An abundance of aliC and aliD genes were identified in saliva using a novel multiplex qPCR to characterize group II non-encapsulated pneumococci with improved specificity.

IF 2.6 4区 生物学 Q3 MICROBIOLOGY
Claire S Laxton, Femke L Toekiran, Tzu-Yi Lin, Beta D Lomeda, Maikel S Hislop, Lance Keller, Orchid M Allicock, Anne L Wyllie
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引用次数: 0

Abstract

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 pspK, or aliC and aliD in their cps 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 pspK, aliD and two regions of aliC. 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 aliC and aliD had low pneumococcal specificity but indicated that targeting two regions of aliC would improve species specificity, without compromising sensitivity. Our novel multiplex assay accurately typed all isolates. When screening saliva, we found a high prevalence of aliC and aliD, even in samples negative for pneumococcal genes lytA and piaB. Isolated colonies which were aliC and aliD 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.

利用一种新的多重qPCR方法,在唾液中鉴定出了丰富的aliC和aliD基因,以提高特异性来表征II群非包膜肺炎球菌。
肺炎球菌监测研究报告,非囊化肺炎球菌(NESp)的患病率正在上升。NESp是链球菌之间遗传交换的重要储存库,包括抗菌素耐药性,并且越来越多地与疾病有关。疾病相关的NESp通常在其cps位点携带毒力基因pspK,或aliC和aliD,而不是胶囊基因。虽然针对cps区域的分子方法被广泛用于包封菌株的血清分型,但用于NESp分类的检测方法很少,这意味着它没有被广泛采用。因此,我们利用这些基因作为目标,建立了一种新的qPCR检测和分类NESp菌株的方法,提高了效率和特异性。我们对402株NESp和其他45株mitis群链球菌的序列进行了生物信息学分析,并建立了针对pspK、aliD和aliC两个区域的多重qpcr。用16株先前鉴定的NESp分离株验证了该方法。然后,我们将该方法应用于从培养富集的唾液中提取的DNA,并分离和表征可疑的NESp菌落,并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aliC和aliD引物特异性较低,但表明靶向aliC的两个区域将提高物种特异性,而不影响敏感性。我们新颖的多重检测方法准确地分型了所有分离株。当筛选唾液时,我们发现aliC和aliD的患病率很高,即使在肺炎球菌基因lytA和piaB阴性的样本中也是如此。aliC和aliD阳性的分离菌落可以通过我们的实验区分为非肺炎球菌链球菌。我们的多重qpcr方法可以有效地筛选甚至是高度多微生物的样本,如唾液,用于NESp基因,以检测和区分潜在的致病性NESp分支与密切相关的mitis-group链球菌。这将有助于更好地了解NESp的真实流行情况及其对肺炎球菌携带和疾病的影响。
本文章由计算机程序翻译,如有差异,请以英文原文为准。
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来源期刊
Microbiology-Sgm
Microbiology-Sgm 生物-微生物学
CiteScore
4.60
自引率
7.10%
发文量
132
审稿时长
3.0 months
期刊介绍: We publish high-quality original research on bacteria, fungi, protists, archaea, algae, parasites and other microscopic life forms. Topics include but are not limited to: Antimicrobials and antimicrobial resistance Bacteriology and parasitology Biochemistry and biophysics Biofilms and biological systems Biotechnology and bioremediation Cell biology and signalling Chemical biology Cross-disciplinary work Ecology and environmental microbiology Food microbiology Genetics Host–microbe interactions Microbial methods and techniques Microscopy and imaging Omics, including genomics, proteomics and metabolomics Physiology and metabolism Systems biology and synthetic biology The microbiome.
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