Mark G. Young

Background: Stool-based molecular tests provide noninvasive options for pediatric tuberculosis (TB) diagnosis but have lower sensitivity compared to sputum-based tests. Untargeted metagenomic next-generation sequencing (mNGS) on stool could improve sensitivity and identify new gene targets for molecular testing. Methods: We performed shotgun mNGS on DNA isolated from stool samples of children undergoing assessment for pulmonary TB in Uganda. We defined the performance of mNGS to identify Mycobacterium tuberculosis (Mtb) against a microbiological reference standard (MRS, TB if sputum Xpert Ultra or culture positive) and a composite reference standard (TB if confirmed or unconfirmed TB). We also compared the accuracy of mNGS against stool-based Xpert Ultra. Finally, we identified enriched genomic loci among Mtb-classified reads. Results: We analyzed 176 stool samples of children with a median age of 3.6 years (interquartile range [IQR], 1–6 years). Against the MRS, the sensitivities of mNGS with positive TB defined as ≥1, 2, or 5 sequence fragments were 35.5% (95% confidence interval [CI] 19%–55%), 25.7% (12%–45%), and 19.4% (7%–37%), respectively, and specificities 92.6% (87%–96%), 97% (93%–99%), and 99.3% (96%–100%). Stool Xpert Ultra had similar sensitivity (22.6%) to stool mNGS considering all samples tested. In a head-to-head comparison, stool mNGS had lower sensitivity than stool Xpert Ultra (38.5% vs 53.8%, difference −15.3%, 95% CI 14–68 vs 25–81). mNGS utilized genes encoding rRNA, virulence proteins, and membrane proteins not targeted in current PCR-based platforms. Conclusions: Metagenomic sequencing of stool DNA did not increase sensitivity of TB detection, but identified novel targets for molecular testing that may support development of more sensitive tests.

Gut microbiota often influence host defense against infection, but this relationship is incompletely understood in wild bumblebees. These critical pollinators host a characteristic core gut microbiota, yet field studies have offered conflicting insights into its association with Crithidia bombi , a prevalent trypanosomid parasite. To address this gap in our knowledge, we conducted an 3 year field survey, profiling the gut microbiota of 638 bumblebees from 9 sympatric species across diverse sites in Maine using 16S rRNA amplicon sequencing and qPCR for C. bombi detection. We confirmed a robust core bumblebee microbiota, identifying novel host-specific phylogenetic associations of bacterial amplicon sequence variants even among closely related host species. C. bombi infection was common and showed significant seasonal increases. We also found spatial variation, with higher prevalence in coastal regions. Crucially, increasing C. bombi infection load was consistently associated with microbiome dysbiosis. This dysbiosis was characterized by a depletion of core bumblebee-associated microbial taxa, notably Apibacter and Gilliamella (previously shown to be protective), and a corresponding increase in opportunistic, environmentally derived microbes like Entomomonas . While the core microbiota’s association with initial pathogen transmission appears minor, its depletion in severe infections strongly supports a correlation to host health in wild bumblebees. IMPORTANCE: The community of microorganisms in close association with an animal, its microbiota, can be important to its health. Understanding how microbiota composition relates to health and disease is an important goal with broad potential implications. Like most animals, bumblebees have a characteristic core gut microbiota. We have conducted a broad survey of bumblebees over 3 years to examine the interactions of microbiota composition with infection by an endemic trypanosomatid parasite. We found that the relative abundances of core microbes were inversely related to infection load, and that increased pathogen load was associated with the prevalence of novel microbes. These results are evidence of strong associations between bumblebees and their core microbiota and suggest a role in providing resistance to severe parasitism.

Low-abundance members of microbial communities are difficult to study in their native habitats, including Escherichia coli, a minor but common inhabitant of the gastrointestinal tract, and key opportunistic pathogen of the urinary tract. While multi-omic analyses have detailed interactions between uropathogenic Escherichia coli (UPEC) and the bladder mediating urinary tract infection (UTI), little is known about UPEC in its pre-infection reservoir, the gastrointestinal tract, partly due to its low relative abundance (E. coli, we develop E. coli PanSelect, which uses probes designed to specifically capture E. coli’s broad pangenome. We demonstrate its ability to enrich diverse E. coli by orders of magnitude, in a mock community and in human stool from a study investigating recurrent UTI (rUTI). Comparisons of transcriptomes between gut E. coli of women with and without history of rUTI suggest rUTI gut E. coli are responding to increased oxygen and nitrate, suggestive of mucosal inflammation, which may have implications for recurrent disease. E. coli PanSelect is well suited for investigations of in vivo E. coli biology in other low-abundance environments, and the framework described here has broad applicability to other diverse, low-abundance organisms.

Low-abundance members of microbial communities are difficult to study in their native habitats. This includes Escherichia coli , a minor, but common inhabitant of the gastrointestinal tract and opportunistic pathogen, including of the urinary tract, where it is the primary pathogen. While multi-omic analyses have detailed critical interactions between uropathogenic Escherichia coli (UPEC) and the bladder that mediate UTI outcome, comparatively little is known about UPEC in its pre-infection reservoir, partly due to its low abundance there (E. coli in gastrointestinal communities, we developed E. coli PanSelect which uses a set of probes designed to specifically recognize and capture E. coli ’s broad pangenome from sequencing libraries. We demonstrated the ability of E. coli PanSelect to enrich, by orders of magnitude, sequencing data from diverse E. coli using a mock community and a set of human stool samples collected as part of a cohort study investigating drivers of recurrent urinary tract infections (rUTI). Comparisons of genomes and transcriptomes between E. coli residing in the gastrointestinal tracts of women with and without a history of rUTI suggest that rUTI gut E. coli are responding to increased levels of oxygen and nitrate, suggestive of mucosal inflammation, which may have implications for recurrent disease. E. coli PanSelect is well suited for investigations of native in vivo biology of E. coli in other environments where it is at low relative abundance, and the framework described here has broad applicability to other highly diverse, low abundance organisms.

The microbiome is increasingly recognized for its complex relationship with host fitness. Bumblebees are host to a characteristic gut microbiome community that is derived and reinforced through social contact between individuals. The bumblebee microbiome is species-poor, and primarily composed from a small number of core taxa that are associated with the greater tribe of corbiculate bees. Experimental findings support a role for the core bumblebee microbiome in resistance to severe infections by a common trypanosomal parasite, Crithidia bombi . However, most studies have been small in scale, often considering just one or two bumblebee species, or making use of commercially-reared bees. To better understand the microbiome diversity of wild populations, we have deeply sampled field populations of ten sympatric species found throughout central and down east Maine in a three-year microbiome field survey. We have used 16S amplicon sequencing to produce microbiome community profiles, and qPCR to screen samples for infections by Crithidia bombi . The breadth of our dataset has enabled us to test for seasonal and interspecific trends in the microbiome community. Controlling for these external sources of variation, we have identified microbial factors associated with infection and parasite load that support the role of the core microbiome in resistance to severe infection.