Staphylococci are important members of the human skin microbiome. Many staphylococcal species and strains are commensals of the healthy skin microbiota, while few play essential roles in skin diseases such as atopic dermatitis. To study the involvement of staphylococci in health and disease, it is essential to determine staphylococcal populations in skin samples beyond the genus and species level. Culture-independent approaches such as amplicon next-generation sequencing (NGS) are time- and cost-effective options. However, their suitability depends on the power of resolution. Here we compare three amplicon NGS schemes that rely on different targets within the genes tuf and rpsK, designated tuf1, tuf2 and rpsK schemes. The schemes were tested on mock communities and on human skin samples. To obtain skin samples and build mock communities, skin swab samples of healthy volunteers were taken. In total, 254 staphylococcal strains were isolated and identified to the species level by MALDI-TOF mass spectrometry. A subset of ten strains belonging to different staphylococcal species were genome-sequenced. Two mock communities with nine and eighteen strains, respectively, as well as eight randomly selected skin samples were analysed with the three amplicon NGS methods. Our results imply that all three methods are suitable for species-level determination of staphylococcal populations. However, the novel tuf2-NGS scheme was superior in resolution power. It unambiguously allowed identification of Staphylococcus saccharolyticus and distinguish phylogenetically distinct clusters of Staphylococcus epidermidis. Powerful amplicon NGS approaches for the detection and relative quantification of staphylococci in human samples exist that can resolve populations to the species and, to some extent, to the subspecies level. Our study highlights strengths, weaknesses and pitfalls of three currently available amplicon NGS approaches to determine staphylococcal populations. Applied to the analysis of healthy and diseased skin, these approaches can be useful to attribute host-beneficial and -detrimental roles to skin-resident staphylococcal species and subspecies.

中文翻译：

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三种扩增子测序方法的比较，以确定人类皮肤上的葡萄球菌种群

葡萄球菌是人体皮肤微生物组的重要成员。许多葡萄球菌物种和菌株是健康皮肤微生物群的共生菌，而很少有人在特应性皮炎等皮肤病中发挥重要作用。为了研究葡萄球菌与健康和疾病的关系，必须确定皮肤样本中超越属和种水平的葡萄球菌种群。扩增子下一代测序 (NGS) 等独立于培养的方法是既省时又经济的选择。然而，它们的适用性取决于分辨率。在这里，我们比较了三种依赖于基因 tuf 和 rpsK 内不同靶标的扩增子 NGS 方案，指定为 tuf1、tuf2 和 rpsK 方案。这些方案在模拟社区和人体皮肤样本上进行了测试。获取皮肤样本，搭建模拟社区，采集了健康志愿者的皮肤拭子样本。通过 MALDI-TOF 质谱法，总共分离并鉴定了 254 株葡萄球菌菌株。属于不同葡萄球菌属的十个菌株的子集进行了基因组测序。使用三种扩增子 NGS 方法分析了分别具有九个和十八个菌株的两个模拟群落以及八个随机选择的皮肤样本。我们的结果意味着所有三种方法都适用于葡萄球菌种群的物种水平测定。然而，新颖的 tuf2-NGS 方案在分辨率上更胜一筹。它明确允许鉴定溶糖葡萄球菌并区分表皮葡萄球菌的系统发育不同簇。存在用于检测和相对定量人类样本中葡萄球菌的强大扩增子 NGS 方法，可以将种群解析为物种，并在某种程度上解析为亚种级别。我们的研究强调了三种目前可用的扩增子 NGS 方法来确定葡萄球菌种群的优势、劣势和缺陷。应用于健康和患病皮肤的分析，这些方法可用于将宿主有益和有害作用归因于皮肤常驻葡萄球菌物种和亚种。