Laxton Claire S, Toekiran Femke L, Lin Tzu-Yi, Lomeda Beta D, Hislop Maikel S, Keller Lance, Allicock Orchid M, Wyllie Anne L
Department of Epidemiology of Microbial Diseases, Yale School of Public Health, New Haven, CT, USA.
Department of Cell and Molecular Biology, Center for Immunology and Microbial Research, University of Mississippi Medical Center, Jackson, MS, USA.
Microbiology (Reading). 2025 Apr;171(4). doi: 10.1099/mic.0.001555.
Pneumococcal surveillance studies are reporting increasing prevalence of non-encapsulated pneumococci (NESp). NESp are an important reservoir for genetic exchange among streptococci, including for antimicrobial resistance, and are increasingly implicated in disease. Disease-associated NESp commonly carries the virulence genes , or and in their locus instead of capsule genes. While molecular methods targeting the cps region are widely used for serotyping encapsulated strains, there are few assays available for the classification of NESp, meaning it is not widely undertaken. Therefore, we exploited these genes as targets for a novel qPCR assay for detecting and classifying NESp strains with improved efficiency and specificity. We conducted bioinformatic analysis on sequences from 402 NESp and 45 other mitis-group streptococci and developed a multiplex-qPCR, targeting , and two regions of . The assay was validated using 16 previously identified NESp isolates. We then applied the assay to DNA extracted from culture-enriched saliva and isolated and characterized suspected NESp colonies, with confirmation by whole genome sequencing. Bioinformatic analyses demonstrated that previously published primers for and had low pneumococcal specificity but indicated that targeting two regions of would improve species specificity, without compromising sensitivity. Our novel multiplex assay accurately typed all isolates. When screening saliva, we found a high prevalence of and , even in samples negative for pneumococcal genes and . Isolated colonies which were and positive could be differentiated as non-pneumococcal streptococci using our assay. Our multiplex-qPCR assay can be used to efficiently screen even highly polymicrobial samples, such as saliva, for NESp genes, to detect and differentiate potentially pathogenic NESp clades from closely related mitis-group streptococci. This will allow for a better understanding of the true prevalence of NESp and its impact on pneumococcal carriage and disease.
肺炎球菌监测研究报告称，非包膜肺炎球菌（NESp）的流行率在不断上升。NESp是链球菌之间基因交换的重要储存库，包括抗菌药物耐药性方面的基因交换，并且越来越多地与疾病相关。与疾病相关的NESp通常在其cps位点携带毒力基因、或，而不是荚膜基因。虽然针对cps区域的分子方法广泛用于对包膜菌株进行血清分型，但可用于NESp分类的检测方法很少，这意味着该分类尚未广泛开展。因此，我们利用这些基因作为新型qPCR检测的靶标，以提高检测和分类NESp菌株的效率和特异性。我们对402株NESp和45株其他缓症链球菌的序列进行了生物信息学分析，并开发了一种多重qPCR，靶向、和的两个区域。该检测方法使用16株先前鉴定的NESp分离株进行了验证。然后，我们将该检测方法应用于从富含培养物的唾液中提取的DNA，并分离和鉴定疑似NESp菌落，通过全基因组测序进行确认。生物信息学分析表明，先前发表的针对和的引物肺炎球菌特异性较低，但表明靶向的两个区域将提高物种特异性，而不影响敏感性。我们的新型多重检测方法准确地对所有分离株进行了分型。在筛查唾液时，我们发现和的流行率很高，即使在肺炎球菌基因和呈阴性的样本中也是如此。使用我们的检测方法，可以将呈阳性的分离菌落鉴定为非肺炎球菌链球菌。我们的多重qPCR检测方法可用于高效筛查甚至是高度多菌的样本，如唾液中的NESp基因，以检测和区分潜在致病的NESp进化枝与密切相关的缓症链球菌。这将有助于更好地了解NESp的真实流行率及其对肺炎球菌携带和疾病的影响。
