College of Animal Science and Technology, Northwest A&F University, Yangling, Shaanxi, People's Republic of China.
Key Laboratory of Animal Genetics, Breeding and Reproduction of Shaanxi Province, Northwest A&F University, Yangling, Shaanxi, People's Republic of China.
J Antimicrob Chemother. 2021 Oct 11;76(11):2765-2773. doi: 10.1093/jac/dkab260.
Antibiotic tolerance allows bacteria to overcome antibiotic treatment transiently and potentially accelerates the emergence of resistance. However, our understanding of antibiotic tolerance at the genetic level during adaptive evolution of Staphylococcus aureus remains incomplete. We sought to identify the mutated genes and verify the role of these genes in the formation of vancomycin tolerance in S. aureus.
Vancomycin-susceptible S. aureus strain Newman was used to induce vancomycin-tolerant isolates in vitro by cyclic exposure under a high concentration of vancomycin (20× MIC). WGS and Sanger sequencing were performed to identify the genetic mutations. The function of mutated genes in vancomycin-tolerant isolates were verified by gene complementation. Other phenotypes of vancomycin-tolerant isolates were also determined, including mutation frequency, autolysis, lysostaphin susceptibility, cell wall thickness and cross-tolerance.
A series of vancomycin-tolerant S. aureus (VTSA) strains were isolated and 18 mutated genes were identified by WGS. Among these genes, pbp4, htrA, stp1, pth and NWMN_1068 were confirmed to play roles in VTSA formation. Mutation of mutL promoted the emergence of VTSA. All VTSA showed no changes in growth phenotype. Instead, they exhibited reduced autolysis, decreased lysostaphin susceptibility and thickened cell walls. In addition, all VTSA strains were cross-tolerant to antibiotics targeting cell wall synthesis but not to quinolones and lipopeptides.
Our results demonstrate that genetic mutations are responsible for emergence of phenotypic tolerance and formation of vancomycin tolerance may lie in cell wall changes in S. aureus.
抗生素耐受使细菌能够暂时克服抗生素治疗，并可能加速耐药性的出现。然而，我们对金黄色葡萄球菌适应性进化过程中抗生素耐受的遗传水平的理解仍不完整。我们试图确定突变基因，并验证这些基因在金黄色葡萄球菌万古霉素耐受形成中的作用。
使用万古霉素敏感的金黄色葡萄球菌菌株 Newman 在高浓度万古霉素（20×MIC）下进行循环暴露，体外诱导万古霉素耐受分离株。进行 WGS 和 Sanger 测序以鉴定遗传突变。通过基因互补验证突变基因在万古霉素耐受分离株中的作用。还确定了万古霉素耐受分离株的其他表型，包括突变频率、自溶、溶葡萄球菌素敏感性、细胞壁厚度和交叉耐受。
分离出一系列万古霉素耐受的金黄色葡萄球菌（VTSA）菌株，并通过 WGS 鉴定了 18 个突变基因。在这些基因中，pbp4、htrA、stp1、pth 和 NWMN_1068 被证实在 VTSA 形成中发挥作用。mutL 的突变促进了 VTSA 的出现。所有 VTSA 均未改变生长表型。相反，它们表现出自溶减少、溶葡萄球菌素敏感性降低和细胞壁增厚。此外，所有 VTSA 菌株均对靶向细胞壁合成的抗生素具有交叉耐药性，但对喹诺酮类和脂肽类药物无交叉耐药性。
我们的结果表明，遗传突变是表型耐受出现的原因，而万古霉素耐受的形成可能在于金黄色葡萄球菌细胞壁的变化。
