Table_1_Insights Into the Evolution of Staphylococcus aureus Daptomycin Resistance From an in vitro Bioreactor Model.xlsx

The extensive use of daptomycin for treating complex methicillin-resistant Staphylococcus aureus infections has led to the emergence of daptomycin-resistant strains. Although genomic studies have identified mutations associated with daptomycin resistance, they have not necessarily provided insight into the evolution and hierarchy of genetic changes that confer resistance, particularly as antibiotic concentrations are increased. Additionally, plate-dependent in vitro analyses that passage bacteria in the presence of antibiotics can induce selective pressures unrelated to antibiotic exposure. We established a continuous culture bioreactor model that exposes S. aureus strain N315 to increasing concentrations of daptomycin without the confounding effects of nutritional depletion to further understand the evolution of drug resistance and validate the bioreactor as a method that produces clinically relevant results. Samples were collected every 24 h for a period of 14 days and minimum inhibitory concentrations were determined to monitor the acquisition of daptomycin resistance. The collected samples were then subjected to whole genome sequencing. The development of daptomycin resistance in N315 was associated with previously identified mutations in genes coding for proteins that alter cell membrane charge and composition. Although genes involved in metabolic functions were also targets of mutation, the common route to resistance relied on a combination of mutations at a few key loci. Tracking the frequency of each mutation throughout the experiment revealed that mutations need not arise progressively in response to increasing antibiotic concentrations and that most mutations were present at low levels within populations earlier than would be recorded based on single-nucleotide polymorphism (SNP) filtering criteria. In contrast, a serial-passaged population showed only one mutation in a gene associated with resistance and provided limited detail on the changes that occur upon exposure to higher drug dosages. To conclude, this study demonstrates the successful in vitro modeling of antibiotic resistance in a bioreactor and highlights the evolutionary paths associated with the acquisition of daptomycin non-susceptibility.

达托霉素（daptomycin）在治疗复杂性耐甲氧西林金黄色葡萄球菌（methicillin-resistant Staphylococcus aureus）感染中的广泛应用，已催生达托霉素耐药菌株的出现。尽管基因组学研究已鉴定出与达托霉素耐药相关的突变，但尚未完全阐明赋予耐药性的遗传变异的演化过程与层级关系，尤其是在抗生素浓度逐步升高的场景下。此外，将细菌置于抗生素环境中进行平板传代的体外分析方法，可能引入与抗生素暴露无关的选择压力。为此，我们构建了连续培养生物反应器模型，使金黄色葡萄球菌（Staphylococcus aureus）菌株N315暴露于逐步升高的达托霉素浓度中，且规避了营养耗竭带来的混杂干扰，以进一步解析耐药性的演化机制，并验证该生物反应器方法可产生具有临床相关性的研究结果。我们在14天的实验周期内每24小时采集样本，并通过最低抑菌浓度（minimum inhibitory concentration, MIC）检测来监测达托霉素耐药性的获得情况。随后对采集的样本开展全基因组测序。N315的达托霉素耐药性发展，与此前已报道的、编码改变细胞膜电荷与组成的蛋白质的基因突变相关。尽管参与代谢功能的基因也成为突变靶点，但常见的耐药演化路径依赖于少数关键基因座的突变组合。追踪实验全程各突变的频率后发现，突变无需随抗生素浓度升高而逐步产生，且多数突变在种群中的出现频率早于基于单核苷酸多态性（single-nucleotide polymorphism, SNP）筛选标准所能检测到的水平。与之相反，连续传代的种群仅在一个耐药相关基因中出现突变，对更高药物剂量暴露下的变化细节揭示有限。综上，本研究成功在生物反应器中实现了抗生素耐药性的体外建模，并阐明了与获得达托霉素非易感表型相关的演化路径。