National Veterinary Assay Laboratory, Ministry of Agriculture, Forestry and Fisheries Kokubunji, Tokyo, Japan ; School of Veterinary Medicine, Rakuno Gakuen University Ebetsu, Hokkaido, Japan.
Front Microbiol. 2013 Mar 14;4:53. doi: 10.3389/fmicb.2013.00053. eCollection 2013.
Understanding the impact of antimicrobial use on the emergence of resistant bacteria is imperative to prevent its emergence. For instance, activation of the AcrAB efflux pumps is responsible for the emergence of antimicrobial-resistant Salmonella strains. Here, we examined the expression levels of acrB and its multiple regulator genes (RamA, SoxS, MarA, and Rob) in 17 field isolates of S. Choleraesuis by using quantitative PCR methods. The expression of acrB increased in eight of the field isolates (P < 0.05). The expression of acrB was associated with that of ramA in one isolate, soxS in one isolate, and both these genes in six isolates. Thereafter, to examine the effect of selected antimicrobials (enrofloxacin, ampicillin, oxytetracycline, kanamycin, and spectinomycin) on the expression of acrB and its regulator genes, mutants derived from five isolates of S. Choleraesuis were selected by culture on antimicrobial-containing plates. The expression of acrB and ramA was higher in the mutants selected using enrofloxacin (3.3-6.3- and 24.5-37.7-fold, respectively), ampicillin (1.8-7.7- and 16.1-55.9-fold, respectively), oxytetracycline (1.7-3.3- and 3.2-31.1-fold, respectively), and kanamycin (1.6-2.2- and 5.6-26.4-fold, respectively), which are AcrAB substrates, than in each of the parental strains (P < 0.05). In contrast, in AcrAB substrate-selected mutants, the expression of soxS, marA, and rob remained similar to that in parental strains. Of the four antimicrobials, the level of ramA expression was significantly higher in the enrofloxacin- and ampicillin-selected mutants than in the oxytetracycline- and kanamycin-selected mutants (P < 0.05), whereas the expression levels of acrB and multiple regulator genes in spectinomycin-selected mutants were similar to those in each parental strain. These data suggest that exposure to antimicrobials that are AcrAB substrates enhance the activation of the AcrAB efflux pump via RamA, but not via SoxS, MarA, or Rob in S. Choleraesuis.
了解抗菌药物使用对抗菌药物耐药性细菌出现的影响对于防止其出现至关重要。例如，AcrAB 外排泵的激活是导致沙门氏菌耐药菌株出现的原因。在这里，我们通过定量 PCR 方法检测了 17 株猪霍乱沙门氏菌田间分离株中 acrB 及其多个调控基因（RamA、SoxS、MarA 和 Rob）的表达水平。结果发现，在 8 株田间分离株中，acrB 的表达增加（P<0.05）。在 1 株分离株中，acrB 的表达与 ramA 相关，在 1 株分离株中与 soxS 相关，在 6 株分离株中与这两个基因均相关。此后，为了检测选定的抗菌药物（恩诺沙星、氨苄西林、土霉素、卡那霉素和大观霉素）对 acrB 及其调控基因表达的影响，我们从 5 株猪霍乱沙门氏菌的分离株中选择了通过在含抗菌药物的平板上培养获得的突变体。在使用恩诺沙星（分别为 3.3-6.3-和 24.5-37.7 倍）、氨苄西林（分别为 1.8-7.7-和 16.1-55.9 倍）、土霉素（分别为 1.7-3.3-和 3.2-31.1 倍）和卡那霉素（分别为 1.6-2.2-和 5.6-26.4 倍）作为 AcrAB 底物选择的突变体中，acrB 和 ramA 的表达均高于相应的亲本菌株（P<0.05）。相比之下，在 AcrAB 底物选择的突变体中，soxS、marA 和 rob 的表达与亲本菌株相似。在这四种抗菌药物中，恩诺沙星和氨苄西林选择的突变体中 ramA 的表达水平明显高于土霉素和卡那霉素选择的突变体（P<0.05），而大观霉素选择的突变体中 acrB 和多个调控基因的表达水平与各自的亲本菌株相似。这些数据表明，暴露于 AcrAB 底物的抗菌药物通过 RamA 而非 SoxS、MarA 或 Rob 增强了 AcrAB 外排泵的激活。
