Rectal swabs in critically-ill patients provide discordant representations of the gut microbiome compared to stool samples: a brief methodologic report.

Abstract

The role of the gut microbiome in critical illness is being actively investigated, but the optimal sampling methods for sequencing studies of gut microbiota remain unknown. Stool samples are generally considered gold-standard but are not practical to obtain in the intensive care unit (ICU), and thus, rectal swabs are often used. However, the reliability of rectal swabs for gut microbiome profiling has not been established in this clinical setting. In this study, we compared 16S rRNA gene sequencing results between rectal swab and stool samples collected at three timepoints in mechanically-ventilated critically-ill adults. Rectal swabs comprised 89% of samples collected at the baseline timepoint, but stool samples became more available at later time-points. Significant differences in alpha and beta-diversity between rectal swabs and stool samples were observed (p<0.003), but these differences were primarily due to baseline samples. Higher relative abundance of Actinobacteria phyla (typically skin microbes) was present in rectal swabs compared to stool samples (p<0.02), a difference that was attenuated overtime. The progressive similarity of rectal swabs and stool samples likely results from increasing stool coating of the rectal vault and direct soiling of the rectal swabs taken at later time points. Therefore, inferences about the role of the gut microbiome in critical illness should be drawn cautiously and take into account the actual type and timing of samples analyzed. Statement of ImportanceRectal swabs are considered reliable alternatives to stool samples for gut microbiome profiling in outpatients or healthy adults, but their reliability in critically-ill patients has not been defined. Because stool sampling is not practical or often feasible in the intensive care unit, we performed a detailed comparison of gut microbial sequencing profiles between rectal swabs vs. stool samples in a longitudinal cohort of critically-ill patients. We identified systematic differences in gut microbial profiles between rectal swabs and stool samples, and highlighted that timing of rectal swabbing had a significant impact on sequencing results. Our methodological findings should inform future investigations of the role of the gut microbiome in critical illness.