Pumbwe Lilian, Chang Abraham, Smith Rachel L, Wexler Hannah M
Greater Los Angeles Veterans Administration Healthcare Systems, Los Angeles, CA 90073, USA.
Microb Drug Resist. 2007 Summer;13(2):96-101. doi: 10.1089/mdr.2007.719.
The RND-family efflux pump gene bmeB5 was previously shown to be overexpressed in metronidazole-resistant laboratory mutants of Bacteroides fragilis. In the present study, we characterized the bmeABC5 genes and an upstream putative TetR-family regulator gene (bmeR5). bmeR5 (645 bp) was located 51 bp upstream of bmeA5 and encoded a 24.9-kDa protein. Deletant strains lacking bmeB5 or bmeR5 were constructed from a wild-type B. fragilis strain ADB77. Strain antimicrobial susceptibility was determined and gene expression was quantified. bmeR5 was overexpressed in Escherichia coli using a 6x-His tag system; BmeR5-His6 was isolated from inclusion bodies and its binding to bmeABC5 promoter regions was determined. BmeR5-His6 bound specifically to the bmeR5-bmeC5 intergenic region (IT1). Deletion of bmeR5 (ADB77DeltabmeR5) resulted in a significant (p < 0.05) increase in expression of bmeA5, bmeB5, and bmeC5, and > two-fold increase in minimum inhibitory concentrations (MICs) of ampicillin, cefoxitin, cefoperazone, ciprofloxacin, imipenem, metronidazole, ethidium bromide, and sodium dodecyl sulfate (SDS). MICs were reduced by the efflux pump inhibitor carbonyl cyanide m-chlorophenyl hydrazone (CCCP). The MICs of ampicillin, cefoperazone, metronidazole, and SDS were reduced by approximately two-fold in ADB77DeltabmeB5. A multidrug (metronidazole)-resistant, nim-negative B. fragilis clinical isolate overexpressed bmeABC5 genes, had a G-->T point mutation in IT1, and significantly reduced binding to BmeR5-His6. These data demonstrate that BmeR5 is a local repressor of bmeABC5 expression and that mutations in IT1 can lead to a derepression and resistance to multiple antimicrobial agents, including metronidazole.
RND家族外排泵基因bmeB5先前已被证明在脆弱拟杆菌的甲硝唑耐药实验室突变体中过表达。在本研究中，我们对bmeABC5基因和一个上游假定的TetR家族调节基因（bmeR5）进行了表征。bmeR5（645 bp）位于bmeA5上游51 bp处，编码一种24.9 kDa的蛋白质。从野生型脆弱拟杆菌菌株ADB77构建了缺失bmeB5或bmeR5的缺失菌株。测定了菌株的抗菌敏感性并对基因表达进行了定量。使用6x-His标签系统在大肠杆菌中过表达bmeR5；从包涵体中分离出BmeR5-His6，并测定其与bmeABC5启动子区域的结合。BmeR5-His6特异性结合bmeR5-bmeC5基因间区域（IT1）。缺失bmeR5（ADB77DeltabmeR5）导致bmeA5、bmeB5和bmeC5的表达显著（p < 0.05）增加，氨苄西林、头孢西丁、头孢哌酮、环丙沙星、亚胺培南、甲硝唑、溴化乙锭和十二烷基硫酸钠（SDS）的最低抑菌浓度（MIC）增加了两倍以上。外排泵抑制剂羰基氰化物间氯苯腙（CCCP）降低了MIC。在ADB77DeltabmeB5中，氨苄西林、头孢哌酮、甲硝唑和SDS的MIC降低了约两倍。一株耐多药（甲硝唑）、nim阴性的脆弱拟杆菌临床分离株过表达bmeABC5基因，在IT1中有一个G→T点突变，并且与BmeR5-His6的结合显著减少。这些数据表明，BmeR5是bmeABC5表达的局部阻遏物，IT1中的突变可导致去阻遏并对包括甲硝唑在内的多种抗菌药物产生耐药性。
