If the COVID-19 pandemic has reminded us of anything, it’s how important identifying and interrupting transmission chains is for controlling outbreaks of infectious diseases. Our paper describes how we used deep sequencing to investigate the variation of a bacterium called Citrobacter rodentium as it spread between mice. We found that we could use the differences in the relative abundance of within-host single nucleotide variants (iSNVs) to infer who infected who more precisely.

Instead of getting into the nitty-gritty of the science, I want to share this project's journey from an initial idea to a publication. It took a long time, a lot of effort, and sometimes disheartening knockbacks before finally finding a home. And as is so often the case with research, there’s so much left to do!

In the late 90s, I was doing my PhD at what was then the Institute of Virology and Environmental Microbiology in Oxford. My project was to make bacteria glow in the dark and use them as biosensors for industrial pollution  (1). In 2000, I left Oxford for Imperial College London, where I swapped industrial pollution for infectious diseases. Now, I was making bacteria glow in the dark to help understand how they cause disease and speed up drug and vaccine discovery and development using biophotonic imaging (2).

One of the bacteria I engineered was C. rodentium, which infects mice, similar to how enterohaemorrhagic Escherichia coli (EHEC) and enteropathogenic E. coli (EPEC) infect people. Using that glowing strain of C. rodentium, my colleague Simon Clare and I showed that laboratory-grown bacteria first infect a patch of lymphoid tissue called the caecal patch before infecting the colon (3). I then showed that C. rodentium rapidly spreads from infected to healthy animals, that bacteria shed in the faeces of infected mice are 1,000 times more infectious than laboratory-grown bacteria, and that those faecal bacteria do not need to infect the caecal patch before infecting the colon (4).

These findings sparked my interest in disease transmission and the need for experimental animal models that included this crucial aspect of the infection cycle. That way, we could better investigate the virulence factors that aid transmission rather than just those that cause disease. We might also be saved from drawing conclusions about infectious diseases that were simply an artefact of the way we did our experiments. Around that time, I met Bill Hanage, and we made these very arguments in an opinion piece published in Nature Microbiology (5).

In 2007, I started my lab at Imperial, switching from C. rodentium to work on Mycobacterium tuberculosis, Staphylococcus aureus, and Streptococcus pyogenes. Funding was flowing, and things were going well. There was just one problem – my partner was offered a job on the other side of the world. So, in 2009, I left Imperial to take up a Sir Charles Hercus Fellowship from the Health Research Council of New Zealand and moved to the University of Auckland.

That same year, Angus Buckling and colleagues published a great review in Nature on experimental evolution (6). They argued that “the next logical step in studying the evolution of microorganisms is to carry out experimental evolution in natural environments”. I immediately thought C. rodentium would be perfect for this. It would let us study how a gut pathogen evolves as it naturally transmits between its mammalian hosts.

In 2010, Bill and I submitted our first funding application to New Zealand’s “blue skies” Marsden Fund, which has ten disciplinary panels and a success rate of around 10%. It also has a two-stage application process. First, you submit a one-page executive summary. About 20% of applications are then invited to the full proposal stage, after which about half will be funded. That year, we tried the Ecology, Evolution and Behaviour panel. We didn’t get past the first round. The following year, we submitted to the Biomedical panel. We got through to the full proposal stage but weren’t funded.

All wasn’t lost, however. Because we’d got through to the second round, my faculty gave us some seed money to start our experiments. With another small grant-in-aid from the Maurice Wilkins Centre, my PhD student Hannah Read began a stripped-down version of our proposed C. rodentium experimental evolution project. I’ve carried on applying for funding to support this work, including six applications to the Marsden Fund over the last ten years. Two made it to the full proposal stage but weren’t funded. I’ve tried other funding bodies, too, but it’s often been too “blue skies” for them. Apart from small grants-in-aid, my only success has been with the Explorer Fund from New Zealand’s Health Research Council. This fascinating little fund operates as a lottery; every application that meets the criteria of being “transformational” has an equal chance of being funded (7). It’s not much money, but it helped.

On a more positive note, in 2017, Bill was awarded an R01 from the National Institutes of Health to study how to infer transmission from genome sequences. His team could now sequence all the evolving C. rodentium samples we had collected during the experiment. That was completed by the middle of 2019, so data processing began shortly before the pandemic struck.

I’m immensely proud to see this work published. For one, it’s helped to show how within-host variation can tell us who infected who. This turned out to be relevant far beyond experiments. When the Delta variant of SARS-CoV-2 caused an outbreak over the Northern Hemisphere's 2021 summer, within-host variation was one of the key pieces of evidence confirming that transmission was possible from vaccinated hosts (8). Shortly after, the CDC updated advice on masking as the pandemic returned with a vengeance. The experiments in this paper were a critical part of the groundwork that enabled such fine-scale detective work. Given that wasn’t even part of the original reason for doing the work, it’s an excellent example of the unanticipated findings of blue skies research.

Meanwhile, in my lab in Auckland, we have a freezer full of evolved bacteria with interesting phenotypes that, hopefully, we’ll one day get funding to investigate more thoroughly.

References:

1. Wiles, S. et al.  Calibration and deployment of custom-designed bioreporters for protecting biological remediation consortia from toxic shock. Environmental Microbiology 7, 260–269 (2005).
2. Andreu, N. et al.  Noninvasive biophotonic imaging for studies of infectious disease. FEMS Microbiology Reviews 35, 360–394 (2011).
3. Wiles, S. et al. Organ specificity, colonization and clearance dynamics in vivo following oral challenges with the murine pathogen Citrobacter rodentium. Cell Microbiol 6, 963–972 (2004).
4. Wiles, S. et al. Emergence of a ‘hyperinfectious’ bacterial state after passage of Citrobacter rodentium through the host gastrointestinal tract. Cell Microbiol 7, 1163–1172 (2005).
5. Wiles, S. et al. Modelling infectious disease — time to think outside the box? Nat Rev Microbiol 4, 307–312 (2006).
6. Buckling, A.  et al. The Beagle in a bottle. Nature 457, 824–829 (2009).
7. Liu, M. et al. The acceptability of using a lottery to allocate research funding: a survey of applicants. Research Integrity and Peer Review 5, 3 (2020).
8. Siddle, K. J. et al. Transmission from vaccinated individuals in a large SARS-CoV-2 Delta variant outbreak. Cell 185, 485-492.e10 (2022).