金黄色葡萄球菌对达托霉素耐药发展的分子机制和适应性代价研究

Meticillin-resistant Staphylococcus aureus (MRSA) remains one of the principal multiple resistant bacterial pathogens causing serious healthcare-associated and community-onset infections.Daptomycin is one of the last defense to treat severe infections caused by MRSA. The cases on daptomycin resistance resulting in clinical failure have been reported. However, the resistance mechanism of daptomycin remains unknown. Our previous study found that the daptomycin-resistant mutants of different S. aureus clonal lineages showed various fitness and different stability in vitro. To confirm the scientific hypothesis "The formation and development of daptomycin-resistant S. aureus are associated with accumulation of genes mutations and influenced by fitness cost and compensatory mutation", we will select the mutants of different clones acquired in our previous study as the study samples and investigate the relationship between the variation of mismatch repair system (mutL/mutS) and the frequency of daptomycin resistance mutation. The phenotypic characterization will be determined, including cell membrane fluidity, cell surface charge, etc. The fitness costs of wild-type isolates and resistance mutants will be detected by growth rate curve, in-vitro and in- vivo competition growth experiments. The evolution and functions of the important gene mutations will be studied by the whole genome sequencing and allelic replacement experiments. The influence of resistance mutations and compensatory mutations on fitness will be identified. This project will clarify molecular mechanism of formation and development of daptomycin-resistant S. aureus, especially the fitness costs of resistance mutants. This study will provide the evidence for prevention and control of MRSA, development of novel antibiotics and resistance reversal.

达托霉素是治疗MRSA引起的重症感染的最后防线之一，目前其耐药形成和发展机制未明。我们前期研究发现，不同种系的金葡菌达托霉素耐药突变子体外适应力（fitness）、稳定性不同。为证实"金葡菌对达托霉素耐药的形成、发展与多个基因渐进突变有关，并受适应性代价（fitness cost）和补偿突变的影响"这一假设，我们以前期获得的不同种系的突变子为研究对象，研究错配修复系统（mutL/mutS）变异和耐药突变频率的关系；确定突变子的表型特征，包括细胞膜流动性、细胞表面电荷等；采用生长速率法、体内、体外竞争生长实验，研究突变子较亲体株的适应性代价；采用全基因组测序、等位基因替换实验，研究重要基因位点突变的演变过程及其功能，鉴定耐药突变和补偿突变对适应性的影响。本研究将系统阐明达托霉素耐药金葡菌形成和发展的分子机制，特别是耐药突变子的适应性代价，为MRSA的防控、新药的研发、耐药性的逆转提供依据。

达托霉素是治疗耐甲氧西林的金黄色葡萄球菌（MRSA）引起的重症感染的最后防线之一，目前其耐药形成和发展机制未明。为证实"金葡菌对达托霉素耐药的形成、发展与多个基因渐进突变有关，并受适应性代价（fitness cost）和补偿突变的影响"这一假设，我们以不同种系的15株金葡菌为研究对象，发现利福平耐药突变频率从1.32×10-9到3.51×10-8不等，从聚类关系来看，菌株突变频率和错配修复系统的序列亲缘无明显对应关系。通过34天连续筛选高突变菌株得到可耐受达托霉素浓度达到16 μg/mL的突变子，突变频率从8.4×10-9至3.5×10-8，突变子的细胞壁厚度显著增加（P <0.001），细胞表面电荷变化显著（P <0.01）。突变株生长速率明显减慢，相对适应性参数fit值分别为0.652、0.808、0.850，突变株血清耐受能力降低，对小鼠致死（致病）能力减弱（P <0.01）。证实突变子可以稳定传代，且具有较大的适应性代价。全基因组测序并比对亲体株与突变子，我们发现不同菌株基因突变位点数量不同，mprF和walK在达托霉素耐药过程中起重要作用，其中walK (L7Q, Y225N)和mprF (G299V, L473I)这四个位点为新发现的突变位点。另外，等位基因替换实验正在进行，以研究重要基因位点突变的演变过程及其功能，鉴定耐药突变和补偿突变对适应性的影响。综上所述，本研究发现大多数金葡菌达托霉素耐药突变频率处于较低水平，且耐药突变子的适应性降低，这意味着临床菌株不易获得对达托霉素耐药性，获得耐药性的菌株也容易被敏感菌替代而发生耐药性逆转。walK (L7Q, Y225N)和mprF (G299V, L473I)为新发现的在达托霉素耐药过程中起重要作用的突变位点。本研究为MRSA的防控、新药的研发、耐药性的逆转提供了新的依据。