Gorzynski Mylene, Week Tiana, Jaramillo Tiana, Dzalamidze Elizaveta, Danelishvili Lia
Department of Biomedical Sciences, Carlson College of Veterinary Medicine, Oregon State University, Corvallis, OR 97331, USA.
Department of Biochemistry & Molecular Biology, Oregon State University, Corvallis, OR 97331, USA.
Microorganisms. 2021 Dec 7;9(12):2527. doi: 10.3390/microorganisms9122527.
subsp. (MAB) is a fast-growing nontuberculous mycobacterium causing pulmonary infections in immunocompromised and immunocompetent individuals. The treatment of MAB infections in clinics is extremely challenging, as this organism is naturally resistant to most available antibiotics. There is limited knowledge on the mechanisms of MAB intrinsic resistance and on the genes that are involved in the tolerance to antimicrobials. To identify the MAB genetic factors, including the components of the cell surface transport systems related to the efflux pumps, major known elements contributing to antibiotic resistance, we screened the MAB transposon library of 2000 gene knockout mutants. The library was exposed at either minimal inhibitory (MIC) or bactericidal concentrations (BC) of amikacin, clarithromycin, or cefoxitin, and MAB susceptibility was determined through the optical density. The 98 susceptible and 36 resistant mutants that exhibited sensitivity below the MIC and resistance to BC, respectively, to all three drugs were sequenced, and 16 mutants were found to belong to surface transport systems, such as the efflux pumps, porins, and carrier membrane enzymes associated with different types of molecule transport. To establish the relevance of the identified transport systems to antibiotic tolerance, the gene expression levels of the export related genes were evaluated in nine MAB clinical isolates in the presence or absence of antibiotics. The selected mutants were also evaluated for their ability to form biofilms and for their intracellular survival in human macrophages. In this study, we identified numerous MAB genes that play an important role in the intrinsic mechanisms to antimicrobials and further demonstrated that, by targeting components of the drug efflux system, we can significantly increase the efficacy of the current antibiotics.
亚种（MAB）是一种快速生长的非结核分枝杆菌，可在免疫功能低下和免疫功能正常的个体中引起肺部感染。临床上治疗MAB感染极具挑战性，因为这种微生物对大多数现有抗生素天然耐药。关于MAB固有耐药机制以及涉及抗菌耐受性的基因的了解有限。为了鉴定MAB的遗传因素，包括与外排泵相关的细胞表面转运系统的组成部分，这些是导致抗生素耐药性的主要已知因素，我们筛选了包含2000个基因敲除突变体的MAB转座子文库。该文库分别暴露于阿米卡星、克拉霉素或头孢西丁的最低抑菌浓度（MIC）或杀菌浓度（BC）下，并通过光密度测定MAB的敏感性。对分别对所有三种药物表现出低于MIC的敏感性和对BC耐药的98个敏感突变体和36个耐药突变体进行测序，发现16个突变体属于表面转运系统，如与不同类型分子转运相关的外排泵、孔蛋白和载体膜酶。为了确定所鉴定的转运系统与抗生素耐受性的相关性，在有或没有抗生素的情况下，评估了9株MAB临床分离株中与输出相关基因的基因表达水平。还评估了所选突变体形成生物膜的能力及其在人巨噬细胞中的细胞内存活能力。在本研究中，我们鉴定了许多在抗菌固有机制中起重要作用的MAB基因，并进一步证明，通过靶向药物外排系统的组成部分，我们可以显著提高当前抗生素的疗效。
