LAUSR

Background In ecology, population density is a key feature of community analysis. Yet in studies of the gut microbiome, bacterial density is rarely reported. Studies of hospitalized patients commonly use rectal swabs for microbiome analysis, yet variation in their bacterial density—and the clinical and methodologic significance of this variation—remains undetermined. We used an ultra-sensitive quantification approach—droplet digital PCR (ddPCR)—to quantify bacterial density in rectal swabs from 118 hospitalized patients. We compared bacterial density with bacterial community composition (via 16S rRNA amplicon sequencing) and clinical data to determine if variation in bacterial density has methodological, clinical, and prognostic significance. Results Bacterial density in rectal swab specimens was highly variable, spanning five orders of magnitude (1.2 × 10 4 –3.2 × 10 9 16S rRNA gene copies/sample). Low bacterial density was strongly correlated with the detection of sequencing contamination (Spearman ρ = − 0.95, p < 10 −16 ). Low-density rectal swab communities were dominated by peri-rectal skin bacteria and sequencing contaminants ( p < 0.01), suggesting that some variation in bacterial density is explained by sampling variation. Yet bacterial density was also associated with important clinical exposures, conditions, and outcomes. Bacterial density was lower among patients who had received piperacillin-tazobactam ( p = 0.017) and increased among patients with multiple medical comorbidities (Charlson score, p = 0.0040) and advanced age ( p = 0.043). Bacterial density at the time of hospital admission was independently associated with subsequent extraintestinal infection ( p = 0.0028), even when controlled for severity of illness and comorbidities. Conclusions The bacterial density of rectal swabs is highly variable, and this variability is of methodological, clinical, and prognostic significance. Microbiome studies using rectal swabs are vulnerable to sequencing contamination and should include appropriate negative sequencing controls. Among hospitalized patients, gut bacterial density is associated with clinical exposures (antibiotics, comorbidities) and independently predicts infection risk. Bacterial density is an important and under-studied feature of gut microbiome community analysis. Video abstract