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

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.

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.

It would be imprudent to ignore the increasing frequency of antimicrobial resistance (AMR) genes in pathogenic bacteria at the human-animal interface. The research we propose will provide critical information about the effects of antimicrobial use in pig populations in and how that can lead to resistance acquisition in human populations and vice versa. This knowledge is vital in designing new strategies of prudent and responsible usage of antimicrobial agents in veterinary and medical health care, in order to cub the development and transmission of AMR.

Globally, pigs are increasingly reliant on antimicrobial use as growth promoters, prophylactically and for the treatment of infectious diseases. There is therefore no doubt that a change in such usage would lead to a profound impact on animals and human occupational welfare. This work would not only drive informed discussions as part of momentum building towards limiting broad usage of these antimicrobial agents for purposes other than treatments of disease that is preceded by a definitive diagnosis, but also has ancillary benefits to development of national and global animal and human health control strategies.

The data output from this study provides rare insight into the natural temporal dynamics of microbiota under varying levels of antimicrobial and dietary selection pressure. The impact of such knowledge goes beyond academic and research curiosity but indeed into realm of understanding health determinants in both humans and animals as a function of the microbiome. I am specifically going to be engaged with adopting and developing in-silico analytical methods on metagenomic and genomic data to identify the most critical microbiome fraction in AMR gene transmission. Such output can also be used to develop cost effective risk based surveillance protocols

There is an ancillary translational benefit with linking E. coli phenotypic antibiotic resistance to genomic and their domicile microbiome characteristic. This specifically refers to the next phase that will identify biologically relevant and targetable mutations in AMR genes. Having worked as a technology scout for the BBSRC funded activation of impact award, these skill-sets ensures that any translational benefits in this study can be extended beyond fundamental research.

There is added value of having direct participation in global policy development at the human-animal interface with the WHO, OIE, & FAO, therefore these linkages with the global health tripartite make it possible to further engage the specific working groups on antimicrobial resistance.
Finally, antimicrobial resistance is a topic of high public interest and the University of Edinburgh is committed to disseminating results through all its media and public engagement channels. The programmme will benefit from my proven skills in public engagement, as demonstrated through my role as the chair of the zoonotic TB at the Union.