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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
(Massey University, 2025-08-25) Dos Anjos Almeida, Valter
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.
Extensive epigenetic modification with large-scale chromosomal and plasmid recombination characterise the Legionella longbeachae serogroup 1 genome
(Springer Nature Limited, 2022-04-06) Slow S; Anderson T; Murdoch DR; Bloomfield S; Winter D; Biggs PJ
Legionella longbeachae is an environmental bacterium that is the most clinically significant Legionella species in New Zealand (NZ), causing around two-thirds of all notified cases of Legionnaires’ disease. Here we report the sequencing and analysis of the geo-temporal genetic diversity of 54 L. longbeachae serogroup 1 (sg1) clinical isolates, derived from cases from around NZ over a 22-year period, including one complete genome and its associated methylome. The 54 sg1 isolates belonged to two main clades that last shared a common ancestor between 95 BCE and 1694 CE. There was diversity at the genome-structural level, with large-scale arrangements occurring in some regions of the chromosome and evidence of extensive chromosomal and plasmid recombination. This includes the presence of plasmids derived from recombination and horizontal gene transfer between various Legionella species, indicating there has been both intra- and inter-species gene flow. However, because similar plasmids were found among isolates within each clade, plasmid recombination events may pre-empt the emergence of new L. longbeachae strains. Our complete NZ reference genome consisted of a 4.1 Mb chromosome and a 108 kb plasmid. The genome was highly methylated with two known epigenetic modifications, m4C and m6A, occurring in particular sequence motifs within the genome.
A comparison of next-generation sequencing protocols for microbial profiling : a thesis submitted in partial fulfillment of the requirements for the degree of Master of Science in Genetics, Massey University, Palmerston North, New Zealand
(Massey University, 2016) Fong, Yang (Richard)
The introduction of massive parallel sequencing has revolutionized analyses of microbial communities. Illumina and other Whole Genome Shotgun Sequencing (WGS) sequencing protocols have promised improved opportunities for investigation of microbial communities. In the present work, we compared and contrasted the findings from different NGS library preparation protocols (Illumina Nextera, Nextera-XT, NEXTFlex PCR-free and Ion-Xpress-400bp) and two sequencing platforms (MiSeq and Ion-Torrent). Short reads were analysed using the rapid database matching software PAUDA and visualization software MEGAN5, which provides a conservative approach for taxonomic identification and functional analyses. In analyses of a Tamaki River water sample, biological inferences were made and compared across platforms and protocols. For even a relatively small number of reads generated on the MiSeq sequencing platform important pathogens were identified in the water sample. Far greater phylogenetic resolution was obtained with WGS sequencing protocols than has been reported in similar studies that have used 16S rDNA Illumina sequencing protocols. TruSeq and Nextera-XT sequencing protocols produced similar results. The latter protocol offered cheaper, and faster results from less DNA starting material. Proteobacteria (alpha, beta and gamma), Actinobacteria and Bacteroidetes were identified as major microbial elements in the Tamaki River sample. Our findings support the emerging view that short read sequence data and enzymatic library prep protocols provide a cost effective tool for evaluating, cataloguing and monitoring microbial species and communities. This is an approach that complements, and provides additional insight to microbial culture “water testing” protocols routinely used for analysing aquatic environments.

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