The dynamics of anitmicrobial resistance genes of the pig and human gut microbiome in Uganda

Horizontal gene transfer among bacteria in the gut microbiomes ensures that Antimicrobial Resistance (AMR) genes, which evolve in one organism can be disseminated to others, including clinically pathogenic bacteria. Until recently, the non-culturable microbiome fraction harbouring the vast majority of AMR genes was unexplored. However, advances in sequencing and bioinformatic techniques have revolutionized our understanding of this eco-system. We can now interrogate the most granular relationships in this microbial fraction and their role in microbial-sharing and between host-exchange of AMR genes. This can be done using targeted sequencing of 16S rRNA and real time qPCR to detect changes in taxa and AMR gene composition and abundance. One can then cost effectively dissect the detected changes using shotgun sequencing, bioinformatics and statistical methods to reveal the underlying dynamics. We therefore propose a natural longitudinal cohort study in Uganda to examine taxa related dynamics of AMR genes and exchange across the human-pig interface in two settings representing different levels of antibiotic selection pressure and use and pig-human interaction.
Specific objectives:
1. Identifying significant changes in taxonomic and AMR genes in gut microbiomes of people and their pigs in peri urban and rural settings over a one-year period using 16SrRNA, AMR gene probes and real time PCR.
2. Conducting forensic investigations of the significant changes detected in objective 1 using sequence based metagenomics on the corresponding gut microbiomes in parallel with whole genome sequencing and phenotypic AMR testing of a sentinel bacterial to understand the underlying dynamics.
3. Predicting, estimating and making inferences on AMR gene occurrence, taxa-linked within and between host exchange and their potential phenotypic effects using bioinformatic, statistical and support vector machine approaches on the data generated in 1.

The proposed research aims to gain fundamental insight into the generation, sharing and transfer of antimicrobial resistance (AMR) genes within and between humans and their pigs under natural settings that represent different levels of antibiotic use/misuse. Defining these dynamics offers the best chance of developing effective control measures and underwrites the efforts toward limiting irrational antibiotic use in both human and animal health care.This study combines the use of current molecular techniques, efficiency in data processing and a setting of epidemiological relevance to detect antibiotic driven changes in the gut bacteria. A forensic investigation of each of these changes would then reveal the fundamental characteristics of AMR genes in the gut and their transfer potential across the pig-human interface. The hypothesis to be tested is that people in contact with pigs in peri-urban settings are at higher risk of acquiring AMR genes across this interface than people in rural settings.The specific objectives are:1.To identify significant changes in taxonomic and AMR genes in gut microbiomes of people and their pigs in peri urban and rural settings over a one-year period using 16SrRNA, AMR gene probes and real time PCR. 2.To conduct forensic investigations of the significant changes detected in objective 1 using sequence based metagenomics on the corresponding gut microbiomes in parallel with whole genome sequencing and phenotypic AMR testing of sentinel bacteria to understand the underlying dynamics. 3.To predict, estimate and make inferences on AMR gene occurrence, taxa-linked within and between host exchange and their potential phenotypic effects using bioinformatic, statistical and support vector machine approaches on the data generated in 1&2.