Enteric nervous system modulation of luminal pH modifies the microbial environment to promote intestinal health.

The enteric nervous system (ENS) controls many aspects of intestinal homeostasis, including parameters that shape the habitat of microbial residents. Previously we showed that zebrafish lacking an ENS, due to deficiency of the sox10 gene, develop intestinal inflammation and bacterial dysbiosis, with an expansion of proinflammatory Vibrio strains. To understand the primary defects resulting in dysbiosis in sox10 mutants, we investigated how the ENS shapes the intestinal environment in the absence of microbiota and associated inflammatory responses. We found that intestinal transit, intestinal permeability, and luminal pH regulation are all aberrant in sox10 mutants, independent of microbially induced inflammation. Treatment with the proton pump inhibitor, omeprazole, corrected the more acidic luminal pH of sox10 mutants to wild type levels. Omeprazole treatment also prevented overabundance of Vibrio and ameliorated inflammation in sox10 mutant intestines. Treatment with the carbonic anhydrase inhibitor, acetazolamide, caused wild type luminal pH to become more acidic, and increased both Vibrio abundance and intestinal inflammation. We conclude that a primary function of the ENS is to regulate luminal pH, which plays a critical role in shaping the resident microbial community and regulating intestinal inflammation.

Host-emitted amino acid cues regulate bacterial chemokinesis to enhance colonization.

Animal microbiomes are assembled predominantly from environmental microbes, yet the mechanisms by which individual symbionts regulate their transmission into hosts remain underexplored. By tracking the experimental evolution of Aeromonas veronii in gnotobiotic zebrafish, we identify bacterial traits promoting host colonization. Multiple independently evolved isolates with increased immigration harbored mutations in a gene we named sensor of proline diguanylate cyclase enzyme (SpdE) based on structural, biochemical, and phenotypic evidence that SpdE encodes an amino-acid-sensing diguanylate cyclase. SpdE detects free proline and to a lesser extent valine and isoleucine, resulting in reduced production of intracellular c-di-GMP, a second messenger controlling bacterial motility. Indeed, SpdE binding to amino acids increased bacterial motility and host colonization. Hosts serve as sources of SpdE-detected amino acids, with levels varying based on microbial colonization status. Our work demonstrates that bacteria use chemically regulated motility, or chemokinesis, to sense host-emitted cues that trigger active immigration into hosts.

Phenotypic Parallelism during Experimental Adaptation of a Free-Living Bacterium to the Zebrafish Gut.

Although animals encounter a plethora of bacterial species throughout their lives, only a subset colonize vertebrate digestive tracts, and these bacteria can profoundly influence the health and development of their animal hosts. However, our understanding of how bacteria initiate symbioses with animal hosts remains underexplored, and this process is central to the assembly and function of gut bacterial communities. Therefore, we used experimental evolution to study a free-living bacterium as it adapts to a novel vertebrate host by serially passaging replicate populations of Shewanella oneidensis through the intestines of larval zebrafish (Danio rerio). After approximately 200 bacterial generations, isolates from evolved populations improved their ability to colonize larval zebrafish during competition against their unpassaged ancestor. Genome sequencing revealed unique sets of mutations in the two evolved isolates exhibiting the highest mean competitive fitness. One isolate exhibited increased swimming motility and decreased biofilm formation compared to the ancestor, and we identified a missense mutation in the mannose-sensitive hemagglutinin pilus operon that is sufficient to increase fitness and reproduce these phenotypes. The second isolate exhibited enhanced swimming motility but unchanged biofilm formation, and here the genetic basis for adaptation is less clear. These parallel enhancements in motility and fitness resemble the behavior of a closely related Shewanella strain previously isolated from larval zebrafish and suggest phenotypic convergence with this isolate. Our results demonstrate that adaptation to the zebrafish gut is complex, with multiple evolutionary pathways capable of improving colonization, but that motility plays an important role during the onset of host association.IMPORTANCE Although animals encounter many bacterial species throughout their lives, only a subset colonize vertebrate digestive tracts, and these bacteria can profoundly influence the health and development of their animal hosts. We used experimental evolution to study a free-living bacterium as it adapts to a novel vertebrate host by serially passaging replicate populations of Shewanella oneidensis through the intestines of larval zebrafish (Danio rerio). Our results demonstrate that adaptation to the zebrafish gut is complex, with multiple evolutionary pathways capable of improving colonization, but that motility plays an important role during the onset of host association.

Scales of persistence: transmission and the microbiome.

Historically microbiomes have been studied on the scale of the individual host, giving little consideration for the role of extra-host microbial populations in microbiome assembly. However, work in recent years has brought to light the importance of inter-host transmission and its influence on microbiome composition and dynamics. We now appreciate that microbiomes do not exist in isolation, but exchange constituents with the microbial communities of other hosts and the environment. Moving forward, fully understanding the role of transmission in microbiome assembly and dynamics will require a high-resolution view of the colonization and persistence patterns of particular microbial lineages (i.e. strains) across individuals and the environment. Yet, accomplishing this level of resolution will be an immense challenge, requiring improved sampling and bioinformatics approaches as well as employment of tractable experimental models. Insight gained from these investigations will contribute to our understanding of microbiome composition and variation, and lead to improved strategies for modulating microbiomes to improve human health.

Experimental bacterial adaptation to the zebrafish gut reveals a primary role for immigration.

All animals live in intimate association with microorganisms that profoundly influence their health and development, yet the traits that allow microorganisms to establish and maintain host associations are not well understood. To date, most investigations aimed at identifying traits required for host association have focused on intrahost niches. Consequently, little is known about the relative contribution of extrahost factors such as environmental growth and survival and immigration into hosts from the external environment, as promoters of host association. To address this, we developed a tractable experimental evolution system that investigates both intra- and extrahost factors contributing to bacterial adaptation to the vertebrate gut. We passaged replicate lines of a zebrafish bacterial isolate, Aeromonas veronii, through populations of germ-free larval zebrafish (Danio rerio), each time using gut-associated Aeromonas populations to inoculate the aquatic environment of the next zebrafish population. We observed rapid increased adaptation to the host in all replicate lines. The initial adaptations present in early-evolved isolates did not increase intrahost fitness but rather enhanced both immigration from the environment and interhost transmission. Only in later-evolved isolates did we find evidence for intrahost-specific adaptations, as demonstrated by comparing their competitive fitness in the host genotype to which they evolved to that in a different genotype. Our results show how selection for bacterial transmission between hosts and their environment can shape bacterial-host association. This work illuminates the nature of selective forces present in host-microbe systems and reveals specific mechanisms of increased host association. Furthermore, our findings demonstrate that the entire host-microbe-environment system must be considered when identifying microbial traits that contribute to host adaptation.

MiniBioReactor Arrays (MBRAs) as a Tool for Studying C. difficile Physiology in the Presence of a Complex Community.

The commensal microbiome plays an important role in the dynamics of Clostridium difficile infection. In this chapter, we describe minibioreactor arrays (MBRAs), an in vitro cultivation system that we developed that allows for C. difficile physiology to be assayed in the presence of complex fecal microbial communities. The small size of the bioreactors within the MBRAs allows for dozens of reactors to be run simultaneously and therefore several different variables can be tested with limited time and cost. When coupled with experiments in animal models of C. difficile infection, MBRAs can provide important insights into C. difficile physiology and pathogenesis.

Cultivation of stable, reproducible microbial communities from different fecal donors using minibioreactor arrays (MBRAs).

BACKGROUND: Continuous-flow culture models are one tool for studying complex interactions between members of human fecal microbiotas because they allow studies to be completed during an extended period of time under conditions where pH, nutrient availability, and washout of waste products and dead cells can be controlled. Because many of the existing well-validated continuous-flow models are large and complex, we were interested in developing a simpler continuous-flow system that would allow microbial community dynamics to be examined in higher throughput while still maintaining complex microbial communities. To this end, we developed minibioreactor arrays (MBRAs), small volume bioreactors (15 ml) that allow simultaneous cultivation of up to 48 microbial communities in a single anaerobic chamber. RESULTS: We used MBRA to characterize the microbial community dynamics of replicate reactors inoculated from three different human fecal donors and reactors seeded with feces pooled from these three donors. We found that MBRA could be used to efficiently cultivate complex microbial communities that were a subset of the initial fecal inoculum (15-25 % of fecal OTUs initially observed). After an initial acclimation period of approximately 1 week, communities in each reactor stabilized and exhibited day-to-day variation similar to that observed in stable mouse fecal communities. Replicate reactors were predominately populated by shared core microbial communities; variation between replicate reactors was primarily driven by shifts in abundance of shared operational taxonomic units (OTUs). Consistent with differences between fecal donors, MBRA communities present in reactors seeded with different fecal samples had distinct composition and structure. CONCLUSIONS: From these analyses, we conclude that MBRAs can be used to cultivate communities that recapitulate key features of human fecal communities and are a useful tool to facilitate higher-throughput studies of the dynamics of these communities.

Epidemic Clostridium difficile strains demonstrate increased competitive fitness compared to nonepidemic isolates.

Clostridium difficile infection is the most common cause of severe cases of antibiotic-associated diarrhea (AAD) and is a significant health burden. Recent increases in the rate of C. difficile infection have paralleled the emergence of a specific phylogenetic clade of C. difficile strains (ribotype 027; North American pulsed-field electrophoresis 1 [NAP1]; restriction endonuclease analysis [REA] group BI). Initial reports indicated that ribotype 027 strains were associated with increased morbidity and mortality and might be hypervirulent. Although subsequent work has raised some doubt as to whether ribotype 027 strains are hypervirulent, the strains are considered epidemic isolates that have caused severe outbreaks across the globe. We hypothesized that one factor that could lead to the increased prevalence of ribotype 027 strains would be if these strains had increased competitive fitness compared to strains of other ribotypes. We developed a moderate-throughput in vitro model of C. difficile infection and used it to test competition between four ribotype 027 clinical isolates and clinical isolates of four other ribotypes (001, 002, 014, and 053). We found that ribotype 027 strains outcompeted the strains of other ribotypes. A similar competitive advantage was observed when two ribotype pairs were competed in a mouse model of C. difficile infection. Based upon these results, we conclude that one possible mechanism through which ribotype 027 strains have caused outbreaks worldwide is their increased ability to compete in the presence of a complex microbiota.