome the problem.Methods:1,bacterial isolates: A total of 20 clinical isolates of Acinetobacter baumannii wereobtained between 2001 and 2005 in Tianjin, and selected resistance tocabapenem(including imipenem and meropenem) isolates for further study.2,Susceptibility testing: The MIC were tested by micro-dilution broth method andinhibition zone were tested by agar dilution to total 13 antibacterial agent3,PCR amplification of bla_OXA-23-like gene: According to the sequence published onGenBank, primers for bla_OXA-23和bla_OXA-27 gene were designed. PCRs werecarried out with forward primer, SQ1 5'-GATGTGTCATAGTATTCGTCG-3', located at-108～-88bp upstream open reading frame and reverse primer, SQ25'-TCACAACAACTAAAAGCACTG-3', located at +930～+950bpdownstream open reading frame. PCR products were analyzed on a 0.8％agarosegel.4,Preparation of bacterial OM and SDS-PAGE5,PCR amplification of carO gene: According to the sequence pulished onGenBank, specific primers for carOgene were designed. PCRs were carried outwith forward primer, OMP1 5'-ATGAAAGTATTACGTGTTTTAGTG-3',located at +1～+24bp and reverse primers, OMP2 5'-TTACCAgTAgAAgTTTACACC-3', located at +724～+744bp. PCR products were analyzed on a 0.8％agarose gel.6,Sequence datas were analyzed of PCR products.Results:1,Antibiotic susceptibility: three isolates AB13, AB24, AB173 resistance toimipenem and meropenem were selected from 20 clinical strains of Acinetobacterbaumannii. Them were resistant to ciprofloxacin ,highly resistant to all theβ-lactams(piperacillin, cefepime, ceftazidime, cefotaxime, cefoperazone,ceftriaxone, cefoperazone/sulbactam), aminoglycosides(including amikacin andgentamicin) and tetracycline, with the exception of quinolone(ofloxacin andlevofloxacin).2,bla_OXA-23-like PCR-DNA sequence: Clinical isolates of Acinetobacter baumanniiAB13, AB24, AB173 gave 1058bp PCR products with the presence of bla_OXAprimers, at the same time ATCC25922 and Sme006 had no PCR product. All thethree products were same DNA sequence, and were identical with bla_OXA-23.3,SDS-PAGE: The OMP patterns of AB173 and AB942 were very similar exceptfor the conspicuous absence of a 29-KDa OMP in the carbapenem-resistantAB173 strain. The OMP patterns of another two isolates AB13 and AB24 weresame to AB942.4,carO gene PCR-DNA sequence: Clinical isolates of Acinetobacter baumanniiAB13, AB24, AB173 and AB942 gave 744bp PCR products with the presence ofspecific primers for carO gene. AB942, AB13 and AB173 showed 81％, 80％,77％identity with carO gene published on GenBank(AY684798) respectively.And the predicted amino acid sequence showed 76％, 72％and 74％identity withCarO published on GenBank(accession no AAV80243).Conclusions: 3 clinical isolates of multidrug-resistant Acinetobacter baumannii allCarded bla_OXA-23, one of them was also accompanied by an outer membrane protein29KDa missing which may relate to the resistant to carbapenem in Acinetobacterbaumannii. CarO gene variations, gene mutation and protein structure and function ofthe relationship needs further study.