Host Clustering of Campylobacter Species and Other Enteric Pathogens in a Longitudinal Cohort of Infants, Family Members and Livestock in Rural Eastern Ethiopia

Background Livestock are recognized as major reservoirs for Campylobacter species and other enteric pathogens, posing substantial infection risks to humans. High prevalence of Campylobacter during early childhood has been linked to environmental enteric dysfunction and stunting, particularly in low-resource settings. Methods A longitudinal study of 106 infants was conducted from December 2020 to June 2022. Monthly stool samples were collected from infants beginning in the first month after birth. Additional stool samples from mothers, siblings, and livestock (goats, cattle, sheep, and chickens) were collected biannually. A subset of 280 samples from Campylobacter positive households with complete metadata were analyzed by shotgun metagenomic sequencing followed by bioinformatic analysis via the CZ-ID metagenomic pipeline (Illumina mNGS Pipeline v7.1). Further statistical analyses in JMP PRO 16 explored the microbiome, emphasizing Campylobacter and other enteric pathogens. Two-way hierarchical clustering and split k-mer analysis examined host structuring, patterns of co-infections and genetic relationships. Principal component analysis was used to characterize microbiome composition across the seven sample types. Results More than 3,844 genera were detected in the 263 samples. Twenty-one dominant Campylobacter species were detected with distinct clustering patterns for humans, ruminants, and broad hosts. The generalist (broad-host) cluster included the most prevalent species, C. jejuni, C. concisus , and C. coli , present across sample types. Among C. jejuni a major cluster involving humans, chickens, and ruminants isolates, was detected, indicating potential zoonotic transmission to infants and mothers. Candidatus C. infans was only detected in human hosts. Campylobacter species from chickens showed strong positive correlations with mothers (r = 0.76), siblings (r = 0.61) and infants (r = 0.54), while no to weak correlation was observed between Campylobacter species from chickens and small ruminants (sheep and goats) with (r = 0.15, r = 0.0, respectively). Co-occurrence analysis revealed a higher likelihood (p > 0.5) of pairs such as C. jejuni with C. coli, C. concisus , and C. showae . Overall microbiome composition was strongly host driven, with two principal components accounting for 62% of the total variation. Analysis of the top 50 most abundant microbial taxa in infant stool revealed a distinct cluster uniquely present in human stool samples and absent in all livestock samples. Hierarchical clustering revealed frequent co-occurrence of C. jejuni with other enteric pathogens such as Salmonella , and Shigella , particularly in human and chicken samples. Additionally, instances of Candidatus C. infans were identified co-occurring with Salmonella and Shigella species in stool samples from infants, mothers, and siblings. Conclusions A comprehensive analysis of Campylobacter diversity in humans and livestock in a low-resource setting, revealed that infants can be exposed to multiple Campylobacter species early in life. C. jejuni is the dominant species with a propensity for co-occurrence with other notable enteric bacterial pathogens, including Salmonella , and Shigella , especially among infants.