on bioRxiv: Using the Wax moth larva Galleria mellonella infection model to detect emerging bacterial pathogens

In the past few years I have worked on and off (i.e. doing pilot experiments, having a grant proposal rejected) on a very simple, but I believe quite powerful, assay to detect pathogenic bacteria in environmental samples. As it stands, microbial water and food safety is based on the quantication of faecal indicator bacteria: more poo-related bacteria in your sample=bad. The problem is that some poo-related bacteria are not as harmful as others, and more importantly, there are some very harmful bacteria that are not associated with poo and thus are completely missed by this approach. Alternative ways to detect pathogens have their own limitations: for instance, molecular markers  are restricted to a (small) set of ‘known knowns’ and are usually costly.

It would be much more useful to directly screen for bacteria able to cause disease, whatever their identity. Visiting student Rafael Hernandez took water and sediment samples and directly injected these in the the wax moth larva Galleria mellonella, a model for the innate immune system. There are many studies that inject particular pathogen strains in Galleria to quantify the rate of  killing, and this has been shown to correlate well with death in infected mice. What is novel about our approach is that we inoculated Galleria not with specific strains but with samples containing entire microbial communities to detect where pathogens might be present.

Most samples from local beaches (water and sediment) we assayed did not result in Galleria death after injection, but for some samples, mortality after overnight incubation at 37C was very high. Rafael could isolate clones from infected Galleria, which then were used to infect Galleria again to confirm that they were the cause of death, and were subsequently whole-genome sequenced. The results were exciting. We found usual suspect E. coli, as well as another well known (but not gastro-intestinal-associated) human pathogen Pseudomonas aeruginosa, both harbouring many virulence and antibiotic resistance genes. However, the most virulent clone was a Proteus mirabilis strain harbouring a Salmonella Genomic Island that has been reported in recent years from human and animal infections but (to my knowledge) not from the natural environment or from the UK. We also found Vibrio injenensis, a species only very recently described from human patients in Korea and not reported from anywhere else. The figure above shows on the left panel Galleria cumulative death (inoculation with 100, 10.000 and 1.000.000 cells) and on the right genome characteristics (virulence genes in blue, antibiotic resistance genes in red) for the four characterised clones.

The combination of climate change, changing farming practices, increased exposure through water-based recreation and rising levels of antibiotic resistance is expected to lead to an increase in hard-to-treat opportunistic infections. The approach described here looks promising to uncover the prevalence and identity of pathogenic bacteria in the environment, including potential emerging pathogens, which is key to assessing environmental transmission risks.

This study is out but not ‘out out’; I have used bioarxiv for the first time to make results accessible before peer-reviewed publication. Read it here:

Using the Wax moth larva Galleria mellonella infection model to detect emerging bacterial pathogens.

Rafael Hernandez, Elze Hesse, Andrea Dowling, Nicola Coyle, Edward Feil, Will Gaze & Michiel Vos

 

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Symposium Penryn 28-31 August 2018: Resolving Microbial Communities at Strain-level Resolution

I have the pleasure to announce a symposium/workshop to be held here at the University of Exeter’s Penryn Campus 28-31 August focusing on strain-level resolution metagenomics.

Metagenomics involves the high-throughput sequencing of random DNA fragments isolated from microbial communities, followed by assembly into longer fragments and assignment of gene functions and taxonomic identities. Novel computational developments are allowing us to increasingly resolve strain- and species level differences from metagenomic data. Rather than viewing communities as ‘bags of genes’, this approach enables us to gain deeper insights into the composition and functioning of microbial communities.The main focus of the workshop will be metagenome-assembled genomes(MAGs) which are operationally defined as metagenomic assemblies binned according to nucleotide composition and depth of coverage across multiple samples.

Training will be delivered by experts Chris Quince (at the University of Warwick and a research fellow at CLIMB, who are co-sponsoring the event) and Murat Eren and Tom Delmont at the University of Chicago. We will also have some presentations, including by Rachel Whitaker from the University of Urbana-Champaign and Jesse Shapiro who is at the University of Montreal. For more information, please have a look at the symposium website.

We strive to to stimulate participation by early-career researchers and to have an equal gender ratio. Please follow this link to register an expression of interestand to send an email expressing how this workshop could benefit your work and what your current position (e.g. PhD student, lecturer) is. At the start of June, you will be notified of the success of your application, and sent a link with payment details and accommodation options. For any specific questions please shoot me an email.

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ExeterMarine

The University of Exeter has a large contingent of researchers involved in Marine Sciences. As this perhaps has been a bit underappreciated in the past, Prof. Brendan Godley is currently leading the charge to tell the world what amazing activities are going on at our university. A new website, ExeterMarine is now up with details about marine projects, researchers and teaching. It features regular blog posts and also has an associated twitter account. Both my colleague at the European Centre for Environment and Human Health Will Gaze and myself originally started out as marine biologists, and still retain a keen interest in the field. There is a strong emphasis on Oceans and Human Health research in the ECEHH (check the website for current projects). There is a lot of relevant marine teaching too, for instance, I will start the undergraduate module “Oceans and Human Health” with head of the ECEHH Prof. Lora Fleming next year (more about that in a later post). In short, Cornwall is a fantastic place for marine science! Above, see the beautiful rugged North coast at Bedruthan Steps, and below some beautiful colour variants of the Dog whelk Nucella lapillus, a very common species here in the intertidal.

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NERC ‘FRESH’ PhD studentship

Applications are open January 15th – February 15th for a PhD studentship “Combining a novel phenotypic virulence screen with genomic approaches to uncover bacterial acquisition of multi-drug resistance and virulence in aquatic environments”. The studentship is organised through a NERC Doctoral Training Centre focusing on interdisciplinary freshwater science formed by the universities of Exeter, Bath, Bristol, Cardiff and Exeter, the Centre for Ecology and Hydrology and the British Geological Survey. The supervisory team will be made up by myself, Prof Will Gaze (ECEHH, Exeter), Prof Ed Feil (Milner Centre for Evolution, Bath) and Dr Jonathan Porter (Environment Agency). I have pasted an Abstract below; more information on the project is available here. I am very excited by the project and if you have been based in the UK for three years or longer and are interested in microbial ecology, genomics, antibiotic resistance and pathogenicity I urge you to apply (details here). We have an excellent supervisory team with an extensive network of collaborators and stakeholders in place. The student will be encouraged to participate in the wide range of professional development opportunities available at the University of Exeter and will be embedded within a multi-disciplinary research group with NERC, MRC and BBSRC PhD students situated within the Medical School laboratory based in the Environment and Sustainability Institute at the Exeter Cornwall Campus.

Project Abstract:

Poor microbiological water quality poses a major threat to environmental and human health and is likely to deteriorate through climate change and changes in land use. Worryingly, the global increase in antimicrobial resistance (AMR) could mean that exposure to contaminated waters leads to infections that are very hard or impossible to treat. The role of the natural environment in the dissemination of, and selection for, AMR through pollution with resistant bacteria, antibiotic residues, biocides and heavy metals is increasingly appreciated. The risk to human health posed by resistant bacteria present in the environment are likely to be significant. There is evidence for a range of pathogens that AMR and virulence genes can be carried on the same mobile genetic elements, and these could potentially spread between species. However, we currently know very little about the diversity and distribution of multidrug-resistant pathogens in aquatic environments.

In this project, we will selectively isolate pathogenic bacteria by directly injecting environmental samples into the Wax Moth larva Galleria mellonella, a well-established model for the mammalian innate immune system. Combining these assays with antibiotic selection and the ability to mobilise plasmids from freshwater microbial communities into model recipient bacteria will enable capture of novel plasmids carrying both AMR and virulence genes. Using both Illumina and Nanopore technologies to sequence chromosomes and plasmids of bacterial clones isolated from Galleria will give detailed information on pathogen identity, origin (i.e. hospital- or environment-associated) and carriage of known virulence- and antibiotic resistance genes. Functional genomics will be employed to identify unknown virulence and antibiotic resistance genes. Prevalence of multi-drug resistance and virulence will be related to catchment scale variables and point and diffuse sources of pollution.

Although microbiologically safe water is considered a fundamental human right, it is not exactly clear what it constitutes and how it is best monitored. This project will employ novel approaches enabling us to characterize the diversity and prevalence of antibiotic resistant pathogenic bacteria and their ecological drivers to ultimately assess human infection risk.

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SMBE satellite meeting on evolution of microbes in natural and experimental populations: synthesis and synergies

A few weeks ago I had the pleasure to be invited to a Society for Molecular Biology and Evolution (SMBE) Meeting aimed to bridge the divide between scientists studying microbial evolution in the lab (mainly through experimental evolution) and scientists studying evolution of microbes ‘in the wild’ (mainly through genome sequencing). As I have found myself in experimental evolution labs studying natural variation, and in a microbial ecology lab attempting experimental evolution, crossing this divide is a topic close to my heart! The meeting was jointly organized by scientist at Tezpur University in India and the University of Bath in the UK and took place in Kaziranga National Park in Assam, a state in the not-so-often visited North East of India. The 30+ hour door-to-door travel was certainly worth it however. Small meetings in relatively secluded venues have the advantage that it is much easier to talk to people. The meeting included several very good talks by young researchers who were awarded travel grants, ranging from topics as diverse as biofilm formation, linkage disequilibrium and plasmid transfer. Each session ended with a general discussion which greatly facilitated trying to bridge research divides and identifying challenges. Included in the meeting was a mini-safari through the park, in which we saw a variety of birds, deer, monkeys and the Indian rhinoceros (Rhinoceros unicornis) from up close! In short, a stimulating meeting: scientifically, socially and culturally!

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Paper out: Are pangenomes adaptive or not?

I have been interested in bacterial pan genomes for some time: apart from acquiring mutations in genes shared between strains, bacteria are able to pick up genes (via several Lateral Gene Transfer processes), duplicate genes and also lose genes. These processes can take place rapidly, and could potentially have large effects on fitness. Together with Adam Eyre-Walker and several co-authors, I wrote an Opinion piece exploring both the rate  at which these accessory genome changes can occur and their evolutionary fate (see this post). Earlier this year, I published a short meta-analysis with Nadia Andreani and Elze Hesse (see this post) explicitly demonstrating that species with genetically more diverse core genomes also have more diverse accessory genomes. This pattern is inconsistent with predominantly strongly deleterious effects and consistent with slightly deleterious or neutral fitness effects. A recent Opinion piece in Nature Microbiology by McInerney etal argued that most observed accessory genome changes are actually adaptive. This prompted Jesse Shapiro to write a short letter to Nature Microbiology contrasting these findings with ours, prompting Adam and myself to write a critique of the McInerney paper and McInerney to write a response. I feel that the evolution of accessory genomes is one of the least understood but most pressing issues in evolutionary microbiology. Hopefully the present discussion will help to stir up debate.

M. Vos and A. Eyre-Walker (2017) Are pangenomes adaptive or not? Nature Microbiology 2, 1576 DOIdoi:10.1038/s41564-017-0067-5 cough

Prospective PhD’s: please see the blog post below!

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PhD opportunities

The BBSRC GW4 Universities Doctoral Training Program is currently advertising PhD projects. This is a slightly complicated process, but basically comes down to prospective students picking a project offered by scientists working at participating universities and institutions and applying to the program. I (Dr Michiel Vos, m.vos@exeter.ac.uk) offer one of these projects as part of a team of  GW4 colleagues (Prof Ed Feil, University of Bath, Dr Ben Temperton, University of Exeter and with collaborator Prof Adam Eyre-Walker, University of Sussex). Below the project description:

Quantifying the rate and fate of bacterial gene content evolution

Bacteria are the most abundant and diverse life forms on earth, vital to the functioning of all ecosystems and to human health and wellbeing. Bacteria rely on a highly diverse array of mechanisms to create genomic changes which, combined with usually enormous population sizes, can result in rapid evolutionary change. Most bacterial species have ‘fluid’ genomes, where a stable core genome is complemented by an accessory set of genes that can be rapidly taken up through lateral transfer and lost through deletion (1). Such Gene Content Changes (GCC) have been estimated to occur at rates similar to nucleotide substitution in the core genome and are likely to have more significant effects on fitness (2). Despite its importance to bacterial evolution, the rate of gene content turnover and the selective effects of GCC are not well-understood. Some researchers argue that most changes have only small effects on fitness (3), whereas others argue that most variation in accessory genes is adaptive (4). As the tempo and mode of bacterial evolution is fundamental to a wide range of fields, from molecular epidemiology to biotechnology, this controversy needs to be resolved. This project takes a comparative genomics approach to 1) explicitly measure the rate of GCC in a range of different bacteria and 2) develop novel tests of selection to quantify the selective effects of gene content change.

  1. Ochman H, Davalos LM. The nature and dynamics of bacterial genomes. Science. 2006; 311(5768):1730-3
  2. Vos M, Hesselman MC, te Beek TA, van Passel MW, Eyre-Walker A. Rates of Lateral Gene Transfer in Prokaryotes: High but Why? Trends in microbiology. 2015; 23(10):598-605
  3. Andreani, E. Hesse and M. Vos. Prokaryote genome fluidity is dependent on effective population size. 2017 ISME J doi:10.1038/ismej.2017.36
  4. McInerney, James O., Alan McNally, and Mary J. O’Connell. “Why prokaryotes have pangenomes.” Nature Microbiology. 2017 28; 2:17040. doi:10.1038/nmicrobiol.2017.40

I am second supervisor (along with Prof Sam Sheppard at Bath) on a project led by Prof Ben Raymond at the University of Exeter:

Clone wars in niche space: exploring the evolutionary and genetic basis for bacterial species

Understanding the forces that shape bacterial genetic variation is a fundamental problem in microbiology.  Classifying bacteria into meaningful species groups is also essential for applied microbiology and ecology. Many bacterial species have been shown to exhibit extensive variation in gene repertoires, where a set of core genes shared by all strains are supplemented with a set of accessory genes that are only present in a subset of strains.  The ability to exploit particular niches is thought to depend on the acquisition of a range of accessory genes, typically acquired via horizontal gene transfer (Vos et al. 2015).  However, genetic variation in core genes shared between different strains is often associated with ecological niche (Raymond et al 2010, Zheng et al 2017)(Figure 1), suggesting that variation in core genes may be ecologically significant.

This project will explore the extent to which core genetic variation arises from neutral genetic drift process and from positive selection in distinct habitats, a question with broad importance for understanding bacterial biology and evolution.  For instance, many isolates of economic and therapeutic importance (Bacillus thuringiensis biopesticides, Escherichia coli probiotics etc) are closely related to isolates capable of causing disease.  In both these cases, humans consume large doses of viable microbes in food or as therapeutic agents.  Understanding the potential of beneficial bacteria to cause harm or acquire harmful genes is particularly important for assessing the safety of these uses.   If core genetic variation limits virulence or niche shifts then the risk of these applications will be substantially reduced  (Raymond & Federici 2017).

Bacillus thuringiensis, in particular, has an excellent safety record and is the most widely applied microbial insecticide, facilitating environmentally friendly mosquito control and pest management.  Nevertheless, disagreements regarding its ecological niche, and it taxonomic status relative to Bacillus cereus, a causative agent of diarrhea, have threatened its continued use in the European Union.  In this project, the student will (1) carry out genome sequencing and phenotypic characterization of isolates from natural populations (2) apply experimental evolution and re-sequencing approaches to look for convergent adaptive mutations in novel niches (3) use CRISPR-Cas9 genome editing to introduce putative adaptive alleles into different genetic backgrounds.

Raymond, B., & Federici, B.A. (2017).FEMS Microbiology and Ecology.  doi: 10.1093/femsec/fix084

Zheng, J., Gao, Q., Liu, L., Liu, H., Wang, Y., Peng, D., Ruan, L., Raymond, B., & Sun., M.  (2017) mBio 8:e 00822-17. doi: 10.1128/mBio.00822-17.

Raymond, B., Wyres, K., Sheppard, S., Ellis, R.J., Wright, D.J., & Bonsall, M.B (2010). PLoS Pathogens 6(5): e1000905.

Vos, M., Hesselman, M., Te Beek, T., van Passel, M.,Eyre-Walker, A. (2015) Trends Microbiol 23.10: 598-605

If this seems of interest, please check the BBSRC SW BioSciences DTP website. The application deadline is Midnight, Monday 4th December 2017.

 

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