Novel inter-disciplinary approaches for identifying and tackling the spread of AntiMicrobial Resistance through Environmental pathways in PAKistan

Lead Research Organisation: University of Warwick
Department Name: Warwick Medical School


Medicine has been transformed by the development of antibiotics, medicines that kill bacteria. These have made deadly bacterial diseases that once killed millions such as tetanus, syphilis, and leprosy, easily treatable. They are given as a preventative measure prior to surgery to prevent infection and they relieve suffering from less dangerous diseases such as strep throat. There is, however, a problem. Bacteria are becoming resistant to antibiotics. This is a result of evolution. The use of antibiotics inevitably selects for bacteria that carry genes that can protect them against these compounds. We call these antibiotic or antibacterial resistance genes (ARGs). Genes are pieces of DNA that make up part of the bacteria's genome or smaller DNA molecules such as plasmids. ARGs provide bacteria with the ability to degrade or excrete antibiotics. They can be exchanged between different species of bacteria allowing these abilities to spread through communities.

The use and misuse of antibiotics is accelerating the rate at which ARGs evolve and spread. Using antibiotics to treat non-bacterial infections or to improve the growth rate of livestock results in large amounts of antibiotics entering the environment, this creates a strong selective pressure leading to many resistant bacteria. This resistance can develop in free-living bacteria that perform important functions in ecosystems, e.g. soils or rivers, but then spread to bacteria that cause disease within humans, pathogens. There are pathogens beginning to appear that are resistant to multiple antibiotics, e.g. multidrug-resistant tuberculosis. If this multi-drug resistance spreads further then diseases that are now treatable will become increasingly deadly. This is a potential global public health disaster.

The problems caused by antibiotic resistance (ABR) will particularly badly impact Low and Middle Income Countries (LMICs), such as Pakistan, that depend upon cheap antibiotics to treat many infectious diseases. Tragically, it is just these countries, which are likely to have the most problems. This is because antibiotics are easily available without prescription over the counter. People take antibiotics to treat non-bacterial diseases or do not take them for long enough, this allows the resistant bacteria to spread and proliferate. This is compounded by manufacturers of antibiotics releasing wastewater contaminated with antibiotics into the environment, farmers using large quantities of antibiotics in intensive livestock rearing, and the fact that human and animal waste is dumped untreated into water sources. All this pollution results in more resistant bacteria evolving. It is unclear, however, which of these factors are most important to address, in order to prevent the spread of ABR.

We have assembled a team of biologists, engineers, social scientists and mathematicians, to better understand ABR in Pakistan and how to combat it. We will conduct a survey of resistance genes across multiple areas within Pakistan, chosen in order to determine what causes them to proliferate and spread, ultimately leading to drug-resistant infections in humans. We will do this by sequencing DNA direct from environmental samples to resolve the genes that are present in the bacteria. We will sample from the environment and from a range of health facilities to reveal how genes are being transmitted from environmental bacteria into pathogens. We will also study the behaviour of people and institutions in Pakistan and determine how that contributes to the scale of the problem. This will allow us to propose ways in which they can reduce the spread of antibiotic resistant genes. These might be changes to how antibiotics are used in health facilities or improved approaches to waste disposal. This will be of great benefit to Pakistan but also - because these genes are capable of spreading between individuals across the world - to other LMICs and us in the UK too.

Technical Summary

The spread of antibacterial resistance (ABR) in human pathogens is a looming public health disaster. The combination of high burden of infectious disease, intensification of agriculture, poor sanitation, political instability, and unregulated antibiotic usage makes Pakistan a particularly high risk country for ABR. It is the sixth most populous nation in the world with over 200 million people but ABR in Pakistan receives far less attention than in India. We will redress this imbalance through an inter-disciplinary research programme. We will conduct a survey of ABR across multiple areas within Pakistan chosen to determine the importance of our hypothesised ABR risk factors: urbanisation, globalisation, intensive agriculture, pollution, and antibiotic usage. They will include the city of Karachi, which combines a high density of human population with both intensive agriculture and pharmaceutical manufacture, but also low-density rural areas. Within these areas we will take samples from a range of environments including health facilities, rivers, sewage, and slurry from intensive animal rearing. These will be subjected to microbiological tests for ABR including metagenomics sequencing, high throughput qPCR, and isolate cultivation. This will enable us to determine the importance of alternative ABR drivers and the rate of transmission into human pathogens. Concurrent, with the biological sampling will be an in-depth health systems study utilising geographical information systems (GIS), to characterise, and map the potential sources of ABR. This will be used to develop intervention strategies based around changes in antibiotic usage or sanitation. These two sources of data will be integrated into predictive epidemiological models of ABR spread allowing the efficacy of the alternate policies to be tested in silico. Finally, the most effective but politically feasible options - as determined by the health policy research component - will be recommended to stakeholders.

Planned Impact

The WHO Global Action Plan on Antimicrobial Resistance relies on the World Health Organisation (WHO), the Food and Agricultural Organisation (FAO) and the World Organisation for Animal Health (OIE), facilitating the preparation of multi-sectorial national AMR action plans. To date, few LMICs have initiated these. This research will impact on the policies and responses for tackling drivers of AMR being developed in LMICs. It is obviously relevant to Pakistan but because of our emphasis on creating connections with India it will have a regional impact too. Furthermore, our focus on the AMR challenges associated with extreme urbanisation, a common problem facing LMICs, will give it a global relevance. It will also be amongst the first studies analysing the policy process relating to AMR in LMICs which is little understood and will be critical to implementation.

The research also has relevance outside of LMICs because movement of microbes across national boundaries makes antimicrobial resistance a truly global problem. The UK, in particular, given its close connections to Pakistan stands to benefit if we can understand and ameliorate the processes generating ABR in that country. In addition, our work will also contribute at the global level by furthering our understanding of the generic processes by which antibacterial resistance genes move between environmental reservoirs.

Specific examples of beneficiaries:

1) International multi-sectoral policy makers that are essential in the response to AMR, including the WHO (country and regional offices), FAO, OIE, Health Security Partners (HSP), Chatham House Centre on Global Health Security (see letter of support)

2) National and regional actors involved in AMR policy formation in Pakistan, India and neighbouring countries such as the Orangi Pilot Project and Urban Resource Centre (see letters of support)

3) In addition to the above, we will seek additional partners from organisations in Pakistan instrumental in influencing regulations and policymaking and / or implementation (both at the central and provincial levels):
a. National Institutes of Health , Islamabad (links to Ministry of Health)
b. Pakistan Agriculture and Research Council
c. Pakistan Health Research Council (see letter of support)
d. Veterinary and Agricultural University
e. Director General Health Services, Sindh
f. Karachi Water and Sewage Board
g. Sindh Agricultural Department
h. Sindh Fisheries and Livestock Department

4) Pharmaceutical multinationals, these are increasingly concerned about pollution and its impact on AMR by their antibiotic suppliers, in part this is due to pressure from investors, see letter of support from Professor Jason Snape (AstraZeneca)


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Description We organised a workshop in Karachi entitled "AMR in Pakistan: a One Health approach" from November 13-14th 2017, that was attended by over 40 participants from multiple sectors including human health, veterinary, environment and the pharmaceuticals industry. This included representatives from organisations involved in antimicrobial resistance (AMR) surveillance in Pakistan, including Dr Farah Sohail (WHO Country Office).

In the pilot study we tested the hypothesis that ABRG abundance and diversity would differ significantly between environments associated with intensive agriculture and residential areas within the city of Karachi, Pakistan. Triplicate sediment samples were collected from three slurry drainage sites from a farm in Landhi Diary Colony, and from three open sewer drainage sites within the residential settlement of Rehri Goth, Karachi. DNA was purified from each sample and long-range endpoint PCR used to amplify entire gene cassettes associated with class I integrons of mobile and chromosomal origins. These were then sequenced using an Oxford Nanopore Minion device, and base calling, barcode demultiplexing and alignment to the CARD database, undertaken in real time, whilst in Pakistan.

Multivariate analysis of normalised ABR gene abundance shows significant differentiation of ABR gene composition both between the cattle colony and residential area (p = 0.003) and between chromosomal and mobile elements (p = 0.001) - see Figure 1. Intriguingly, the individual gene most associated with the cattle colony was a Carbapenemase 9 gene (p = 0.002). These results were confirmed by qPCR analysis of the major gene classes, which revealed a greater prevalence
of NDM genes in the residential samples (p = 1.7e-05), and a higher abundance of the colistin degrading MCR-1 gene in the cattle colony (p = 4.9e-03). The latter fits with our observation that intramammary injections of amoxicillin and colistin are used to treat mastitis on the farms. This is disturbing given that colistin is used in humans as an antibiotic of last resort. Our focus on agriculture as a driver was confirmed by a higher prevalence of Integrons in the cattle colony. These preliminary results confirm that even between two sites that are just 5 km away in Karachi, NMDS showing normalised AMR gene abundance as a function of sample site the ABRG profile can differ substantiallayn,drgefnletictpinosgititohne different land use.
Exploitation Route We are hopeful that the network of collaborators we established in Pakistan as a result of the award will enable us to obtain funding in future to pursue the study of AMR in Pakistan.
Sectors Agriculture, Food and Drink,Environment

Title Novel use of minION for diagnostics 
Description Novel approach to diagnostics for typing pathogens in faecal samples directly 
Type Of Material Technology assay or reagent 
Year Produced 2017 
Provided To Others? No  
Impact New approach for direct typing of a pathovar or spoligotype directly from cells in a faecal or other samples for example soil, blood, tissue. This will allow rapid identification of pathogens in the environment and help track dissemination pathways. 
Description Collaboration with National University of Sciences and Technology (NUST) - Islamabad Pakistan 
Organisation National University of Sciences and Technology
Country Pakistan 
Sector Academic/University 
PI Contribution We organised a two-day workshop at the National University of Science and Technology (NUST) in Islamabad Pakistan (Feburary 12th-13th) 2019. This discussed the issues of antibacterial resistance in Pakistan and developiong countries more generally. It was hosted by Prof Imran Hashmi. Following the workshop we discussed the possibility of joint grant proposals and future collaborations in research and teaching.
Collaborator Contribution NUST hosted the workshop, found a source of funding through the Pakistan Higher Education Council and arranged everything. In the ongoing collaboration they will provide potential Masters and PhD students for collaborative work on antibiotic resistance in Pakistan.
Impact The workshop "Tackling the spread of antibacterial resistance through environmental pathways in Pakistan" was attended by students, scientists, and policy makers. As such it raised the profile of the issues of AMR in Pakistan. Something that the ongoing collaboration will strengthen.
Start Year 2018
Description Online workshop for local science and public sector workers in Islamabad regarding diagnostics of pathogens and what is qPCR 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Postgraduate students
Results and Impact We produced a video and also provided online zoom workshop session describing qPCR and providing technical as well as general information for diagnosis of pathogens in various scenarios.
Year(s) Of Engagement Activity 2021