Department of Microbiology, University of Washington, Seattle, Washington, USA.
Department of Microbiology, University of Washington, Seattle, Washington, USA
Appl Environ Microbiol. 2019 Nov 14;85(23). doi: 10.1128/AEM.01866-19. Print 2019 Dec 1.
While microbiome studies have focused on diversity at the species level or higher, bacterial species in microbiomes are represented by different, often multiple, strains. These strains could be clonally and phenotypically very different, making assessment of strain content vital to a full understanding of microbiome function. This is especially important with respect to antibiotic-resistant strains, the clonal spread of which may be dependent on competition between them and susceptible strains from the same species. The pandemic, multidrug-resistant, and highly pathogenic subclone ST131-H30 (H30) is of special interest, as it has already been found persisting in the gut and bladder in healthy people. In order to rapidly assess clonal diversity, we developed a novel method based on deep sequencing of two loci used for sequence typing, along with an algorithm for analysis of the resulting data. Using this method, we assessed fecal and urinary samples from healthy women carrying H30 and were able to uncover considerable diversity, including strains with frequencies at <1% of the population. We also found that, even in the absence of antibiotic use, H30 could completely dominate the gut and, especially, urine of healthy carriers. Our study offers a novel tool for assessing a species' clonal diversity (clonobiome) within the microbiome, which could be useful in studying the population structure and dynamics of multidrug-resistant and/or highly pathogenic strains in their natural environments. Bacterial species in the microbiome are often represented by multiple genetically and phenotypically different strains, making insight into subspecies diversity critical to a full understanding of the microbiome, especially with respect to opportunistic pathogens. However, methods allowing efficient high-throughput clonal typing are not currently available. This study combines a conventional typing method with deep amplicon sequencing to allow analysis of many samples concurrently. While our method was developed for , it may be adapted for other species, allowing microbiome researchers to assess clonal strain diversity in natural samples. Since assessment of subspecies diversity is particularly important for understanding the spread of antibiotic resistance, we applied our method to the study of a pandemic multidrug-resistant clone. The results we present suggest that this clone could be highly competitive in healthy carriers and that the mechanisms of colonization by such clones need to be studied.
虽然微生物组研究主要集中在物种水平或更高水平的多样性上，但微生物组中的细菌物种由不同的、通常是多个菌株代表。这些菌株在克隆和表型上可能非常不同，因此评估菌株含量对于全面了解微生物组的功能至关重要。这在评估抗生素耐药菌株时尤其重要，这些菌株的克隆传播可能取决于它们与同一物种的敏感菌株之间的竞争。具有特殊意义的是，具有流行、多药耐药性和高度致病性的 ST131-H30 (H30) 亚克隆，因为它已经在健康人群的肠道和膀胱中持续存在。为了快速评估克隆多样性，我们开发了一种基于用于序列分型的两个基因座的深度测序的新方法，以及一种用于分析所得数据的算法。使用这种方法，我们评估了携带 H30 的健康女性的粪便和尿液样本，能够揭示出相当大的多样性，包括频率低于人群 1%的菌株。我们还发现，即使没有使用抗生素，H30 也可以完全主导健康携带者的肠道，特别是尿液。我们的研究提供了一种评估微生物组中物种克隆多样性（克隆生物组）的新工具，这对于研究自然环境中多药耐药和/或高度致病性菌株的种群结构和动态可能很有用。微生物组中的细菌物种通常由多个遗传和表型不同的菌株代表，因此深入了解亚种多样性对于全面了解微生物组至关重要，尤其是对于机会性病原体。然而，目前还没有允许高效高通量克隆分型的方法。本研究将传统的分型方法与深度扩增子测序相结合，允许同时分析多个样本。虽然我们的方法是为 ST131 开发的，但它可以适应其他物种，使微生物组研究人员能够在自然样本中评估克隆菌株多样性。由于评估亚种多样性对于理解抗生素耐药性的传播尤为重要，因此我们将我们的方法应用于对一种流行的多药耐药 ST131 克隆的研究。我们提出的结果表明，该克隆在健康携带者中可能具有很强的竞争力，需要研究此类克隆的定植机制。
