Machado Diana, Coelho Tatiane S, Perdigão João, Pereira Catarina, Couto Isabel, Portugal Isabel, Maschmann Raquel De Abreu, Ramos Daniela F, von Groll Andrea, Rossetti Maria L R, Silva Pedro A, Viveiros Miguel
Unidade de Microbiologia Médica, Global Health and Tropical Medicine, Instituto de Higiene e Medicina Tropical, Universidade Nova de LisboaLisboa, Portugal.
Programa de Pós-Graduação em Biologia Celular e Molecular, Centro de Biotecnologia, Fundação Universidade Federal do Rio GrandePorto Alegre, Brazil.
Front Microbiol. 2017 Apr 27;8:711. doi: 10.3389/fmicb.2017.00711. eCollection 2017.
Numerous studies show efflux as a universal bacterial mechanism contributing to antibiotic resistance and also that the activity of the antibiotics subject to efflux can be enhanced by the combined use of efflux inhibitors. Nevertheless, the contribution of efflux to the overall drug resistance levels of clinical isolates of is poorly understood and still is ignored by many. Here, we evaluated the contribution of drug efflux plus target-gene mutations to the drug resistance levels in clinical isolates of . A panel of 17 clinical strains were characterized for drug resistance associated mutations and antibiotic profiles in the presence and absence of efflux inhibitors. The correlation between the effect of the efflux inhibitors and the resistance levels was assessed by quantitative drug susceptibility testing. The bacterial growth/survival vs. growth inhibition was analyzed through the comparison between the time of growth in the presence and absence of an inhibitor. For the same mutation conferring antibiotic resistance, different MICs were observed and the different resistance levels found could be reduced by efflux inhibitors. Although susceptibility was not restored, the results demonstrate the existence of a broad-spectrum synergistic interaction between antibiotics and efflux inhibitors. The existence of efflux activity was confirmed by real-time fluorometry. Moreover, the efflux pump genes , and were shown to be overexpressed in the presence of antibiotics, demonstrating the contribution of these efflux pumps to the overall resistance phenotype of the clinical isolates studied, independently of the genotype of the strains. These results showed that the drug resistance levels of multi- and extensively-drug resistant clinical strains are a combination between drug efflux and the presence of target-gene mutations, a reality that is often disregarded by the tuberculosis specialists in favor of the almost undisputed importance of antibiotic target-gene mutations for the resistance in .
大量研究表明，外排是细菌产生抗生素耐药性的一种普遍机制，而且受外排作用影响的抗生素活性可通过联合使用外排抑制剂来增强。然而，外排在结核分枝杆菌临床分离株总体耐药水平中的作用却鲜为人知，且仍被许多人忽视。在此，我们评估了药物外排加靶基因突变对结核分枝杆菌临床分离株耐药水平的影响。对一组17株结核分枝杆菌临床菌株在有和无外排抑制剂的情况下进行了与耐药相关突变及抗生素谱特征分析。通过定量药敏试验评估外排抑制剂的作用与耐药水平之间的相关性。通过比较有抑制剂和无抑制剂时的生长时间来分析细菌生长/存活与生长抑制情况。对于赋予抗生素耐药性的相同突变，观察到不同的最低抑菌浓度（MIC），且发现外排抑制剂可降低不同的耐药水平。尽管药敏性未恢复，但结果表明抗生素与外排抑制剂之间存在广谱协同相互作用。通过实时荧光法证实了外排活性的存在。此外，外排泵基因mdtA、mdtC和mdtG在有抗生素存在时显示过表达，这表明这些外排泵对所研究的结核分枝杆菌临床分离株的总体耐药表型有贡献，与菌株的基因型无关。这些结果表明，多重耐药和广泛耐药的结核分枝杆菌临床菌株的耐药水平是药物外排与靶基因突变存在的综合结果，而这一现实往往被结核病专家所忽视，因为他们更倾向于认为抗生素靶基因突变对结核分枝杆菌耐药性具有几乎无可争议的重要性。
