Rectal Swabs from Critically Ill Patients Provide Discordant Representations of the Gut Microbiome Compared to Stool Samples

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 the reference 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 the ICU setting. In this study, we compared 16S rRNA gene sequencing results between rectal swab and stool samples collected at three time points from mechanically ventilated critically ill adults. Rectal swabs comprised 89% of the samples collected at the baseline time point, but stool samples became more extensively available at later time points. Significant differences in alpha-diversity and beta-diversity between rectal swabs and stool samples were observed, but these differences were primarily due to baseline samples. Higher relative abundances of members of the Actinobacteria phylum (typically skin microbes) were present in rectal swabs than in stool samples (P = 0.05), a difference that was attenuated over time. The progressively increasing similarity of rectal swabs and stool samples likely resulted from increasing levels of 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 should take into account the type and timing of samples analyzed. IMPORTANCE Rectal swabs have been proposed as potential alternatives to stool samples for gut microbiome profiling in outpatients or healthy adults, but their reliability in assessment of critically ill patients has not been defined. Because stool sampling is not practical and often not feasible in the intensive care unit, we performed a detailed comparison of gut microbial sequencing profiles between rectal swabs and 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 demonstrated that the timing of the rectal swab sampling had a significant impact on sequencing results. Our methodological findings should provide valuable information for the design and interpretation of future investigations of the role of the gut microbiome in critical illness.

Metagenomic identification of severe pneumonia pathogens in mechanically-ventilated patients: a feasibility and clinical validity study

“…From enrolled subjects, we collected ETAs per our research protocol within the first 48 h from intubation (baseline samples) and then on day 5 post-intubation if the patient remained on mechanical ventilation in the ICU [19,22]. For the purposes of comparing ETA sequencing results with clinical microbiologic cultures, we utilized baseline samples in 20/22 subjects, because baseline samples were closer to the timing of clinical microbiologic specimen acquisition for diagnosis of index pneumonia (not ventilator-associated pneumonia [VAP]).…”

“…To further validate the results of Nanopore sequencing for bacterial DNA, we performed standard 16S rRNA gene (V4 region) PCR amplification and sequencing on the Illumina MiSeq Platform as a reference method for bacterial DNA sequencing [26]. We processed 16S sequences using an in-house pipeline developed by the University of Pittsburgh Center for Medicine and the Microbiome (CMM) [22,[27][28][29][30][31]. Samples that generated fewer than 100 bacterial reads were excluded from further analyses.…”

Plasma (1→3) β-d-glucan levels are associated with host inflammatory responses and predict adverse clinical outcomes in critical illness

“…Clinical cohort and sample collection: From enrolled subjects, we collected blood samples for centrifugation and separation of plasma and serum. We also collected noninvasive biospecimens for study of lower respiratory tract microbiota with endotracheal aspirates (ETA) and we analyzed stool samples for study of the intestinal microbiota, as previously described (48,49).…”

“…Classifications were performed without knowledge of biomarker data. We retrospectively classified subjects as having ARDS per established criteria (49), at risk for ARDS due to the presence of direct (pneumonia or aspiration) or indirect (e.g. extra-pulmonary sepsis or acute pancreatitis) lung injury risk factors (51) but lacking ARDS diagnostic criteria, acute respiratory failure without risk factors for ARDS, or acute on chronic respiratory failure.…”

On the robustness of inference of association with the gut microbiota in stool, swab and mucosal tissue samples

“…Rectal swab may be used when stool samples are not practical to obtain, for example in the intensive care unit, and may collect a combination of both luminal and mucosal communities [8]. While stool and mucosal samples are generally distinct, there are mixed findings on the similarity between stool and swab samples [9–11]. Thus, different biospecimen types may be needed to sample microorganisms residing in different niches or to reflect different physiological conditions.…”