Towards the generation of gnotobiotic larvae as a tool to investigate the influence of the microbiome on the development of the amphibian immune system.

The immune equilibrium model suggests that exposure to microbes during early life primes immune responses for pathogen exposure later in life. While recent studies using a range of gnotobiotic (germ-free) model organisms offer support for this theory, we currently lack a tractable model system for investigating the influence of the microbiome on immune system development. Here, we used an amphibian species (Xenopus laevis) to investigate the importance of the microbiome in larval development and susceptibility to infectious disease later in life. We found that experimental reductions of the microbiome during embryonic and larval stages effectively reduced microbial richness, diversity and altered community composition in tadpoles prior to metamorphosis. In addition, our antimicrobial treatments resulted in few negative effects on larval development, body condition, or survival to metamorphosis. However, contrary to our predictions, our antimicrobial treatments did not alter susceptibility to the lethal fungal pathogen Batrachochytrium dendrobatidis (Bd) in the adult life stage. While our treatments to reduce the microbiome during early development did not play a critical role in determining susceptibility to disease caused by Bd in X. laevis, they nevertheless indicate that developing a gnotobiotic amphibian model system may be highly useful for future immunological investigations. This article is part of the theme issue 'Amphibian immunity: stress, disease and ecoimmunology'.

Diluted Fecal Community Transplant Restores Clostridioides difficile Colonization Resistance to Antibiotic-Perturbed Murine Communities.

Fecal communities transplanted into individuals can eliminate recurrent Clostridioides difficile infection (CDI) with high efficacy. However, this treatment is only used once CDI becomes resistant to antibiotics or has recurred multiple times. We sought to investigate whether a fecal community transplant (FCT) pretreatment could be used to prevent CDI altogether. We treated male C57BL/6 mice with either clindamycin, cefoperazone, or streptomycin and then inoculated them with the microbial community from untreated mice before challenge with C. difficile. We measured colonization and sequenced the V4 region of the 16S rRNA gene to understand the dynamics of the murine fecal community in response to the FCT and C. difficile challenge. Clindamycin-treated mice became colonized with C. difficile but cleared it naturally and did not benefit from the FCT. Cefoperazone-treated mice became colonized by C. difficile, but the FCT enabled clearance of C. difficile. In streptomycin-treated mice, the FCT was able to prevent C. difficile from colonizing. We then diluted the FCT and repeated the experiments. Cefoperazone-treated mice no longer cleared C. difficile. However, streptomycin-treated mice colonized with 1:102 dilutions resisted C. difficile colonization. Streptomycin-treated mice that received an FCT diluted 1:103 became colonized with C. difficile but later cleared the infection. In streptomycin-treated mice, inhibition of C. difficile was associated with increased relative abundance of a group of bacteria related to Porphyromonadaceae and Lachnospiraceae. These data demonstrate that C. difficile colonization resistance can be restored to a susceptible community with an FCT as long as it complements the missing populations. IMPORTANCE Antibiotic use, ubiquitous with the health care environment, is a major risk factor for Clostridioides difficile infection (CDI), the most common nosocomial infection. When C. difficile becomes resistant to antibiotics, a fecal microbiota transplant from a healthy individual can effectively restore the gut bacterial community and eliminate the infection. While this relationship between the gut bacteria and CDI is well established, there are no therapies to treat a perturbed gut community to prevent CDI. This study explored the potential of restoring colonization resistance to antibiotic-induced susceptible gut communities. We described the effect that gut bacterial community variation has on the effectiveness of a fecal community transplant for inhibiting CDI. These data demonstrated that communities susceptible to CDI can be supplemented with fecal communities but that the effectiveness depended on the structure of the community following the perturbation. Thus, a reduced bacterial community may be able to recover colonization resistance in patients treated with antibiotics.

An Osmotic Laxative Renders Mice Susceptible to Prolonged Clostridioides difficile Colonization and Hinders Clearance.

Antibiotics are a major risk factor for Clostridioides difficile infections (CDIs) because of their impact on the microbiota. However, nonantibiotic medications such as the ubiquitous osmotic laxative polyethylene glycol 3350 (PEG 3350) also alter the microbiota. Clinicians also hypothesize that PEG helps clear C. difficile. But whether PEG impacts CDI susceptibility and clearance is unclear. To examine how PEG impacts susceptibility, we treated C57BL/6 mice with 5-day and 1-day doses of 15% PEG in the drinking water and then challenged the mice with C. difficile 630. We used clindamycin-treated mice as a control because they consistently clear C. difficile within 10 days postchallenge. PEG treatment alone was sufficient to render mice susceptible, and 5-day PEG-treated mice remained colonized for up to 30 days postchallenge. In contrast, 1-day PEG-treated mice were transiently colonized, clearing C. difficile within 7 days postchallenge. To examine how PEG treatment impacts clearance, we administered a 1-day PEG treatment to clindamycin-treated, C. difficile-challenged mice. Administering PEG to mice after C. difficile challenge prolonged colonization up to 30 days postchallenge. When we trained a random forest model with community data from 5 days postchallenge, we were able to predict which mice would exhibit prolonged colonization (area under the receiver operating characteristic curve [AUROC] = 0.90). Examining the dynamics of these bacterial populations during the postchallenge period revealed patterns in the relative abundances of Bacteroides, Enterobacteriaceae, Porphyromonadaceae, Lachnospiraceae, and Akkermansia that were associated with prolonged C. difficile colonization in PEG-treated mice. Thus, the osmotic laxative PEG rendered mice susceptible to C. difficile colonization and hindered clearance. IMPORTANCE Diarrheal samples from patients taking laxatives are typically rejected for Clostridioides difficile testing. However, there are similarities between the bacterial communities from people with diarrhea and those with C. difficile infections (CDIs), including lower diversity than the communities from healthy patients. This observation led us to hypothesize that diarrhea may be an indicator of C. difficile susceptibility. We explored how osmotic laxatives disrupt the microbiota's colonization resistance to C. difficile by administering a laxative to mice either before or after C. difficile challenge. Our findings suggest that osmotic laxatives disrupt colonization resistance to C. difficile and prevent clearance among mice already colonized with C. difficile. Considering that most hospitals recommend not performing C. difficile testing on patients taking laxatives, and laxatives are prescribed prior to administering fecal microbiota transplants via colonoscopy to patients with recurrent CDIs, further studies are needed to evaluate if laxatives impact microbiota colonization resistance in humans.

The Initial Gut Microbiota and Response to Antibiotic Perturbation Influence Clostridioides difficile Clearance in Mice.

The gut microbiota has a key role in determining susceptibility to Clostridioides difficile infections (CDIs). However, much of the mechanistic work examining CDIs in mouse models uses animals obtained from a single source. We treated mice from 6 sources (2 University of Michigan colonies and 4 commercial vendors) with clindamycin, followed by a C. difficile challenge, and then measured C. difficile colonization levels throughout the infection. The microbiota were profiled via 16S rRNA gene sequencing to examine the variation across sources and alterations due to clindamycin treatment and C. difficile challenge. While all mice were colonized 1 day postinfection, variation emerged from days 3 to 7 postinfection with animals from some sources colonized with C. difficile for longer and at higher levels. We identified bacteria that varied in relative abundance across sources and throughout the experiment. Some bacteria were consistently impacted by clindamycin treatment in all sources of mice, including Lachnospiraceae, Ruminococcaceae, and Enterobacteriaceae To identify bacteria that were most important to colonization regardless of the source, we created logistic regression models that successfully classified mice based on whether they cleared C. difficile by 7 days postinfection using community composition data at baseline, post-clindamycin treatment, and 1 day postinfection. With these models, we identified 4 bacterial taxa that were predictive of whether C. difficile cleared. They varied across sources (Bacteroides) or were altered by clindamycin (Porphyromonadaceae) or both (Enterobacteriaceae and Enterococcus). Allowing for microbiota variation across sources better emulates human interindividual variation and can help identify bacterial drivers of phenotypic variation in the context of CDIs.IMPORTANCEClostridioides difficile is a leading nosocomial infection. Although perturbation to the gut microbiota is an established risk, there is variation in who becomes asymptomatically colonized, develops an infection, or has adverse infection outcomes. Mouse models of C. difficile infection (CDI) are widely used to answer a variety of C. difficile pathogenesis questions. However, the interindividual variation between mice from the same breeding facility is less than what is observed in humans. Therefore, we challenged mice from 6 different breeding colonies with C. difficile We found that the starting microbial community structures and C. difficile persistence varied by the source of mice. Interestingly, a subset of the bacteria that varied across sources were associated with how long C. difficile was able to colonize. By increasing the interindividual diversity of the starting communities, we were able to better model human diversity. This provided a more nuanced perspective of C. difficile pathogenesis.

Uncovering the Core Microbiome and Distribution of Palmerolide in Synoicum adareanum Across the Anvers Island Archipelago, Antarctica.

Polar marine ecosystems hold the potential for bioactive compound biodiscovery, based on their untapped macro- and microorganism diversity. Characterization of polar benthic marine invertebrate-associated microbiomes is limited to few studies. This study was motivated by our interest in better understanding the microbiome structure and composition of the ascidian, Synoicum adareanum, in which palmerolide A (PalA), a bioactive macrolide with specificity against melanoma, was isolated. PalA bears structural resemblance to a hybrid nonribosomal peptide-polyketide that has similarities to microbially-produced macrolides. We conducted a spatial survey to assess both PalA levels and microbiome composition in S. adareanum in a region of the Antarctic Peninsula near Anvers Island (64° 46'S, 64° 03'W). PalA was ubiquitous and abundant across a collection of 21 ascidians (3 subsamples each) sampled from seven sites across the Anvers Island Archipelago. The microbiome composition (V3-V4 16S rRNA gene sequence variants) of these 63 samples revealed a core suite of 21 bacterial amplicon sequence variants (ASVs)-20 of which were distinct from regional bacterioplankton. ASV co-occurrence analysis across all 63 samples yielded subgroups of taxa that may be interacting biologically (interacting subsystems) and, although the levels of PalA detected were not found to correlate with specific sequence variants, the core members appeared to occur in a preferred optimum and tolerance range of PalA levels. These results, together with an analysis of the biosynthetic potential of related microbiome taxa, describe a conserved, high-latitude core microbiome with unique composition and substantial promise for natural product biosynthesis that likely influences the ecology of the holobiont.

The Proton Pump Inhibitor Omeprazole Does Not Promote Clostridioides difficile Colonization in a Murine Model.

Proton pump inhibitor (PPI) use has been associated with microbiota alterations and susceptibility to Clostridioides difficile infections (CDIs) in humans. We assessed how PPI treatment alters the fecal microbiota and whether treatment promotes CDIs in a mouse model. Mice receiving a PPI treatment were gavaged with 40 mg of omeprazole per kg of body weight during a 7-day pretreatment phase, the day of C. difficile challenge, and the following 9 days. We found that mice treated with omeprazole were not colonized by C. difficile When omeprazole treatment was combined with a single clindamycin treatment, one cage of mice remained resistant to C. difficile colonization, while the other cage was colonized. Treating mice with only clindamycin followed by challenge resulted in C. difficile colonization. 16S rRNA gene sequencing analysis revealed that omeprazole had minimal impact on the structure of the murine microbiota throughout the 16 days of omeprazole exposure. These results suggest that omeprazole treatment alone is not sufficient to disrupt microbiota resistance to C. difficile infection in mice that are normally resistant in the absence of antibiotic treatment.IMPORTANCE Antibiotics are the primary risk factor for Clostridioides difficile infections (CDIs), but other factors may also increase a person's risk. In epidemiological studies, proton pump inhibitor (PPI) use has been associated with CDI incidence and recurrence. PPIs have also been associated with alterations in the human intestinal microbiota in observational and interventional studies. We evaluated the effects of the PPI omeprazole on the structure of the murine intestinal microbiota and its ability to disrupt colonization resistance to C. difficile We found omeprazole treatment had minimal impact on the murine fecal microbiota and did not promote C. difficile colonization. Further studies are needed to determine whether other factors contribute to the association between PPIs and CDIs seen in humans or whether aspects of murine physiology may limit its utility to test these types of hypotheses.