Department of Microbiology, University of Washington School of Medicine, Seattle, Washington, USA.
Department of Pathology and Laboratory Medicine, Cedars-Sinai Medical Center, Los Angeles, California, USA.
JCI Insight. 2021 Dec 22;6(24):e152472. doi: 10.1172/jci.insight.152472.
A hallmark of chronic bacterial infections is the long-term persistence of 1 or more pathogen species at the compromised site. Repeated detection of the same bacterial species can suggest that a single strain or lineage is continually present. However, infection with multiple strains of a given species, strain acquisition and loss, and changes in strain relative abundance can occur. Detecting strain-level changes and their effects on disease is challenging because most methods require labor-intensive isolate-by-isolate analyses, and thus, only a few cells from large infecting populations can be examined. Here, we present a population-level method for enumerating and measuring the relative abundance of strains called population multi-locus sequence typing (PopMLST). The method exploits PCR amplification of strain-identifying polymorphic loci, next-generation sequencing to measure allelic variants, and informatic methods to determine whether variants arise from sequencing errors or low-abundance strains. These features enable PopMLST to simultaneously interrogate hundreds of bacterial cells that are cultured en masse from patient samples or are present in DNA directly extracted from clinical specimens without ex vivo culture. This method could be used to detect epidemic or super-infecting strains, facilitate understanding of strain dynamics during chronic infections, and enable studies that link strain changes to clinical outcomes.
慢性细菌感染的一个标志是在受损部位长期存在 1 种或多种病原体。同一细菌物种的反复检测表明，单一菌株或谱系持续存在。然而，可能会发生感染给定物种的多种菌株、菌株获得和丧失以及菌株相对丰度的变化。检测菌株水平的变化及其对疾病的影响具有挑战性，因为大多数方法需要费力的逐个分离物分析，因此，只能从大量感染群体中检查少数细胞。在这里，我们提出了一种称为群体多位点序列分型 (PopMLST) 的用于计数和测量菌株相对丰度的群体水平方法。该方法利用针对菌株鉴定多态性位点的 PCR 扩增、下一代测序来测量等位基因变体，以及信息学方法来确定变体是来自测序错误还是低丰度菌株。这些特征使 PopMLST 能够同时检测从患者样本中大规模培养的数百个细菌细胞，或直接从临床标本中提取的 DNA 中存在的细菌细胞，而无需体外培养。该方法可用于检测流行菌株或超级感染菌株，有助于了解慢性感染期间的菌株动态，并能够将菌株变化与临床结果联系起来的研究。
