Simultaneous Analysis of Bacterial and Fungal Communities in Oral Samples from Intubated Patients in Intensive Care Unit

Abstract

Intubated patients in intensive care units (ICUs) too frequently contract ventilator-associated pneumonia or Candida infections. Oropharyngeal microbes are believed to play an important etiologic role. This study was undertaken to determine whether next-generation sequencing (NGS) can be used to simultaneously analyze bacterial and fungal communities. Buccal samples were collected from intubated ICU patients. Primers targeting the V1-V2 region of bacterial 16S rRNA and the internal transcribed spacer 2 (ITS2) region of fungal 18S rRNA were used. V1-V2, ITS2, or mixed V1-V2/ITS2 primers were used to prepare an NGS library. Bacterial and fungal relative abundances were comparable for V1-V2, ITS2, or mixed V1-V2/ITS2 primers, respectively. A standard microbial community was used to adjust the relative abundances to theoretical abundance, and NGS and RT-PCR-adjusted relative abundances showed a high correlation. Using mixed V1-V2/ITS2 primers, bacterial and fungal abundances were simultaneously determined. The constructed microbiome network revealed novel interkingdom and intrakingdom interactions, and the simultaneous detection of bacterial and fungal communities using mixed V1-V2/ITS2 primers enabled analysis across two kingdoms. This study provides a novel approach to simultaneously determining bacterial and fungal communities using mixed V1-V2/ITS2 primers.

EnsembleSeq: A workflow towards real-time, rapid and simultaneous multi-kingdom amplicon sequencing for holistic and cost-effective microbiome research at scale

“…This approach, here termed as "multikingdom amplicon sequencing" may potentially lower the time, effort and cost of microbial community evaluation while also simultaneously characterizing the co-occurring endogenous residents of the human microbiota. A few recent reports, though using short reads and a small number of samples, have in fact provided encouraging evidence towards potential to concomitantly capture both bacteriome and mycobiome using amplicon sequencing (31,34). Current literature evidence however seems to indicate that a majority of non-bacteriome (primarily mycobiota) studies are done in silos (e.g., only mycobiome profiling of oral cavity) (5,14,20,22,35,36) with a handful of short read microbiome studies attempting both bacterial and fungal amplicon sequencing for the targeted samples, often as independent libraries or even independently sequenced bioprojects (31,33,(37)(38)(39).…”