The objective of this experiment was to determine if microbial inoculum composition alters the rumen microbial ecology and performance of pre-weaned dairy calves. Twenty Holstein bull calves were removed from their dam at birth, fed 3.8 L colostrum within 1-4 hours after birth, and housed individually. Calves were fed pasteurized milk 3x/d and offered a texturized calf starter ad libitum at 6d of age. A randomized complete block design with repeated measures and a 2 x 2 factorial arrangement of treatments was used to evaluate responses. Treatments were administered by stomach intubation and included: 50 mL autoclaved rumen fluid (RF), 50 mL bacterial-enriched RF (BE), 50 mL protozoal-enriched RF (PE); or 50 mL of each BE and PE inoculum. A rumen content composite was collected from 4 rumen fistulated, lactating Holstein cows. BE inocula were microscopically confirmed to be free of ciliate protozoa before inoculation, while PE contained 2.9±2.2 x 105 protozoa/mL. RF was collected from the calves and inocula were administered once/week at 3-6 weeks of age by stomach intubation. pH and total volatile fatty acid concentration of the PE inocula were greater than BE inocula (P<0.001). PE inoculum (3.7±0.5 mM) had lower NH3 concentrations than BE inoculum (6.8 mM). The bacterial community structure and composition differed by inocula type (P<0.01). Animal performance was not altered by inocula type. All calves were microscopically free of rumen ciliates at 3 weeks of age and calves that did not receive PE remained ciliate-free. Ciliate protozoa were observed in RF from 6, 8, and 6 PE calves (n=10) at week 4, 5, and 6, respectively. Ruminal NH3 was lower in PE calves (3.3 vs 6.8±1.0 mM), while ruminal butyrate molar percent was greater in BE calves (10.8 vs 8.3±0.8). Rumen bacterial diversity measures did not differ by treatment. Individual calf bacterial communities from treated calves became temporarily similar to the inocula, but these communities diverged from the inocula prior to the last inoculation. This study provides new information about two types of rumen-derived inocula and insight into the challenges of directing the rumen microbial environment in the pre-weaned calf.

The human pathogen Clostridioides difficile causes pseudomembranous colitis through release of the potent TcdA and TcdB toxins. These toxins lack canonical N-terminal secretion signal sequences for translocation across the bacterial membrane, suggesting alternate mechanisms of release including targeted secretion mediated by the holin-like protein TcdE and passive release from cell lysis. Here, we show how the lack of the TcdE holin profoundly reduces toxin secretion in the high toxin-producing strains UK1 and VPI10463, independently of cell lysis. Remarkably, tcdE deletion in either strain rescued highly susceptible gnotobiotic mice from lethal infection. ΔtcdE-infected mice demonstrated undetectable levels of toxin acutely and long-term survival, despite active toxin gene expression and cytoplasmic accumulation in mutant strains. These findings demonstrate the dominant role of the TcdE holin in toxin secretion in vivo and host disease and confirm the use of a conserved and non-lytic holin-mediated toxin secretion mechanism among toxigenic species of Clostridia.

Gut microbiota are essential to nutrient metabolism and the maintenance of hindgut health. The characterization of faecal bacterial communities from healthy individuals is important for the establishment of baseline data that can be compared to periods of gut dysbiosis. Diet is a key determinant of the faecal microbial community structure and generation of volatile fatty acids, a main energy source for the host. While rhinoceroses are herbivores, black rhinoceroses are browsers and white rhinoceroses are grazers. The objective of our study was to characterize and compare diets, faecal bacterial communities, nutrients and metabolites between and amongst Southern white rhinoceroses and Southern black rhinoceroses (n = 3 rhinos/species) managed at Disney's Animal Kingdom^®. Faecal bacterial communities were similar between individual white rhinos and dissimilar between species and individual black rhinos. Faecal butyrate and propionate molar proportions and concentrations were greater in black rhinos than white rhinos, whereas lactate was greater in white rhinos. The Shannon diversity, total operational taxonomic units, and relative abundance of Firmicutes were greater in white than black rhinos. The relative abundance of Proteobacteria in faeces from black rhinos was 3‐fold greater than from white rhinos. One black rhino had a greater relative abundance of Verrucomicrobia (7.45 ± 1.31%) than all other individual rhinos (0.01–1.37%). White rhinoceroses demonstrated similar abundances of bacterial phyla and communities between one another and by individual, while black rhinoceroses were more dissimilar by individual. The dissimilarities between black rhinos were suspected to be due to total diet consumption variability, including browse diversity, and lack of direct contact. In contrast, the white rhinos commingled (i.e. nose‐to‐nose contact) and consumed similar amounts of hay, pellets and training items. These results suggest that species‐specific diets and the individual contribute to differences in faecal bacterial communities, nutrients and metabolites between black and white rhinos housed at the same institution.

Clostridioides difficile is the leading cause of healthcare associated infections. The Pathogenicity Locus (PaLoc) toxins TcdA and TcdB promote host disease. These toxins lack canonical N-terminal signal sequences for translocation across the bacterial membrane, suggesting alternate mechanisms of release, which have included targeted secretion and passive release from cell lysis. While the holin TcdE has been implicated in TcdA and TcdB release, its role in vivo remains unknown. Here, we show profound reductions in toxin secretion in tcdE mutants in the highly virulent strains UK1 (epidemic ribotype 027, Clade 3) and VPI10463 (ribotype 087, Clade 1). Notably, tcdE deletion in either strain rescued highly susceptible gnotobiotic mice from lethal infection by reducing acute extracellular toxin to undetectable levels, limiting mucosal damage, and enabling long-term survival, in spite of continued toxin gene expression in tcdE mutants. Our findings confirm TcdE’s critical functions in vivo for toxin secretion and C. difficile virulence.

Clostridioides difficile (CD) is a sporulating and toxin-producing nosocomial pathogen that opportunistically infects the gut, particularly in patients with depleted microbiota after antibiotic exposure. Metabolically, CD rapidly generates energy and substrates for growth from Stickland fermentations of amino acids, with proline being a preferred reductive substrate. To investigate the in vivo effects of reductive proline metabolism on C . difficile’s virulence in an enriched gut nutrient environment, we evaluated wild-type and isogenic ΔprdB strains of ATCC43255 on pathogen behaviors and host outcomes in highly susceptible gnotobiotic mice. Mice infected with the ΔprdB mutant demonstrated extended survival via delayed colonization, growth and toxin production but ultimately succumbed to disease. In vivo transcriptomic analyses demonstrated how the absence of proline reductase activity more broadly disrupted the pathogen’s metabolism including failure to recruit oxidative Stickland pathways, ornithine transformations to alanine, and additional pathways generating growth-promoting substrates, contributing to delayed growth, sporulation, and toxin production. Our findings illustrate the central role for proline reductase metabolism to support early stages of C. difficile colonization and subsequent impact on the pathogen’s ability to rapidly expand and cause disease.

The characterization of microbial communities by metagenomic approaches has been enhanced by recent improvements in short-read sequencing efficiency and assembly algorithms. We describe the results of adding long-read sequencing to the mix of technologies used to assemble a highly complex cattle rumen microbial community, and compare the assembly to current short read-based methods applied to the same sample. Contigs in the long-read assembly were 7-fold longer on average, and contained 7-fold more complete open reading frames (ORF), than the short read assembly, despite having three-fold lower sequence depth. The linkages between long-read contigs, provided by proximity ligation data, supported identification of 188 novel viral-host associations in the rumen microbial community that suggest cross-species infectivity of specific viral strains. The improved contiguity of the long-read assembly also identified 94 antimicrobial resistance genes, compared to only seven alleles identified in the short-read assembly. Overall, we demonstrate a combination of experimental and computational methods that work synergistically to improve characterization of biological features in a highly complex rumen microbial community.