Genetic diversity of microbes and its impacts on host switching : a thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy in Veterinary Science at Massey University, Palmerston North, New Zealand

Abstract

Land use around forests, leading to habitat overlap, increases the risk of anthroponotic and zoonotic pathogen/microorganism transmission between humans, livestock, and wildlife. This scenario is exemplified by Buhoma, a village where most residents are primarily subsistence farmers who raise livestock and cultivate crops near the Bwindi Impenetrable National Park (BINP) in Southwestern Uganda, creating an ideal interface for understanding these dynamics. Given the significance of this geographic location and the underrepresentation of African populations in global microbiome research, this project aims to characterise the gut microbiome diversity and potential novel bacterial species, as well as microorganism sharing within a multi-host system in a rural area with documented interactions between humans, livestock, and wildlife. For microbial detection, DNA extracted from 553 faecal samples collected from humans, gorillas, and livestock was processed and sequenced to analyse microbial species communities in different hosts using shotgun high-throughput sequencing and bioinformatics tools. The analysis of 2,411 bacterial metagenome-assembled genomes (MAGs) confirmed the presence of numerous putative novel bacterial species in the gut microbiomes of the Ugandan hosts studied in this project. While many species were host-specific, others were detected across multiple host gut microbiomes from Buhoma and BINP, and in different host samples from other geographic locations (by comparing the MAGs generated in this thesis with other publicly available genomes). Among the bacterial MAGs assembled in this project, genera such as Prevotella and Treponema—considered markers of the gut microbiome in individuals with traditional agricultural lifestyles—and Campylobacter—which comprises species that are leading causes of bacterial gastroenteritis globally—were further investigated. These genera were found to contain numerous putative novel species, contributing to our knowledge of the human and animal gut microbiomes and enhancing our understanding of the species associated with them. The results of this study reveal a high diversity of previously unknown microbes recovered from human, gorilla, and livestock gut microbiomes collected simultaneously at the same wild rural interface.

The Treponema genus includes species that inhabit a wide range of hosts and types of microbiotas. In the Ugandan host samples, 75 Treponema MAGs were reconstructed, enabling the identification of sixteen intestinal species, including thirteen putative novel species. Three of the detected species were found in human samples from this study, as well as in publicly available Treponema genomes recovered from animals such as pigs, cattle, wild boars, roe deer, and goats. This detection underscores the potential for interspecies transmission and the establishment of these bacteria in different hosts. Overall, these findings highlight the complex interplay between humans, animals, and their gut microbiomes, emphasising the need for further research to elucidate the implications for public health.

Prevotella species are prevalent and abundant components of microbial communities associated with mammals. This bacterial genus influences individual clinical and metabolic responses to dietary changes and overall health conditions. In humans, Prevotella species have been identified in various body sites such as the skin, oral cavity, vagina, and gastrointestinal tract. In the gut microbiome, they are commonly linked with diets abundant in plant-based foods and are particularly prevalent among agriculturalist populations, where they tend to dominate the microbiota. In the investigation of this genus, 37 MAGs were assembled, all belonging to yet-to-be-described species. Phylogenetic analysis revealed that they represented fifteen putative novel intestinal Prevotella species. The nimJ gene, which confers resistance to metronidazole (a nitroimidazole-class drug), was detected in one of the human intestinal Prevotella species. Given the widespread use of antibiotics like metronidazole for treating both humans and animals in regions of Uganda, including Buhoma, the presence of this gene provides evidence that Prevotella species can acquire resistance to commonly used nitroimidazole-class drugs in areas where their use is prevalent. Of the seven putative novel species identified in human gut microbiomes, two were also present in publicly available genomes in the gut microbiomes from pigs and rhesus macaques living in different geographic locations, providing evidence that the same Prevotella intestinal species can inhabit multiple hosts. Overall, these findings expand knowledge of intestinal Prevotella by introducing numerous previously unknown species, offering a species-level perspective for future studies on Prevotella’s impact on health.

Campylobacter species are leading causes of bacterial gastroenteritis worldwide, yet their diversity and transmission dynamics at wildlife–livestock–human interfaces remain poorly characterised. I investigated Campylobacter diversity among sympatric mountain gorillas, livestock, and humans in Uganda, assembling 44 Campylobacter MAGs representing seven species—including five putative novel taxa. Three novel species were found in mountain gorillas, livestock harboured C. vicugnae (goats) and C. sp017646085 (cattle), while human gut microbiomes revealed Candidatus Campylobacter infans (not associated with disease) and C. sp900539255, which was significantly enriched in clinical samples (p=0.001) and displayed unique sulfur and nitrogen metabolic pathways. Antimicrobial resistance genes, including blaOXA-471_1, were detected in Ca. C. infans MAGs. Overall, our findings demonstrate high Campylobacter diversity at the human–animal interface in Buhoma and highlight the importance of expanding reference databases for accurate surveillance and effective public health interventions.

In summary, this thesis introduces a substantial amount of new information on gut microbiome bacterial species in gorillas, livestock, and humans living at a wild-livestock-human interface. It also provides detailed insights into three bacterial genera present in the gut microbiomes of human populations, as well as other mammals such as gorillas, cattle, and goats.