Role of the efflux proteins in multidrug resistant Haemophilus influenzae

在这项研究中，我们试图确定acrAB外排系统在多药耐药流感嗜血杆菌中的作用。为了研究外排蛋白在流感嗜血杆菌耐药性中的作用，我们获得了acrAB类基因中断突变体。14元和15元大环内酯类化合物对这些突变体的最低抑菌浓度是亲本菌株的4到16倍。另一方面，16元大环内酯类化合物对这些突变株的最低抑菌浓度为32~，是对亲本菌株的敏感倍数。与流感嗜血杆菌AcrAB系统相关的外膜蛋白尚未得到确切的鉴定。因此，我们对流感嗜血杆菌的外膜蛋白进行了计算机全基因组分析，最终发现了HI1462蛋白。然后将卡那霉素抗性小盒插入到HI1462基因中，获得了HI1462突变株。大环内酯类和氨基糖苷类抗生素对HI1462突变株的MIC与HI0894和HI0895缺失突变株的MIC完全相同。我们制备了针对AcrAB和HI1462蛋白的抗体，用于检测流感嗜血杆菌临床分离株的表达。我们的研究表明，AcrAB-HI1462系统在流感嗜血杆菌ATCC和野生型菌株中正常表达。这种外排系统在野生型菌株中的表达仅使MIC略有增加。然而，这并不意味着这种多药外排系统永远不会对有机体的耐药表型做出重大贡献。

In this study, we tried to determine the role of this acrAB efflux system in the multidrug resistant Haemophilus influenzae. We obtained the acrAB-like gene disruption mutants to study the function of efflux proteins in the resistance of H. influenzae. The MICs of 14- and 15-membered macrolides against these mutants are 4 to 16-fold more susceptible than against parent strain. On the other hand, the MICs of 16-membered macrolides against these mutants are 32 to 64-fold more susceptible than against parent strain. An outer membrane protein associated with the AcrAB system in H. influenzae has not yet been firmly identified. Therefore, we checked the outer membrane protein by computer analysis of whole genome of H. influenzae, and finally found HI1462 protein. And then we got HI1462 disrupted mutant by inserting the kanamycin resistance cartridge into HI1462 gene. The MICs of macrolide and aminoglycoside antibiotics against HI1462 disrupted mutant of H. influenzae are completely similar to MICs of both HI0894- and HI0895-deletion mutants. We made the antibody against AcrAB and HI1462 proteins to examine the expression in clinical isolates of H. influenzae. Our study showed that AcrAB-HI1462 system is normally expressed in H. influenzae ATCC and also wild-type strains. The expression of this efflux system in the wild-type strains produced only a small increase in the MIC. This does not mean, however, that this multidrug efflux system can never make a major contribution to the resistance phenotypes of the organism.