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

Lead Research Organisation: University of Edinburgh
Department Name: The Roslin Institute

Abstract

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.

Technical Summary

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.

Planned Impact

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.

Publications

10 25 50
 
Description In 2018, my fellowship and preliminary field work output was used as leverage to successfully bid for a training grant from the Department of health and social care administered by Mott MacDonald to target Antimicrobial stakeholders surveillance in Uganda. This training award is valued at a little over a half a million pounds and the we had potential to bid for further another country such Malawi and Tanzania at the end of this year. Indeed 2019 under my Fleming Scholarship Deputy Directorship, the University of Edinburgh was awarded the bid to roll out the actives of training AMR stake holders in Malawi(~£520K), these include personal in charge of AMR surveillance at the Ministry of Health, Ministry of Agriculture, National diagnostic laboratories as well as the National drug authorities. These efforts prepare developing countries to put in place strategies to combat AMR. In the same year, as a direct follow on with my BBSRC Fellowship, i was awarded a Wellcome Trust Institutional Strategic Fund(~£31K) to develop two Bioinformatic tools to support investigations of longitudinal dynamics in Microbiomes. In December 2019 i was awarded the Royal Society & African Academy of Science Future Leaders - African Independent Research (FLAIR) Fellowships, which i unfortunately it had to decline due to personal and career direction reasons.
First Year Of Impact 2019
Sector Agriculture, Food and Drink,Communities and Social Services/Policy,Education,Healthcare
Impact Types Societal,Policy & public services

 
Description A blue-print for One-health AMR surveillance in Uganda
Geographic Reach Africa 
Policy Influence Type Influenced training of practitioners or researchers
 
Description Agritech- catalyst PigBoost: Sustainable data-driven pig production for Uganda
Amount £520,000 (GBP)
Organisation Innovate UK 
Sector Public
Country United Kingdom
Start 01/2020 
End 03/2021
 
Description Determining milk microbiome and AMR gene dynamics in dairy cattle under different drying-off conditions
Amount £2,941 (GBP)
Funding ID RI8024 Bbsrc-Bb/P013732/1-Isp 17/22- Muwonge A 
Organisation University of Edinburgh 
Sector Academic/University
Country United Kingdom
Start 12/2018 
End 12/2019
 
Description Developing dairy-specific molecular screening tools to quantify the within herd dynamics of antimicrobial resistance
Amount £9,090 (GBP)
Organisation Hannah Research Foundation 
Sector Charity/Non Profit
Country United Kingdom
Start 04/2019 
End 05/2020
 
Description Edinburgh Global: Interdisciplinary workshop on peri-urban production
Amount £13,000 (GBP)
Organisation Edinburgh College 
Sector Academic/University
Country United Kingdom
Start 03/2018 
End 05/2018
 
Description Environmental and Economic Impacts of Improved Antibiotics Stewardship in Poultry Systems
Amount £647,418 (GBP)
Funding ID BB/T004436/1 
Organisation Biotechnology and Biological Sciences Research Council (BBSRC) 
Sector Public
Country United Kingdom
Start 07/2019 
End 05/2022
 
Description Fleming fellowship fund for Uganda
Amount £510,000 (GBP)
Organisation Mott Macdonald UK Ltd 
Sector Private
Country United Kingdom
Start 01/2019 
End 08/2020
 
Description Fleming fellowships for Malawi
Amount £520,000 (GBP)
Organisation Mott Macdonald UK Ltd 
Sector Private
Country United Kingdom
Start 02/2020 
End 10/2021
 
Description GCRF Impact Accelerator Award: Lipid antigen extract as a novel vaccine and comparative diagnostic for bovine tuberculosis
Amount £30,000 (GBP)
Organisation Biotechnology and Biological Sciences Research Council (BBSRC) 
Sector Public
Country United Kingdom
Start 08/2017 
End 02/2018
 
Description GCRF Impact Accelerator: A genomic map for bovine tuberculosis susceptibility in Bos indicus
Amount £30,000 (GBP)
Organisation Biotechnology and Biological Sciences Research Council (BBSRC) 
Sector Public
Country United Kingdom
Start 08/2017 
End 03/2018
 
Description Wellcome Trust Institutional Strategic Support Fund-University of Edinburgh
Amount £31,700 (GBP)
Funding ID IS3-R1.09 19/20 
Organisation Wellcome Trust 
Sector Charity/Non Profit
Country United Kingdom
Start 03/2020 
End 03/2021
 
Title 16S rRNA gene metabarcoding 
Description During this PhD project, JP developed and validated a 16S rRNA gene metabarcoding method for the study of complex microbial communities. This method has been taken forward into further research within SRUC, The Roslin Institute/R(D)SVS and JP is currently collaborating with several PhD students and research fellows doing microbiome research and this method has been used in several projects. 
Type Of Material Biological samples 
Year Produced 2017 
Provided To Others? Yes  
Impact Due to the good performance of this methodology which was revealed through validation (i.e. sequencing a mock bacterial community and reagent-only controls), other researchers are enquiring about this method and using it in their own research. 
 
Description Canadian Big data centre University of Laval 
Organisation University of Laval
Country Canada 
Sector Academic/University 
PI Contribution I am currently working with the Jacques Corbiel's group at the University of Laval. His group is providing capacity and training for me with regards to analysing big data. We are the fore collaborating on methodology as i contribute a rare insight into metagenomic dynamics in a resource limited setting.
Collaborator Contribution The have given me access to one of Canada's super computer for my computing component.
Impact None as yet
Start Year 2017
 
Description Collaboration: The dynamics of antimicrobial resistance gene prevalence on a commercial pig farm: implications for policy 
Organisation University of Edinburgh
Country United Kingdom 
Sector Academic/University 
PI Contribution I am collaborating with data analysis of this project, which give me the opportunity to develop the bioinformatic pipelines needed for analysing my data when it comes. My first publication on AMR from pigs will be published using this data.
Collaborator Contribution This would primarily be computing infra-struction to process the data. It takes about 1.6TB of space to per sample through the pipeline i am using so this sort of data would require significant robust computing infrastructure
Impact Bio-informatic pipeline
Start Year 2017
 
Description Developing tools for estimating AMR in developing Countries 
Organisation Yale University
Department School of Public Health
Country United States 
Sector Hospitals 
PI Contribution This group has supported my UKRI- Future Leader Fellowship application. They will be providing advance training for human microbial analysis. Through this group i also have access to the largest consortia of experts on AMR in the United State https://arlg.org this networking expertise would be much needed when the next fellowship is funded
Collaborator Contribution The contribution has mainly been in Kind and mostly intellectual input for developing the next program of research
Impact This has only just started, but i have used it write an application to the Soulsby Foundation to support efforts of developing an framework for estimating AMR in developing countries (Makerere University in Uganda, Edinburgh and Cambridge University in UK and Yale University in United States)
Start Year 2019
 
Description Fleming Fellowship partnership for Malawi 
Organisation Government of Malawi
Country Malawi 
Sector Public 
PI Contribution This collaboration, is similar to the one in Uganda. The application that attracted these funds is build on the one in Uganda which was developed around my fellowship. I am the deputy director for these fellowships at the University of Edinburgh
Collaborator Contribution Malawi will be supported by the Fleming fund where we shall train stakeholders in AMR, the grant is ~£520,000
Impact This only started in January 2020, therefore we do not have any outcomes as yet
Start Year 2019
 
Description Flemings AMR training programme Uganda 
Organisation University of Edinburgh
Department Edinburgh Infectious Diseases
Country United Kingdom 
Sector Academic/University 
PI Contribution I am co-PI on an application to the Fleming's fund administered by Mott MacDonald on behalf of the UK department of Health and social care. This grant is for training AMR stakeholders in Uganda. This application is developed directly on the basis of my fellowship in Uganda
Collaborator Contribution Edinburgh Infectious disease is the umbrella through which the University of Edinburgh applied to become a host institution for the Fleming's fund country training programme. The mentors for the training programme for the AMR stakeholders from Uganda(Mentees) are chose from the pool of experts in the Edinburgh infectious disease umbrella
Impact We organised a fellow's workshop in Kampala in January this year to kick start the training activities https://www.ed.ac.uk/edinburgh-infectious-diseases/news/news/edinburgh-host-fleming-fund-fellows
Start Year 2018
 
Description Pigboost consortium- Uganda 
Organisation AbacusBio
Country New Zealand 
Sector Private 
PI Contribution This is an Agritec catalyst funded by Innovate UK. This projects builds on the work i do in Uganda and it is based in the same districts. It aims at developing data driven solution to support sustaibable pig production in Uganda
Collaborator Contribution The three members are primary contributing expertise in breeding, quantitative data management and capture, farmer landscape mapping. This work provide the dept for me to understand the factors the drive the use of antibiotics in my study areas
Impact It has just started
Start Year 2020
 
Description Pigboost consortium- Uganda 
Organisation Makerere University
Country Uganda 
Sector Academic/University 
PI Contribution This is an Agritec catalyst funded by Innovate UK. This projects builds on the work i do in Uganda and it is based in the same districts. It aims at developing data driven solution to support sustaibable pig production in Uganda
Collaborator Contribution The three members are primary contributing expertise in breeding, quantitative data management and capture, farmer landscape mapping. This work provide the dept for me to understand the factors the drive the use of antibiotics in my study areas
Impact It has just started
Start Year 2020
 
Description Pigboost consortium- Uganda 
Organisation Vetline Services
Country Uganda 
Sector Private 
PI Contribution This is an Agritec catalyst funded by Innovate UK. This projects builds on the work i do in Uganda and it is based in the same districts. It aims at developing data driven solution to support sustaibable pig production in Uganda
Collaborator Contribution The three members are primary contributing expertise in breeding, quantitative data management and capture, farmer landscape mapping. This work provide the dept for me to understand the factors the drive the use of antibiotics in my study areas
Impact It has just started
Start Year 2020
 
Description I gave a talk at the 4th National AMR conference in Uganda 
Form Of Engagement Activity A formal working group, expert panel or dialogue
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Professional Practitioners
Results and Impact 118 medical, veterinary, Pharmacists attend this Annual AMR conference in Uganda. We discussed strategies on how to develop a one health AMR surveillance platform for Uganda which would serve as a blue print for LMICs
Year(s) Of Engagement Activity 2019
URL https://www.iuiu.ac.ug/amr2019/
 
Description RHET Food and Farming Event 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Schools
Results and Impact This event was organised by RHET Lothian to educate school children about agriculture and getting food to the table, as well as engagement with scientific research organisations (such as The Roslin Institute) to describe the kind of work that is carried out by scientists. Several schools from the Lothians were invited to the event, and The Roslin Institute stand had the theme of "cells" and the different types of cells that could be visualised under the microscope. The school children were very engaged with the activities and asked many questions about our research, so it was a fantastic event.
Year(s) Of Engagement Activity 2018
 
Description Visit and talk to AMR surveillance institution for Uganda 
Form Of Engagement Activity Participation in an open day or visit at my research institution
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Professional Practitioners
Results and Impact The talk was attended by surveillance stakeholder from the Uganda National Health laboratory services, Ministry of Health, Ministry of Agriculture, academia and health professionals. There after i visited the Uganda National Health laboratory services head quarters in Luzira, Kampala. The reason of this interaction is because the Award from BBSRC is build on a human-animal interface so linking my work to the mandated stakeholder institution provides the best chance for the outputs informing policy in Uganda
Year(s) Of Engagement Activity 2018,2019