Development and evaluation of a culture-free microbiota profiling platform (MYcrobiota) for clinical diagnostics

Microbiota profiling has the potential to greatly impact on routine clinical diagnostics by detecting DNA derived from live, fastidious, and dead bacterial cells present within clinical samples. Such results could potentially be used to benefit patients by influencing antibiotic prescribing practices or to generate new classical-based diagnostic methods, e.g., culture or PCR. However, technical flaws in 16S rRNA gene next-generation sequencing (NGS) protocols, together with the requirement for access to bioinformatics, currently hinder the introduction of microbiota analysis into clinical diagnostics. Here, we report on the development and evaluation of an “end-to-end” microbiota profiling platform (MYcrobiota), which combines our previously validated micelle PCR/NGS (micPCR/NGS) methodology with an easy-to-use, dedicated bioinformatics pipeline. The newly designed bioinformatics pipeline processes micPCR/NGS data automatically and summarizes the results in interactive, but simple web reports. In order to explore the utility of MYcrobiota in clinical diagnostics, 47 clinical samples (40 “damaged skin” samples and 7 synovial fluids) were investigated using routine bacterial culture as comparator. MYcrobiota confirmed the presence of bacterial DNA in 37/37 culture-positive samples and detected bacterial taxa in 2/10 culture-negative samples. Moreover, 36/38 potentially relevant aerobic bacterial taxa and 3/3 mixtures of anaerobic bacteria were identified using culture and MYcrobiota, with the sensitivity and specificity being 95%. Interestingly, the majority of the 448 bacterial taxa identified using MYcrobiota were not identified using culture, which could potentially have an impact on clinical decision-making. Taken together, the development of MYcrobiota is a promising step towards the introduction of microbiota analysis into clinical diagnostic laboratories.

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