Within-host evolution of a gut pathobiont facilitates liver translocation.

Gut commensal bacteria with the ability to translocate across the intestinal barrier can drive the development of diverse immune-mediated diseases1-4. However, the key factors that dictate bacterial translocation remain unclear. Recent studies have revealed that gut microbiota strains can adapt and evolve throughout the lifetime of the host5-9, raising the possibility that changes in individual commensal bacteria themselves over time may affect their propensity to elicit inflammatory disease. Here we show that within-host evolution of the model gut pathobiont Enterococcus gallinarum facilitates bacterial translocation and initiation of inflammation. Using a combination of in vivo experimental evolution and comparative genomics, we found that E. gallinarum diverges into independent lineages adapted to colonize either luminal or mucosal niches in the gut. Compared with ancestral and luminal E. gallinarum, mucosally adapted strains evade detection and clearance by the immune system, exhibit increased translocation to and survival within the mesenteric lymph nodes and liver, and induce increased intestinal and hepatic inflammation. Mechanistically, these changes in bacterial behaviour are associated with non-synonymous mutations or insertion-deletions in defined regulatory genes in E. gallinarum, altered microbial gene expression programs and remodelled cell wall structures. Lactobacillus reuteri also exhibited broadly similar patterns of divergent evolution and enhanced immune evasion in a monocolonization-based model of within-host evolution. Overall, these studies define within-host evolution as a critical regulator of commensal pathogenicity that provides a unique source of stochasticity in the development and progression of microbiota-driven disease.

Development of mass allelic exchange, a technique to enable sexual genetics in Escherichia coli.

Despite dramatic advances in genomics, connecting genotypes to phenotypes is still challenging. Sexual genetics combined with linkage analysis is a powerful solution to this problem but generally unavailable in bacteria. We build upon a strong negative selection system to invent mass allelic exchange (MAE), which enables hybridization of arbitrary (including pathogenic) strains of Escherichia coli. MAE reimplements the natural phenomenon of random cross-overs, enabling classical linkage analysis. We demonstrate the utility of MAE with virulence-related gain-of-function screens, discovering that transfer of a single operon from a uropathogenic strain is sufficient for enabling a commensal E. coli to form large intracellular bacterial collections within bladder epithelial cells. MAE thus enables assaying natural allelic variation in E. coli (and potentially other bacteria), complementing existing loss-of-function genomic techniques.

A set of powerful negative selection systems for unmodified Enterobacteriaceae.

Creation of defined genetic mutations is a powerful method for dissecting mechanisms of bacterial disease; however, many genetic tools are only developed for laboratory strains. We have designed a modular and general negative selection strategy based on inducible toxins that provides high selection stringency in clinical Escherichia coli and Salmonella isolates. No strain- or species-specific optimization is needed, yet this system achieves better selection stringency than all previously reported negative selection systems usable in unmodified E. coli strains. The high stringency enables use of negative instead of positive selection in phage-mediated generalized transduction and also allows transfer of alleles between arbitrary strains of E. coli without requiring phage. The modular design should also allow further extension to other bacteria. This negative selection system thus overcomes disadvantages of existing systems, enabling definitive genetic experiments in both lab and clinical isolates of E. coli and other Enterobacteriaceae.

Purine and carbohydrate availability drive Enterococcus faecalis fitness during wound and urinary tract infections.

IMPORTANCE: Although E. faecalis is a common wound pathogen, its pathogenic mechanisms during wound infection are unexplored. Here, combining a mouse wound infection model with in vivo transposon and RNA sequencing approaches, we identified the E. faecalis purine biosynthetic pathway and galactose/mannose MptABCD phosphotransferase system as essential for E. faecalis acute replication and persistence during wound infection, respectively. The essentiality of purine biosynthesis and the MptABCD PTS is driven by the consumption of purine metabolites by E. faecalis during acute replication and changing carbohydrate availability during the course of wound infection. Overall, our findings reveal the importance of the wound microenvironment in E. faecalis wound pathogenesis and how these metabolic pathways can be targeted to better control wound infections.

Interspecies commensal interactions have nonlinear impacts on host immunity.

The impacts of individual commensal microbes on immunity and disease can differ dramatically depending on the surrounding microbial context; however, the specific bacterial combinations that dictate divergent immunological outcomes remain largely undefined. Here, we characterize an immunostimulatory Allobaculum species from an inflammatory bowel disease patient that exacerbates colitis in gnotobiotic mice. Allobaculum inversely associates with the taxonomically divergent immunostimulatory species Akkermansia muciniphila in human-microbiota-associated mice and human cohorts. Co-colonization with A. muciniphila ameliorates Allobaculum-induced intestinal epithelial cell activation and colitis in mice, whereas Allobaculum blunts the A.muciniphila-specific systemic antibody response and reprograms the immunological milieu in mesenteric lymph nodes by blocking A.muciniphila-induced dendritic cell activation and T cell expansion. These studies thus identify a pairwise reciprocal interaction between human gut bacteria that dictates divergent immunological outcomes. Furthermore, they establish a generalizable framework to define the contextual cues contributing to the "incomplete penetrance" of microbial impacts on human disease.

Complete Genome Sequence of the Original Escherichia coli Isolate, Strain NCTC86.

Escherichia coli is the most well-studied bacterium and a common colonizer of the lower mammalian gastrointestinal tract. We report here the complete genome sequence of the original Escherichia coli isolate, strain NCTC86, which was described by Theodor Escherich, for whom the genus is named.

Application and Optimization of relE as a Negative Selection Marker for Making Definitive Genetic Constructs in Uropathogenic Escherichia coli.

Studies of Uropathogenic Escherichia coli (UPEC) pathogenesis have relied heavily on genetic manipulation to understand virulence factors. We applied a recently reported positive-negative selection system to create a series of unmarked, scarless FimH mutants that show identical phenotypes to previously reported marked FimH mutants; these are now improved versions useful for definitive assignment of phenotypes to FimH mutations. We also increased the efficiency of this system by designing new primer sites, which should further improve the efficiency and convenience of using negative selection in UTI89, other UPEC, and other Enterobacteriaceae.