Elucidating the complex microbial interactions in biological environments requires the identification and characterization of not only the bacterial component but also the eukaryotic viruses, bacteriophage, and fungi. In a proof of concept experiment, next generation sequencing approaches, accompanied by the development of novel computational and bioinformatics tools, were utilized to examine the evolution of the microbial ecology of the avian trachea during the growth of a healthy commercial broiler flock. The flock was sampled weekly, beginning at placement and concluding at 49 days, the day before processing. Metagenomic sequencing of DNA and RNA was utilized to examine the bacteria, virus, bacteriophage, and fungal components during flock growth. The utility of using a metagenomic approach to study the avian respiratory virome was confirmed by detecting the dysbiosis in the avian respiratory virome of broiler chickens diagnosed with infection with infectious laryngotracheitis virus. This study provides the first comprehensive analysis of the ecology of the avian respiratory microbiome and demonstrates the feasibility for the use of this approach in future investigations of avian respiratory diseases.

Clostridioides difficile infection (CDI) is the leading cause of antibiotic and healthcare-associated infective diarrhea. It is linked to 365,000 infections and causes approximately 20,000 deaths annually in the US. CDI is mediated by the action of toxin B (TcdB) in humans. Monoclonal antibodies (mAbs) that neutralize TcdB function have shown protection against CDI in preclinical models and more recently in the clinic. Several regions on the TcdB gene have been targeted by therapeutic mAbs including the glucosyltransferase (GTD) and the C-terminal combined repetitive oligopeptides (CROPS) domains. We have developed AZD5148, an anti-TcdB mAb targeting a region within the GTD. To understand its strain coverage, we examined sequence variations of GTD including the AZD5148 epitope in contemporary C. difficile strains utilizing a public data repository. 8,085 assembled C. difficile genomes collected between 2015-2022 were obtained from Enterobase. These were annotated with Prokka to identify the TcdB gene coding sequence, and subsequently aligned to the R20291 strain reference. Phylogeny was determined with ParSnp and visualized using iTol. From the aligned sequences, variants were identified using an in-house tool (RADAR). A majority of the C. difficile genomes deposited were from North America, specifically the United States. Ribotype (RT) information was available for 9.6% of the genomes with RT-078 being the most common. Phylogenetic analysis did not reveal any patterns by geography or collection year, though clades tended to be associated with ribotypes. Degree of conservation was calculated at each TcdB genomic residue and was evaluated both regionally and temporally. We found GTD to be highly conserved in US isolates as well as globally. Additionally, among contact residues for AZD5148, any variations observed were single, and were seen for positions 323 (Y-H), 329 (G-E), 349 (V-A), and 350 (L-I). These findings have important implications for drug development including for AZD5148. Specifically, our findings reveal that the GTD domain of the TcdB gene has lower variance globally over the last 7 years making it a promising target for a C. difficile TcdB neutralizing mAb. Kelly Mulholland, PhD, AstraZeneca: Employee|AstraZeneca: Stocks/Bonds Victoria Godfrey, MS, AstraZeneca: Employee|AstraZeneca: Stocks/Bonds Ann Marie Stanley, PhD, AstraZeneca: Employee|AstraZeneca: Stocks/Bonds Tyler Brady, MS, AstraZeneca: Employee|AstraZeneca: Stocks/Bonds Bret R. Sellman, PhD, AstraZeneca: Employee|AstraZeneca: Stocks/Bonds Christine Tkaczyk, PhD, AstraZeneca: Employee|AstraZeneca: Stocks/Bonds Vancheswaran Gopalakrishnan, PhD, AstraZeneca: Employee|AstraZeneca: Stocks/Bonds