Commensal-pathogen dynamics structure disease outcomes during Clostridioides difficile colonization.

Gastrointestinal colonization by Clostridioides difficile is common in healthcare settings and ranges in clinical presentation from asymptomatic carriage to lethal C. difficile infection (CDI). We used a systems biology approach to investigate why patients colonized with C. difficile have a range of outcomes. Microbiota-humanization of germ-free mice with fecal samples from toxigenic C. difficile carriers revealed a spectrum of virulence among clade 1 lineages and identified commensal Blautia associated with markers of non-pathogenic colonization. Using gnotobiotic mice engrafted with defined human microbiota, we observed strain-specific CDI severity across clade 1 strains. Yet, mice engrafted with a higher diversity community were protected from severe disease across all strains without suppression of C. difficile colonization. These results indicate that when colonization resistance has been breached without overt infection, commensals can attenuate a diversity of virulent strains without inhibiting pathogen colonization, providing insight into determinants of stable C. difficile carriage.

BoGH13ASus from Bacteroides ovatus represents a novel α-amylase used for Bacteroides starch breakdown in the human gut.

Members of the Bacteroidetes phylum in the human colon deploy an extensive number of proteins to capture and degrade polysaccharides. Operons devoted to glycan breakdown and uptake are termed polysaccharide utilization loci or PUL. The starch utilization system (Sus) is one such PUL and was initially described in Bacteroides thetaiotaomicron (Bt). BtSus is highly conserved across many species, except for its extracellular α-amylase, SusG. In this work, we show that the Bacteroides ovatus (Bo) extracellular α-amylase, BoGH13ASus, is distinguished from SusG in its evolutionary origin and its domain architecture and by being the most prevalent form in Bacteroidetes Sus. BoGH13ASus is the founding member of both a novel subfamily in the glycoside hydrolase family 13, GH13_47, and a novel carbohydrate-binding module, CBM98. The BoGH13ASus CBM98-CBM48-GH13_47 architecture differs from the CBM58 embedded within the GH13_36 of SusG. These domains adopt a distinct spatial orientation and invoke a different association with the outer membrane. The BoCBM98 binding site is required for Bo growth on polysaccharides and optimal enzymatic degradation thereof. Finally, the BoGH13ASus structure features bound Ca2+ and Mn2+ ions, the latter of which is novel for an α-amylase. Little is known about the impact of Mn2+ on gut bacterial function, much less on polysaccharide consumption, but Mn2+ addition to Bt expressing BoGH13ASus specifically enhances growth on starch. Further understanding of bacterial starch degradation signatures will enable more tailored prebiotic and pharmaceutical approaches that increase starch flux to the gut.

Microbiome-targeting therapies in the neonatal intensive care unit: safety and efficacy.

Microbiome-targeting therapies have received great attention as approaches to prevent disease in infants born preterm, but their safety and efficacy remain uncertain. Here we summarize the existing literature, focusing on recent meta-analyses and systematic reviews that evaluate the performance of probiotics, prebiotics, and/or synbiotics in clinical trials and studies, emphasizing interventions for which the primary or secondary outcomes were prevention of necrotizing enterocolitis, late-onset sepsis, feeding intolerance, and/or reduction in hospitalization length or all-cause mortality. Current evidence suggests that probiotics and prebiotics are largely safe but conclusions regarding their effectiveness in the neonatal intensive care unit have been mixed. To address this ambiguity, we evaluated publications that collectively support benefits of probiotics with moderate to high certainty evidence in a recent comprehensive network meta-analysis, highlighting limitations in these trials that make it difficult to support with confidence the routine, universal administration of probiotics to preterm infants.

Gut pathogen colonization precedes bloodstream infection in the neonatal intensive care unit.

Bacterial bloodstream infections (BSIs) resulting in late-onset sepsis affect up to half of extremely preterm infants and have substantial morbidity and mortality. Bacterial species associated with BSIs in neonatal intensive care units (NICUs) commonly colonize the preterm infant gut microbiome. Accordingly, we hypothesized that the gut microbiome is a reservoir of BSI-causing pathogenic strains that increase in abundance before BSI onset. We analyzed 550 previously published fecal metagenomes from 115 hospitalized neonates and found that recent ampicillin, gentamicin, or vancomycin exposure was associated with increased abundance of Enterobacteriaceae and Enterococcaceae in infant guts. We then performed shotgun metagenomic sequencing on 462 longitudinal fecal samples from 19 preterm infants (cases) with BSI and 37 non-BSI controls, along with whole-genome sequencing of the BSI isolates. Infants with BSI caused by Enterobacteriaceae were more likely than infants with BSI caused by other organisms to have had ampicillin, gentamicin, or vancomycin exposure in the 10 days before BSI. Relative to controls, gut microbiomes of cases had increased relative abundance of the BSI-causing species and clustered by Bray-Curtis dissimilarity according to BSI pathogen. We demonstrated that 11 of 19 (58%) of gut microbiomes before BSI, and 15 of 19 (79%) of gut microbiomes at any time, harbored the BSI isolate with fewer than 20 genomic substitutions. Last, BSI strains from the Enterobacteriaceae and Enterococcaceae families were detected in multiple infants, indicating BSI-strain transmission. Our findings support future studies to evaluate BSI risk prediction strategies based on gut microbiome abundance in hospitalized preterm infants.

Single-molecule dynamics of surface lipoproteins in bacteroides indicate similarities and cooperativity.

The gut microbiota comprises hundreds of species with a composition shaped by the available glycans. The well-studied starch utilization system (Sus) is a prototype for glycan uptake in the human gut bacterium Bacteroides thetaiotaomicron (Bt). Each Sus-like system includes outer-membrane proteins, which translocate glycan into the periplasm, and one or more cell-surface glycoside hydrolases, which break down a specific (cognate) polymer substrate. Although the molecular mechanisms of the Sus system are known, how the Sus and Sus-like proteins cooperate remains elusive. Previously, we used single-molecule and super-resolution fluorescence microscopy to show that SusG is mobile on the outer membrane and slows down in the presence of starch. Here, we compare the dynamics of three glycoside hydrolases: SusG, Bt4668, and Bt1760, which target starch, galactan, and levan, respectively. We characterized the diffusion of each surface hydrolase in the presence of its cognate glycan and found that all three enzymes are mostly immobile in the presence of the polysaccharide, consistent with carbohydrate binding. Moreover, experiments in glucose versus oligosaccharides suggest that the enzyme dynamics depend on their expression level. Furthermore, we characterized enzyme diffusion in a mixture of glycans and found that noncognate polysaccharides modify the dynamics of SusG and Bt1760 but not Bt4668. We investigated these systems with polysaccharide mixtures and genetic knockouts and found that noncognate polysaccharides modify hydrolase dynamics through some combination of nonspecific protein interactions and downregulation of the hydrolase. Overall, these experiments extend our understanding of how Sus-like lipoprotein dynamics can be modified by changing carbohydrate conditions and the expression level of the enzyme.