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

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.

MinION™ Nanopore Sequencing of Skin Microbiome 16S and 16S-23S rRNA Gene Amplicons

Human skin microbiome dysbiosis can have clinical consequences. Characterizing taxonomic composition of bacterial communities associated with skin disorders is important for dermatological advancement in both diagnosis and novel treatments. This study aims to analyze and improve the accuracy of taxonomic classification of skin bacteria with MinION™ nanopore sequencing using a defined skin mock community and a skin microbiome sample. We compared the Oxford Nanopore Technologies recommended procedures and concluded that their protocols highly bias the relative abundance of certain skin microbiome genera, most notably a large overrepresentation of Staphylococcus and underrepresentation of Cutibacterium and Corynebacterium. We demonstrated that changes in the amplification protocols improved the accuracy of the taxonomic classification for these three main skin bacterial genera. This study shows that MinION™ nanopore could be an efficient technology for full-length 16S rRNA sequencing; however, the analytical advantage is strongly influenced by the methodologies. The suggested alternatives in the sample processing improved characterization of a complex skin microbiome community using MinION™ nanopore sequencing.

Metabolomic Analysis of Skin Malodor-Associated Compounds and Structure-based Prediction of Acetolactate Synthase Selective Sulfonylurea Inhibitors

Bromhidrosis is characterized as a chronic condition related to malodor from the skin. The underlying etiology is from bacterial decompositions of glandular secretion products. However, specific pathways and metabolites for the disease are yet to be investigated. Here, twenty-eight metabolites, including fifteen major sweat constituents and thirteen compounds emitted from malodor-producing skin bacteria, were subjected to the metabometric analysis using Metaboanalyst. Different pathways in the butanoate metabolism revealed that acetolactate synthase (ALS) in skin Staphylococcus epidermidis (S. epidermidis) bacteria are catalyzing pyruvate to several malodor compounds like diacetyl. In the docking studies of the sulfonylurea-ALS interaction, five selected sulfonylureas, which originally were developed for the treatment of diabetes mellitus type 2, showed different binding free energies (ΔG) from chlorimuron ethyl - a well-known ALS sulfonylurea inhibitor. Amongst five sulfonylureas, gliquidone and glisoxepide were found to have free energy differences that were lower than or equal to chlorimuron ethyl, revealing their high affinities to ALS. In the future, further investigations of gliquidone and glisoxepide against ALS in skin bacteria would be crucial in repurposing these two sulfonylureas as new anti-bromhidrosis drugs.

Canine pyoderma: mecA persists autogenous bacterin formulation from meticillin-resistant Staphylococcus pseudintermedius (MRSP) and S. aureus (MRSA)

Objective Autogenous Staphylococcus pseudintermedius bacterins can reduce prescribing of antimicrobials in the management of canine recurrent pyoderma. However, increasing prevalence of meticillin-resistant, mecA-positive S. pseudintermedius (MRSP) raises concern over dispersal of mecA through bacterin therapy. We investigated the presence and integrity of mecA in bacterin formulations after manufacturing. Material and methods Twenty clinical isolates (12 MRSP, 7 MR-S. aureus, 1 meticillin-susceptible SP) were investigated. Pellets from overnight growth were washed 3 times with 0.5 % phenol saline, followed by addition of 0.1 ml 10 % formal-saline to 10 ml phenol-saline. Sterility was confirmed, and DNA extracted using both a standard genomic extraction kit and one recommended for formalin-fixed tissue samples (FFPE). The presence of mecA was determined after PCR and its integrity examined in 5 randomly selected samples after sequencing. Results In all bacterins from meticillin-resistant isolates, mecA was detected following FFPE extraction; products aligned fully to a reported mecA sequence. After standard DNA extraction, mecA was seen in 16/19 samples. Conclusion Persistence of mecA in MRSP bacterins suggests that dispersal of this important resistance mediator through therapy may be possible. While the ability of skin bacteria to uptake naked DNA remains unclear, it seems prudent to only formulate autogenous bacterins from mecA-negative S. pseudintermedius to avoid unnecessary spread of mecA.

Social Behavior, Community Composition, Pathogen Strain, and Host Symbionts Influence Fungal Disease Dynamics in Salamanders

The emerging fungal pathogen, Batrachochytrium dendrobatidis (Bd), which can cause a fatal disease called chytridiomycosis, is implicated in the collapse of hundreds of host amphibian species. We describe chytridiomycosis dynamics in two co-occurring terrestrial salamander species, the Santa Lucia Mountains slender salamander, Batrachoseps luciae, and the arboreal salamander, Aneides lugubris. We (1) conduct a retrospective Bd-infection survey of specimens collected over the last century, (2) estimate present-day Bd infections in wild populations, (3) use generalized linear models (GLM) to identify biotic and abiotic correlates of infection risk, (4) investigate susceptibility of hosts exposed to Bd in laboratory trials, and (5) examine the ability of host skin bacteria to inhibit Bd in culture. Our historical survey of 2,866 specimens revealed that for most of the early 20th century (~1920–1969), Bd was not detected in either species. By the 1990s the proportion of infected specimens was 29 and 17% (B. luciae and A. lugubris, respectively), and in the 2010s it was 10 and 17%. This was similar to the number of infected samples from contemporary populations (2014–2015) at 10 and 18%. We found that both hosts experience signs of chytridiomycosis and suffered high Bd-caused mortality (88 and 71% for B. luciae and A. lugubris, respectively). Our GLM revealed that Bd-infection probability was positively correlated with intraspecific group size and proximity to heterospecifics but not to abiotic factors such as precipitation, minimum temperature, maximum temperature, mean temperature, and elevation, or to the size of the hosts. Finally, we found that both host species contain symbiotic skin-bacteria that inhibit growth of Bd in laboratory trials. Our results provide new evidence consistent with other studies showing a relatively recent Bd invasion of amphibian host populations in western North America and suggest that the spread of the pathogen may be enabled both through conspecific and heterospecific host interactions. Our results suggest that wildlife disease studies should assess host-pathogen dynamics that consider the interactions and effects of multiple hosts, as well as the historical context of pathogen invasion, establishment, and epizootic to enzootic transitions to better understand and predict disease dynamics.

Circulating Antibodies to Skin Bacteria Detected by Serological Lateral Flow Immunoassays Differentially Correlated With Bacterial Abundance

The serological lateral flow immunoassay (LFIA) was used to detect circulating antibodies to skin bacteria. Next-generation sequencing analysis of the skin microbiome revealed a high relative abundance of Cutibacterium acnes but low abundance of Staphylococcus aureus and Corynebacterium aurimucosum on human facial samples. Yet, results from both LFIA and antibody titer quantification in 96-well microplates illustrated antibody titers that were not correspondent, and instead negatively correlated, to their respective abundance with human blood containing higher concentrations of antibodies to both S. aureus and C. aurimucosum than C. acnes. Acne vulgaris develops several unique microbial and cellular features, but its correlation with circulating antibodies to bacteria in the pilosebaceous unit remains unknown. Results here revealed that antibodies to C. acnes and S. aureus were approximately 3-fold higher and 1.5-fold lower, respectively, in acne patients than in healthy subjects. Although the results can be further validated by larger sample sizes, the proof-of-concept study demonstrates a newfound discrepancy between the abundance of skin bacteria and amounts of their corresponding antibodies. And in light of acne-correlated amplified titers of specific anticommensal antibodies, we highlight that profiling these antibodies in the pilosebaceous unit by LFIAs may provide a unique signature for monitoring acne vulgaris.