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.
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.
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.