School of Biology, Georgia Institute of Technology, Atlanta, Georgia, USA; Applied Bioinformatics Laboratory, Atlanta, Georgia, USA; PanAmerican Bioinformatics Institute, Cali, Valle del Cauca, Colombia.
Pathology and Laboratory Medicine, Emory University School of Medicine, Atlanta, Georgia, USA; Antibiotic Resistance Center, Emory University School of Medicine, Atlanta, Georgia, USA.
mSphere. 2016 Jul 20;1(4). doi: 10.1128/mSphere.00094-16. eCollection 2016 Jul-Aug.
The increased prevalence of vancomycin-intermediate Staphylococcus aureus (VISA) is an emerging health care threat. Genome-based comparative methods hold great promise to uncover the genetic basis of the VISA phenotype, which remains obscure. S. aureus isolates were collected from a single individual that presented with recurrent staphylococcal bacteremia at three time points, and the isolates showed successively reduced levels of vancomycin susceptibility. A population genomic approach was taken to compare patient S. aureus isolates with decreasing vancomycin susceptibility across the three time points. To do this, patient isolates were sequenced to high coverage (~500×), and sequence reads were used to model site-specific allelic variation within and between isolate populations. Population genetic methods were then applied to evaluate the overall levels of variation across the three time points and to identify individual variants that show anomalous levels of allelic change between populations. A successive reduction in the overall levels of population genomic variation was observed across the three time points, consistent with a population bottleneck resulting from antibiotic treatment. Despite this overall reduction in variation, a number of individual mutations were swept to high frequency in the VISA population. These mutations were implicated as potentially involved in the VISA phenotype and interrogated with respect to their functional roles. This approach allowed us to identify a number of mutations previously implicated in VISA along with allelic changes within a novel class of genes, encoding LPXTG motif-containing cell-wall-anchoring proteins, which shed light on a novel mechanistic aspect of vancomycin resistance. IMPORTANCE The emergence and spread of antibiotic resistance among bacterial pathogens are two of the gravest threats to public health facing the world today. We report the development and application of a novel population genomic technique aimed at uncovering the evolutionary dynamics and genetic determinants of antibiotic resistance in Staphylococcus aureus. This method was applied to S. aureus cultures isolated from a single patient who showed decreased susceptibility to the vancomycin antibiotic over time. Our approach relies on the increased resolution afforded by next-generation genome-sequencing technology, and it allowed us to discover a number of S. aureus mutations, in both known and novel gene targets, which appear to have evolved under adaptive pressure to evade vancomycin mechanisms of action. The approach we lay out in this work can be applied to resistance to any number of antibiotics across numerous species of bacterial pathogens.
耐万古霉素中间金黄色葡萄球菌（VISA）的流行率不断上升，是一种新出现的健康威胁。基于基因组的比较方法具有很大的潜力来揭示 VISA 表型的遗传基础，而这种遗传基础仍然不清楚。从一名在三个时间点出现复发性葡萄球菌菌血症的个体中收集了金黄色葡萄球菌分离株，这些分离株的万古霉素敏感性逐渐降低。采用群体基因组学方法比较了患者金黄色葡萄球菌分离株在三个时间点的万古霉素敏感性逐渐降低的情况。为此，对患者分离株进行了高覆盖率（约 500 倍）测序，并使用序列读数来模拟分离种群内和种群间的特定等位基因变异。然后应用群体遗传学方法评估三个时间点的总体变异水平，并确定种群间等位基因变化异常的个体变体。在三个时间点观察到总体群体基因组变异水平的连续降低，这与抗生素治疗导致的群体瓶颈一致。尽管这种总体变异降低，但许多个体突变在 VISA 群体中被横扫至高频。这些突变被认为与 VISA 表型有关，并对其功能作用进行了研究。这种方法使我们能够鉴定出以前与 VISA 相关的突变以及一类新型基因内的等位基因变化，这些基因编码含有 LPXTG 基序的细胞壁锚定蛋白，这揭示了万古霉素耐药性的一个新的机制方面。重要性 细菌病原体对抗生素的耐药性的出现和传播是当今世界公共卫生面临的最严重威胁之一。我们报告了一种新的群体基因组技术的开发和应用，旨在揭示金黄色葡萄球菌中抗生素耐药性的进化动态和遗传决定因素。该方法应用于从一名患者中分离的金黄色葡萄球菌培养物，该患者随着时间的推移对万古霉素抗生素的敏感性降低。我们的方法依赖于下一代基因组测序技术提供的更高分辨率，它使我们能够发现许多金黄色葡萄球菌突变，这些突变发生在已知和新的基因靶标中，似乎是在逃避万古霉素作用机制的适应性压力下进化的。我们在这项工作中提出的方法可以应用于多种细菌病原体对抗生素的耐药性。
