Worms and Bugs - Quantifying Infection Dynamics in Microcosms

Lead Research Organisation: University of Cambridge
Department Name: Veterinary Medicine


Despite continual progress in medicine, infectious diseases pose an unabated threat not only to humans but also to domestic and wild animals. Epidemiologists rely increasingly on mathematical models to analyse and predict the outcome of epidemic outbreaks. However, those models are still far from perfect and are based on many assumptions that cannot always be validated. One of the remaining black boxes in our understanding of infectious disease dynamics is transmission. On the one hand, new technologies have allowed microbiologists to gain increasingly detailed knowledge of the molecular interactions between pathogens and their hosts, informing on the within-host dynamics of infection. On the other hand, the roles of environment and host behaviour on the spread of epidemics is better understood, with the recent hindsight from SARS, pandemic influenza or foot-and-mouth disease outbreaks. But there remains a major gap between those two scales. In particular, we are still far from being able to predict the epidemic potential of a pathogen just based on measures of its growth and spread within an individual host. Reconciling those two levels is also an essential step to improve our understanding of pathogen evolution: because different factors may favour within-host growth and between-host transmission, we are likely to misjudge the selective pressures acting on the whole life-cycle of pathogens. This can have practical implications for the management of vaccination and drugs. In order to help fill those gaps, I propose to set up a new experimental host-pathogen system where individual-level and population dynamics can be measured and used to design and validate integrated mathematical models. Experiments on animals, while essential to gain specific knowledge on chosen pathogens, are limited in scope by technical and ethical issues. My project will use the free-living nematode worm Caenorhabditis elegans, which has been studied by biologists throughout the world for half a century. The worms can be infected in the lab with various microbes, either specific parasites of C. elegans or foodborne pathogens of humans and animals such as Salmonella. Using microscopes, it is possible to keep track of the numbers of infected and uninfected nematodes in a microcosm (an experimental population maintained in a large Petri dish), but also measure the development of infection and its effects in individual worms. Data from these experimental epidemics will be used to design and parameterise mathematical models that simulate population dynamics. The models can then be used to predict the outcomes of different experiments, which can then be carried out in the lab to validate the models. The aim is to establish the quantitative links between individual-level measurements and epidemic spread. For example, if we observe that variations in resources affect the ability of worms to resist or survive infection, can we predict how that will modify the circulation of the pathogen in the population? We will also assess the competitive abilities of different pathogen lines: some pathogens might have a higher growth rate within individual hosts but a lower transmission ability (for example if they kill their host too quickly). Which genotype 'wins' (i.e. spreads across a host population) will depend on a combination of factors, which can be measured and combined into a mathematical framework. These questions are important to understand the ecology and evolution of infectious diseases in natural populations. While they have been studied theoretically for many years, the application to real systems has remained difficult because of our lack of understanding of the detailed mechanisms of infection dynamics at the interface of individuals and populations. This project offers a unique opportunity to reconcile those different levels of investigation and test some fundamental assumptions of mathematical models that had not been validated before.

Technical Summary

This proposal is for a 30-month systems-based study with three main components: - Establish experimental populations of the nematode Caenorhabditis elegans infected with various pathogens, and measure the dynamics of infection at the individual and population levels. - Design and validate mathematical models for the population dynamics of nematodes and their pathogens. - Fit these mathematical models to experimental data using Bayesian statistical models. Nematode populations will be maintained on agar plates and fed on a choice of non-pathogenic bacteria. The number of individuals in different development stages (larvae and adults) can be assessed using a stereomicroscope. Individual life-history traits (fecundity and longevity) can be measured by isolating nematodes. As a first step, we will parameterise simple population dynamic models under different environmental conditions (feed and temperature) in different genotypes of C. elegans. We will then inoculate nematodes in isolation or in small groups with selected pathogens, namely: Microbacterium nematophilum (bacteria), Nematocida parisii (microsporidia) and Salmonella enterica Typhimurium (bacteria). By direct microscopy observation (including fluorescent markers), we will determine how infection affects individual life-history traits in different conditions and will measure transmission between individuals. Different strains of C. elegans will be compared. The data will be used to design mathematical models for the population dynamics of different host-pathogen combinations. The key stage of the project will aim to validate and parameterise those models by monitoring the demography and spread of infection in nematode populations. Individual measurements will provide prior parameter estimates for Bayesian models. Once validated, those models will be used to formulate predictions for experimental competition between multiple strains of nematodes or pathogens.

Planned Impact

Throughout this project, I will take an active role to achieve societal impacts as explained in the attached Impact Statement. Here I outline who will benefit, how they will benefit and what I will do to achieve impact. (a) Who: general public. How: Restore confidence in quantitative research on public health and environment. What: Demonstrate in a visual way how simple experiments can be combined with mathematical models to develop analytical and predictive tools for infectious disease dynamics. (b) Who: Schoolchildren. How: Education in health and environment. What: Work with local association to educate students about the diverse functions of microorganisms. (c) Who: Policy makers. How: Use of improved and more reliable mathematical models for infectious disease dynamics. What: In the long run, contribute towards the refinement and improvement of evidence-based policies for the control of infectious diseases.


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Description This project broke new ground in our understanding of the fate of pathogenic bacteria in the environment. We focused our attention on Pseudomonas aeruginosa and Salmonella enterica, two opportunistic pathogens which can infect a wide range of animals and plants. There is concern about the ability of these pathogens to persist in the environment (soil or water streams) but little is known about their ecology. Recent studies had suggested that other microorganisms found in the soil, such as nematodes (microscopic worms), could act as vectors for those bacteria. In this grant we combined laboratory experiments and mathematical models to measure the interactions between the free-living nematode C. elegans and the bacterial pathogens S. enterica and P. aeruginosa. Whereas other groups had studied these interactions at the molecular level, we took an ecological approach and asked how the worms and the bacteria would affect each other's ability to grow and spread. Our work revealed complex interactions, some of which challenged previously held views.
First, we showed that, even though S. enterica was known to shorten the lifespan of C. elegans, this was more than made up by an increase in the reproduction of the worm. Detailed measurements on individual worms enabled us to calibrate mathematical models for the population dynamics of nematodes feeding on different bacteria. We predicted that after 5 days, populations of worms feeding in S, enterica could reach 20 times the size of worms feeding in P. aeruginosa. This was successfully confirmed experimentally. These results have been presented at international conferences and submitted for publication to the journal Ecology.
The second part of our objectives was to determine whether these bacteria, which are both prey and parasites of C. elegans, would actually benefit from their interactions with the nematodes. We successfully measured the accumulation of bacteria within the intestine of C. elegans and their transmission (by faecal-oral route) between nematodes. Interestingly, P. aeruginosa is more efficient than S. enterica at colonising worms and achieves better transmission. Given our above finding that P. aeruginosa negatively affects the reproduction of its host, this raises questions about the longer-term net effect on the spread of these bacteria in the environment. We hope to address this in a future project.
Finally, we are now completing Objectives 2 and 3 using genetically engineered strains of S. enterica. Even though it has been known for several years that S. enterica (and other bacteria) can accumulate in the intestine of C. elegans, the respective roles of ingestion, bacterial replication and shedding have never been elucidated. Our results indicate that the turnover of live bacteria in the intestine of C. elegans is much higher than previously thought: for most of the life of C. elegans, the resident population of S. enterica is constantly replaced within less than 24 hours. Only in older worms did we observe longer persistence of bacteria. We have started investigating the effects of this process on the competition between S. enterica and P. aeruginosa (Objective 3).
Exploitation Route There are two main areas of applications for these findings: environmental microbiology and antibiotics. First, as more information is becoming available on the natural history of soil-dwelling nematodes, it is possible to investigate their interactions with pathogenic bacteria in experimental conditions closer to those found in their natural environments. This could be paired with field surveys in arable land. We have started pilot studies to determine the potential of such approaches.
A second application of our findings is the improvement of experimental models for the study of antibiotics and antimicrobial resistance (AMR). There is a worrying disconnect between in vitro studies of AMR, where bacteria are grown in liquid broth, and clinical studies which cannot observe the dynamics of bacteria in vivo. As C. elegans is rapidly becoming an established invertebrate animal model for bacterial pathogenesis, there is an opportunity to use this system to study AMR "in the context of the host" (which is one of the current funding priorities for the MRC) without having to resort to vertebrate animals. Our work is for the first time quantifying the dynamics of bacterial population dynamics within C. elegans, and we have started measuring the effects of antibiotics on these.
Sectors Agriculture, Food and Drink,Environment,Pharmaceuticals and Medical Biotechnology

URL http://aem.asm.org/content/80/17/5153.full
Description Bacterial strain - Pseudomonas aeruginosa PAO1 
Organisation University of Liverpool
Country United Kingdom of Great Britain & Northern Ireland (UK) 
Sector Academic/University 
PI Contribution none
Collaborator Contribution They provided bacterial strains for our experiments.
Impact The strains were used for experiments which have formed the base of publications. This has been acknowledged in Diaz and Restif (2014, Appl Env Microbiol.) and in the following manuscript (Diaz et al) submitted to Ecology.
Start Year 2013
Description Evolutionary Biology of Caenorhabditis and other Nematodes 14-17 June 2014 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Other academic audiences (collaborators, peers etc.)
Results and Impact Anaid Diaz presented her latest results to around 200 worldwide experts.

Several participants approached us, and we are planning to develop a new collaboration with a team in Paris about natural pathogens of Caenorhabditis nematodes.
Year(s) Of Engagement Activity 2014
URL https://registration.hinxton.wellcome.ac.uk/display_info.asp?id=390
Description Talk at British Society for Parasitology Conference, 2013 - AD 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Other academic audiences (collaborators, peers etc.)
Results and Impact Anaid Diaz (PDRA) gave an oral presentation of her research as part of the Meeting of the British Society for Parasitology held in Bristol in April 2013. The talk was attended by around 100 scientists.

No impact to date.
Year(s) Of Engagement Activity 2013
Description Workshops on microbes and ecology - OR and AD 
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 Olivier Restif and Anaid Diaz ran three 2-hour workshops for year-4 students at Barrow Primary School, Suffolk, attended by 24 students.

- Workshop1 (30/04/2012): natural selection; measurement and scale (using dissecting microscope); setting up compost experiments.

- Workshop 2 (21/05/2012): Board game on predation; observation of insects under microscope.

- Workshop 3 (13/06/2012): computer-based simulation of population dynamics; measurements and observations from compost experiments.

These workshops were very popular with the children, who were able to use microscopes for the first time. The contents of the workshops were prepared in close collaboration with the teacher, to ensure a good fit with the science curriculum. In particular, this offered a unique opportunity for children to develop their scientific enquiry. The activities enabled the teacher and his student to extend their previous activities on the theme of "habitats". After the series of workshops, the class carried on monitoring the contents of compost prepared during the workshops.
Year(s) Of Engagement Activity 2012