Contribution of bacterial and host factors to pathogen "blooming" in a gnotobiotic mouse model for Salmonella enterica serovar Typhimurium-induced enterocolitis.

Inflammation has a pronounced impact on the intestinal ecosystem by driving an expansion of facultative anaerobic bacteria at the cost of obligate anaerobic microbiota. This pathogen "blooming" is also a hallmark of enteric Salmonella enterica serovar Typhimurium (S. Tm) infection. Here, we analyzed the contribution of bacterial and host factors to S. Tm "blooming" in a gnotobiotic mouse model for S. Tm-induced enterocolitis. Mice colonized with the Oligo-Mouse-Microbiota (OMM12), a minimal bacterial community, develop fulminant colitis by day 4 after oral infection with wild-type S. Tm but not with an avirulent mutant. Inflammation leads to a pronounced reduction in overall intestinal bacterial loads, distinct microbial community shifts, and pathogen blooming (relative abundance >50%). S. Tm mutants attenuated in inducing gut inflammation generally elicit less pronounced microbiota shifts and reduction in total bacterial loads. In contrast, S. Tm mutants in nitrate respiration, salmochelin production, and ethanolamine utilization induced strong inflammation and S. Tm "blooming." Therefore, individual Salmonella-specific inflammation-fitness factors seem to be of minor importance for competition against this minimal microbiota in the inflamed gut. Finally, we show that antibody-mediated neutrophil depletion normalized gut microbiota loads but not intestinal inflammation or microbiota shifts. This suggests that neutrophils equally reduce pathogen and commensal bacterial loads in the inflamed gut.

Ecophysiology and interactions of a taurine-respiring bacterium in the mouse gut.

Taurine-respiring gut bacteria produce H2S with ambivalent impact on host health. We report the isolation and ecophysiological characterization of a taurine-respiring mouse gut bacterium. Taurinivorans muris strain LT0009 represents a new widespread species that differs from the human gut sulfidogen Bilophila wadsworthia in its sulfur metabolism pathways and host distribution. T. muris specializes in taurine respiration in vivo, seemingly unaffected by mouse diet and genotype, but is dependent on other bacteria for release of taurine from bile acids. Colonization of T. muris in gnotobiotic mice increased deconjugation of taurine-conjugated bile acids and transcriptional activity of a sulfur metabolism gene-encoding prophage in other commensals, and slightly decreased the abundance of Salmonella enterica, which showed reduced expression of galactonate catabolism genes. Re-analysis of metagenome data from a previous study further suggested that T. muris can contribute to protection against pathogens by the commensal mouse gut microbiota. Together, we show the realized physiological niche of a key murine gut sulfidogen and its interactions with selected gut microbiota members.

Pulsed antibiotic treatments of gnotobiotic mice manifest in complex bacterial community dynamics and resistance effects.

Bacteria can evolve to withstand a wide range of antibiotics (ABs) by using various resistance mechanisms. How ABs affect the ecology of the gut microbiome is still poorly understood. We investigated strain-specific responses and evolution during repeated AB perturbations by three clinically relevant ABs, using gnotobiotic mice colonized with a synthetic bacterial community (oligo-mouse-microbiota). Over 80 days, we observed resilience effects at the strain and community levels, and we found that they were correlated with modulations of the estimated growth rate and levels of prophage induction as determined from metagenomics data. Moreover, we tracked mutational changes in the bacterial populations, and this uncovered clonal expansion and contraction of haplotypes and selection of putative AB resistance-conferring SNPs. We functionally verified these mutations via reisolation of clones with increased minimum inhibitory concentration (MIC) of ciprofloxacin and tetracycline from evolved communities. This demonstrates that host-associated microbial communities employ various mechanisms to respond to selective pressures that maintain community stability.

Enhanced cultured diversity of the mouse gut microbiota enables custom-made synthetic communities.

Microbiome research needs comprehensive repositories of cultured bacteria from the intestine of mammalian hosts. We expanded the mouse intestinal bacterial collection (www.dsmz.de/miBC) to 212 strains, all publicly available and taxonomically described. This includes strain-level diversity, small-sized bacteria, and previously undescribed taxa (one family, 10 genera, and 39 species). This collection enabled metagenome-educated prediction of synthetic communities (SYNs) that capture key functional differences between microbiomes, notably identifying communities associated with either resistance or susceptibility to DSS-induced colitis. Additionally, nine species were used to amend the Oligo-Mouse Microbiota (OMM)12 model, yielding the OMM19.1 model. The added strains compensated for phenotype differences between OMM12 and specific pathogen-free mice, including body composition and immune cells in the intestine and associated lymphoid tissues. Ready-to-use OMM stocks are available for future studies. In conclusion, this work improves our knowledge of gut microbiota diversity in mice and enables functional studies via the modular use of isolates.

Unravelling the collateral damage of antibiotics on gut bacteria.

Antibiotics are used to fight pathogens but also target commensal bacteria, disturbing the composition of gut microbiota and causing dysbiosis and disease1. Despite this well-known collateral damage, the activity spectrum of different antibiotic classes on gut bacteria remains poorly characterized. Here we characterize further 144 antibiotics from a previous screen of more than 1,000 drugs on 38 representative human gut microbiome species2. Antibiotic classes exhibited distinct inhibition spectra, including generation dependence for quinolones and phylogeny independence for ß-lactams. Macrolides and tetracyclines, both prototypic bacteriostatic protein synthesis inhibitors, inhibited nearly all commensals tested but also killed several species. Killed bacteria were more readily eliminated from in vitro communities than those inhibited. This species-specific killing activity challenges the long-standing distinction between bactericidal and bacteriostatic antibiotic classes and provides a possible explanation for the strong effect of macrolides on animal3-5 and human6,7 gut microbiomes. To mitigate this collateral damage of macrolides and tetracyclines, we screened for drugs that specifically antagonized the antibiotic activity against abundant Bacteroides species but not against relevant pathogens. Such antidotes selectively protected Bacteroides species from erythromycin treatment in human-stool-derived communities and gnotobiotic mice. These findings illluminate the activity spectra of antibiotics in commensal bacteria and suggest strategies to circumvent their adverse effects on the gut microbiota.

E. coli enhance colonization resistance against Salmonella Typhimurium by competing for galactitol, a context-dependent limiting carbon source.

The composition of intrinsic microbial communities determines if invading pathogens will find a suitable niche for colonization and cause infection or be eliminated. Here, we investigate how commensal E. coli mediate colonization resistance (CR) against Salmonella Typhimurium (S. Tm). Using synthetic bacterial communities, we show that the capacity of E. coli Mt1B1 to block S. Tm colonization depends on the microbial context. In an infection-permissive context, E. coli utilized a high diversity of carbon sources and was unable to block S. Tm invasion. In mice that were stably colonized by twelve phylogenetically diverse murine gut bacteria (OMM12), establishing a protective context, E. coli depleted galactitol, a substrate otherwise fueling S. Tm colonization. Here, Lachnospiraceae, capable of consuming C5 and C6 sugars, critically contributed to CR. We propose that E. coli provides CR by depleting a limited carbon source when in a microbial community adept at removing simple sugars from the intestine.

Monitoring and contamination incidence of gnotobiotic experiments performed in microisolator cages.

With the increased interest in the microbiome research, gnotobiotic animals and techniques emerged again as valuable tools to investigate functional effects of host-microbe and microbe-microbe interactions. The increased demand for gnotobiotic experiments has resulted in the greater need for housing systems for short-term maintenance of gnotobiotic animals. During the last six years, the gnotobiotic facility of the Hannover Medical School has worked intensively with different housing systems for gnotobiotic animals. Here, we report our experience in handling, contamination incidence, and monitoring strategies that we apply for controlling gnotobiotic experiments. From our experience, the risk of introducing contaminants to animals housed in microisolator cages is higher than in isolators. However, with strict operating protocols, the contamination rate in these systems can be minimized. In addition to spore-forming bacteria and fungi from the environment, spore-forming bacteria from defined bacterial communities used in experiments represent the major risk for contamination of gnotobiotic experiments performed in microisolator cages. The presence/absence of contaminants in germ-free animals can be easily monitored by preparation of wet mounts and Gram staining of fecal samples. Contaminants in animals colonized with specific microorganisms need to be tracked with methods such as next-generation sequencing. However, when using PCR-based methods it is important to consider that relatively small amounts of bacterial DNA detected likely originates from food, bedding, or reagents and is not to be interpreted as true contamination.

The gut bacterium Extibacter muris produces secondary bile acids and influences liver physiology in gnotobiotic mice.

Extibacter muris is a newly described mouse gut bacterium which metabolizes cholic acid (CA) to deoxycholic acid (DCA) via 7α-dehydroxylation. Although bile acids influence metabolic and inflammatory responses, few in vivo models exist for studying their metabolism and impact on the host. Mice were colonized from birth with the simplified community Oligo-MM12 with or without E. muris. As the metabolism of bile acids is known to affect lipid homeostasis, mice were fed either a low- or high-fat diet for eight weeks before sampling and analyses targeting the gut and liver. Multiple Oligo-MM12 strains were capable of deconjugating primary bile acids in vitro. E. muris produced DCA from CA either as pure compound or in mouse bile. This production was inducible by CA in vitro. Ursodeoxycholic, chenodeoxycholic, and ß-muricholic acid were not metabolized under the conditions tested. All gnotobiotic mice were stably colonized with E. muris, which showed higher relative abundances after HF diet feeding. The presence of E. muris had minor, diet-dependent effects on Oligo-MM12 communities. The secondary bile acids DCA and surprisingly LCA and their taurine conjugates were detected exclusively in E. muris-colonized mice. E. muris colonization did not influence body weight, white adipose tissue mass, liver histopathology, hepatic aspartate aminotransferase, or blood levels of cholesterol, insulin, and paralytic peptide (PP). However, proteomics revealed shifts in hepatic pathways involved in amino acid, glucose, lipid, energy, and drug metabolism in E. muris-colonized mice. Liver fatty acid composition was substantially altered by dietary fat but not by E. muris.In summary, E. muris stably colonized the gut of mice harboring a simplified community and produced secondary bile acids, which affected proteomes in the liver. This new gnotobiotic mouse model can now be used to study the pathophysiological role of secondary bile acids in vivo.

The Spatial Heterogeneity of the Gut Limits Predation and Fosters Coexistence of Bacteria and Bacteriophages.

The ecological dynamics underlying the coexistence between antagonistic populations of bacteria and their viruses, bacteriophages (phages), in the mammalian gut microbiota remain poorly understood. We challenged a murine synthetic bacterial community with phages to study the factors allowing phages-bacteria coexistence. Coexistence was not dependent on the development of phage-resistant clones nor on the ability of phages to extend their host range. Instead, our data suggest that phage-inaccessible sites in the mucosa serve as a spatial refuge for bacteria. From there, bacteria disseminate in the gut lumen where they are predated by luminal phages fostering the presence of intestinal phage populations. The heterogeneous biogeography of microbes contributes to the long-term coexistence of phages with phage-susceptible bacteria. This observation could explain the persistence of intestinal phages in humans as well as the low efficiency of oral phage therapy against enteric pathogens in animal models and clinical trials.

Composition of the Intestinal Microbiota Determines the Outcome of Virus-Triggered Colitis in Mice.

The intestinal microbiota is a complex ecosystem implicated in host health and disease. Inflammatory bowel disease (IBD) is a multifactorial chronic disorder of the gastrointestinal mucosa. Even though the exact mechanisms are still unknown, the intestinal microbiota is crucial in IBD development. We previously showed that murine norovirus (MNV) induces colitis in the Il10-deficient (Il10-/-) mouse model of IBD in a microbiota-dependent manner. Thus, in this study we analyzed whether distinct minimal bacterial consortia influence the outcome of MNV-triggered colitis in Il10-/- mice. Gnotobiotic Il10-/- mice associated with Oligo-Mouse-Microbiota 12 (OMM12) or Altered Schaedler Flora (ASF) developed little to no inflammatory lesions in the colon and cecum. MNV infection exacerbated colitis severity only in ASF-colonized mice, but not in those associated with OMM12. Four weeks after MNV infection, inflammatory lesions in ASF-colonized Il10-/- mice were characterized by epithelial hyperplasia, infiltration of inflammatory cells, and increased barrier permeability. Co-colonization of ASF-colonized Il10-/- mice with segmented filamentous bacteria (SFB) abolished MNV-induced colitis, whereas histopathological scores in SFB-OMM12-co-colonized mice stayed unchanged. Moreover, SFB only colonized mice associated with ASF. The SFB-mediated protective effects in ASF-colonized mice involved enhanced activation of intestinal barrier defense mechanisms and mucosal immune responses in the chronic and acute phase of MNV infection. SFB colonization strengthened intestinal barrier function by increasing expression of tight junction proteins, antimicrobial peptides and mucus. Furthermore, SFB colonization enhanced the expression of pro-inflammatory cytokines such as Tnfα, Il1ß, and Il12a, as well as the expression of the regulatory cytokine Tgfß. Altogether, our results showed that MNV-triggered colitis depends on the microbial context.

Mucispirillum schaedleri Antagonizes Salmonella Virulence to Protect Mice against Colitis.

The microbiota and the gastrointestinal mucus layer play a pivotal role in protection against non-typhoidal Salmonella enterica serovar Typhimurium (S. Tm) colitis. Here, we analyzed the course of Salmonella colitis in mice lacking a functional mucus layer in the gut. Unexpectedly, in contrast to mucus-proficient littermates, genetically deficient mice were protected against Salmonella-induced gut inflammation in the streptomycin colitis model. This correlated with microbiota alterations and enrichment of the bacterial phylum Deferribacteres. Using gnotobiotic mice associated with defined bacterial consortia, we causally linked Mucispirillum schaedleri, currently the sole known representative of Deferribacteres present in the mammalian microbiota, to host protection against S. Tm colitis. Inhibition by M. schaedleri involves interference with S. Tm invasion gene expression, partly by competing for anaerobic electron acceptors. In conclusion, this study establishes M. schaedleri, a core member of the murine gut microbiota, as a key antagonist of S. Tm virulence in the gut.

Reproducible Colonization of Germ-Free Mice With the Oligo-Mouse-Microbiota in Different Animal Facilities.

The Oligo-Mouse-Microbiota (OMM12) is a recently developed synthetic bacterial community for functional microbiome research in mouse models (Brugiroux et al., 2016). To date, the OMM12 model has been established in several germ-free mouse facilities world-wide and is employed to address a growing variety of research questions related to infection biology, mucosal immunology, microbial ecology and host-microbiome metabolic cross-talk. The OMM12 consists of 12 sequenced and publically available strains isolated from mice, representing five bacterial phyla that are naturally abundant in the murine gastrointestinal tract (Lagkouvardos et al., 2016). Under germ-free conditions, the OMM12 colonizes mice stably over multiple generations. Here, we investigated whether stably colonized OMM12 mouse lines could be reproducibly established in different animal facilities. Germ-free C57Bl/6J mice were inoculated with a frozen mixture of the OMM12 strains. Within 2 weeks after application, the OMM12 community reached the same stable composition in all facilities, as determined by fecal microbiome analysis. We show that a second application of the OMM12 strains after 72 h leads to a more stable community composition than a single application. The availability of such protocols for reliable de novo generation of gnotobiotic rodents will certainly contribute to increasing experimental reproducibility in biomedical research.

Candida: Platelet Interaction and Platelet Activity in vitro.

Over the last 2 decades, platelets have been recognized as versatile players of innate immunity. The interaction of platelets with fungal pathogens and subsequent processes may critically influence the clinical outcome of invasive mycoses. Since the role of platelets in Candida infections is poorly characterized and controversially discussed, we studied interactions of human platelets with yeast cells, (pseudo-)hyphae, biofilms and secretory products of human pathogenic Candida species applying platelet rich plasma and a whole blood model. Incubation of Candida with platelets resulted in moderate mutual interaction with some variation between different species. The rate of platelets binding to -Candida (pseudo-) hyphae and candidal biofilm was comparably low as that to the yeast form. Candida-derived secretory products did not affect platelet activity - neither stimulatory nor inhibitory. The small subset of platelets that bound to Candida morphotypes was consequently activated. However, this did not result in reduced growth or viability of the different Candida species. A whole blood model simulating in vivo conditions confirmed platelet activation in the subpopulation of Candida-bound platelets. Thus, the inability of platelets to efficiently react on Candida presence might favor fungal survival in the blood and contribute to high morbidity of Candida sepsis.

Complete Genome Sequencing of the Mouse Intestinal Isolate Escherichia coli Mt1B1.

Escherichia coli Mt1B1, a mouse isolate, is a facultative anaerobic bacterium which was shown to counteract Salmonella enterica serovar Typhimurium infection in a mouse model. In the present study, we describe the complete genome sequence of E. coli Mt1B1, composed of a 5.1-Mb chromosome and a 62.6-kb plasmid.

Tolerance towards gentamicin is a function of nutrient concentration in biofilms of patient-isolated Staphylococcus epidermidis.

Staphylococcus epidermidis is a biofilm-forming bacterial strain that can cause major problems as an agent of nosocomial infections. Bacteria in biofilms are shielded from the environment and can survive high doses of antibiotics. We here test the antibiotic susceptibility of Staphylococcus epidermidis to rising gentamicin concentrations in optimal growth conditions as used in routine bacteriology laboratories with low nutrient situations as suggested to be found in clinical situations. We found that gentamicin-resistant Staphylococcus epidermidis biofilms survived in the absence of external nutrient supply in PBS. While addition of gentamicin sulfate significantly reduced the pH value of all used media and solutions, this acidification did not alter survival of bacteria in the biofilm. We found a statistically significant and dose-dependent reduction of survival in low nutrient situations using gentamicin sulfate in three out of four patient isolates of Staphylococcus epidermidis which have been tested to be gentamicin-resistant under optimal growth conditions. Supporting the original profiling, survival in full media under the same antibiotic dosages was not significantly reduced. Our data here show that antibiotic resistance is a function of the provided nutrient concentration. Antibiotic resistance profiling should consider variations in nutrient availability.