The bacterial density of clinical rectal swabs is highly variable, correlates with sequencing contamination, and predicts patient risk of extraintestinal infection

Abstract

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 × 104–3.2 × 109 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.

Gut microbiome dysbiosis in antibiotic-treated COVID-19 patients is associated with microbial translocation and bacteremia

“…Taken together, our findings support a scenario in which gut-to-blood translocation of microorganisms following microbiome dysbiosis leads to dangerous BSIs during COVID-19, a complication seen in other immunocompromised patients, including patients with cancer 22 , 26 , 27 , 83 , acute respiratory distress syndrome 84 , and in ICU patients receiving probiotics 85 . We suggest that investigating the underlying mechanism behind our observations will inform the judicious application of antibiotics and immunosuppressives in patients with respiratory viral infections and increase our resilience to pandemics.…”

“…Our observation that the type of bacteria that entered the bloodstream was enriched in the associated stool samples is a wellcharacterized phenomenon in cancer patients 22,26,27 , especially during chemotherapy-induced leukocytopenia when patients are severely immunocompromised 20,53 . COVID-19 patients are also immunocompromised and frequently incur lymphopenia, rendering them susceptible to secondary infections 79 .…”

“…In immunocompromised cancer patients, blooms of Enterococcaceae and Gram-negative proteobacteria can lead to gut dominations by few or single species [22][23][24][25] . Such gut domination events are dangerous to these patients because they are associated with increased risk of translocation of antibiotic-resistant bacteria from the gut into the blood stream 22,26,27 . Bacterial co-infection can also cause life-threatening complications in patients with severe viral infections 10,11,28 ; therefore, antibacterial agents were administered empirically to nearly all critically ill suspected COVID-19 patients since the incidence of bacterial superinfection was unknown early during the pandemic 4,29 .…”

In critically ill patients, anti-anaerobic antibiotics increase risk of adverse clinical outcomes

“…We performed a secondary analysis of bacterial community data generated for a previously published study [ 49 , 50 ]. We characterised the bacterial density and community composition of bacteria on rectal swabs collected from 116 hospitalised patients, all of whom were within our larger retrospective cohort.…”

“…Although all patients in our subcohort were eventually intubated and treated with antibiotics prior to hour 72 of mechanical ventilation, some patients did not receive antibiotics prior to admission rectal swab and some had rectal swabs acquired on medical floors before intubation (and were later transferred to the ICU). Patient characteristics and microbiome analysis have been previously reported [ 49 , 50 ].…”