PhD opportunity: the role of natural antimicrobials in selection for antibiotic resistance

img_5998The NERC 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, offer one of these projects as part of a team of  GW4 colleagues (Prof Will Gaze, University of Exeter, Prof Ed Feil, University of Bath and Dr Ruth Airs, Plymouth Marine Laboratory). Below the project description:

The increasing prevalence of antimicrobial resistance (AMR) in bacteria is one of the most pressing problems in global health care. Selection for antibiotic resistance occurs through antibiotic use in the clinic and community and contamination of the environment with heavy metals, biocides and antibiotic residues. However, AMR mechanisms are ancient and their presence in environments with no or minimal exposure to human activity indicates that not all AMR selection is anthropogenic. It is here proposed to test for the first time whether naturally produced antimicrobials can select for resistance to clinical antibiotics. Virtually all organisms, from bacteria to humans, produce antimicrobial compounds. Selection for resistance to these antimicrobials has the potential to also result in resistance to clinical antibiotics when the same bacterial pathways are targeted. Although the ubiquity of interactions between bacteria and antimicrobial producers offers great potential for the molecular diversification of AMR mechanisms, the extent of cross-resistance between naturally produced antimicrobials and clinical antibiotics has remained virtually unstudied.

Seaweeds form a diverse and abundant component of coastal ecosystems and commonly exhibit antimicrobial activity. Seaweed species are colonized by distinct bacterial assemblages, and this process is at least in part mediated by a high diversity of exuded secondary metabolites. Recent research in my lab (Colclough etal in prep) has demonstrated that Staphylococcus aureus strains that were more resistant to clinical antibiotics were on average also more resistant to seaweed extracts. The fact that seaweeds can select for bacteria that are resistant to their metabolites and the observation of cross-resistance between seaweed antimicrobials and clinical antibiotics suggests that there is potential for seaweeds to select for AMR. This PhD project will 1) test whether distinct seaweeds harbour distinct antibiotic resistance genes using metagenomic sequencing, 2) test whether reservoirs of resistance genes can be transferred from seaweed-associated bacterial metagenomes to opportunistic pathogens using functional metagenomics and 3) identify human pathogens on seaweeds and test whether they are more resistant to antibiotics than conspecifics from other environmental reservoirs using whole genome sequencing.

It is critical to understand natural processes governing selection for AMR in the environment. It has been estimated that there are over six million exposure events to cephalosporin-resistant E. coli through recreational use of coastal bathing water in England and Wales alone, demonstrating the potential risks of environmental reservoirs of AMR to human health. This project will allow a first insight into the potential of species interactions to select for AMR mechanisms that confer cross-resistance to clinically relevant antibiotics.

To be clear: the project below is funded only when YOU decide to select this particular (and excellent) project AND are offered entry to the PhD program. See the DTP site for eligibility details etc and the University of Exeter postgraduate research page to apply. The deadline for application is January 6th 2017.

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The Transition from Class Room to a Year of Research

A guest post my BSc student Michael Tadesse who joined us last year for a lab project:

In the academic year 2015-2016, I undertook a Professional Training Year (PTY) at the European Centre for Environment and Human Health (ECEHH) in Cornwall, UK under supervsion of Dr Michiel Vos. I went into my PTY hoping to gain a better idea of what a career in research would look like. During my second year, I was contemplating pursuing a PhD. So along with the boost a PTY would give to my CV, I would also find out whether I could handle the commitment of a PhD. In this post I will try to reflect on my placement, my problems in adjusting to a new environment and my experiences gathering data once I settled in properly. mtMy activities were based on a previous PTY project that screened a variety of seaweeds for antibacterial potential. We now wanted to test whether our extracts are more, less or equally effective on pathogens that are resistant or susceptible to clinical antibiotics. This depends on whether the mode of action of natural antimicrobials is independent from genes conferring clinical antibiotic resistance. Several resistance mechanisms, such as  those involving drug uptake and efflux, are prone to cause cross-resistance, where an organism displays increased resistance to a second drug. The opposite process is also possible: collateral sensitivity means that an organism that has developed resistance to one drug can display increased sensitivity to a second drug. Promising candidates could be further identified using mass spectrometry to elucidate the nature of compounds in collaboration with another lab, adding a mechanistic component to the project and the opportunity to gain experience in organic chemistry methods. I first created several E. coli mutants using one host strain and several plasmids with resistance genes and GFP or mCherry fluorescence tags.  I obtained them from my lab mate, Dr Uli Klumper. I introduced these plasmids using electroporation or selective mating. The transformed cells were then plated out in selective agar containing the respective antibiotic. Successful mutant colonies were then selected through fluorescence microscopy. This seemed pretty straightforward but I had problems creating the necessary electro-competent host cells or introducing the plasmids. Fortunately, in the end, I managed to find solutions for every obstacle with the help of Uli. Secondly, eleven strains of resistant and susceptible Staphylococcus aureus were obtained from Dr Ruth Massey (University of Bath, UK). Each resistant strain had a susceptible counterpart and the two strains only differed in their respective resistance second part of this project was testing my seaweed extracts against these strains. I performed simple Kirby-Bauer disc diffusion assays. I soaked assay discs in seaweed extract for 24 hours, and dried them in a laminar flow hood for 15 minutes. The discs were then placed on agar plates that were mixed with a E. coli or S. aureus strain. Positive control (imipenem, 4 mg/l) and negative control (60% methanol) discs were soaked and dried in the same way.  The plates were incubated at 37°C. After 18 hours, zones of inhibition (areas with no visible bacterial growth) were measured in three different directions for each disc. Unfortunately, the E.coli plates showed no inhibition. However, there was more activity against S. aureus and instances of collateral sensitivity and co-resistance could be observed in some strains (see graphs below). There were two extracts, made from F. lumbricalis and C. baccata that showed activity against all strains.


Antibiotic resistance versus seaweed susceptibility. Halo size in disc diffusion assay for 11 seaweed species on S. aureus strain sets consisting of susceptible and resistant strains. Error bars represent standard deviation.

All in all, this year has taught me that patience and inventiveness is key in life science. Also, ne upside of these mishaps was that I was forced to try and learn several basic molecular microbiology techniques. This will definitely be of great use to me in the future, especially during my final year dissertation project. I started this year as an undergrad with a general knowledge of medical science. However, my experience of the academic research environment was limited to lab practicals and one small scale attempt at a controlled trial. So I felt it was necessary for my development that I had a taste of what happened at the forefront of medical/life science. I came out of it a more mature professional, better versed in the scientific method and scientific communication. Also, being part of a vibrant research group allowed me meet several researchers from varying fields. More importantly, despite my many adversities I still have retained a strong interest in pursuing an academic research career but now with the added knowledge of the pitfalls this career direction entails. Finally, I would like to thank my supervisor Dr Michiel Vos for his guidance and patience, Dr Uli Klümper for tolerating my incessant advice seeking and everyone else in the lab for providing such a relaxed and welcoming atmosphere.

Michael Tadesse

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Re-post: Pitch: a Guide to the Bacteria and Archaea

The blog is a bit inactive at the moment and I hope to turn that around soon. In the mean time, I decided to re-post a blog entry from early last year to hopefully breath new life into this idea. I had been in touch with a publisher but the conditions were not right to pursue this book idea. hopefully there are other publishers or avenues available. Any comments are more than welcome in any case!



Background: inspired by a post by Jonathan Eisen on his blog ‘the tree of life’, I wrote a little post on ‘a Field Guide to the Bacteria’ last year. Both our posts were about the rather megalomaniacal (in a good way) idea of sampling, sequencing and identifying bacteria from the environment in ‘real time’. So rather than walking through the field with binoculars identifying birds, walking (anywhere) with a small sequencing device+computer and identifying microbes. This is obviously quite futuristic, but I have been thinking about a guide book to bacteria (from now on read ‘bacteria’ to mean both bacteria and archaea) that would be perfectly feasible. I think it is a very cool idea, but it would be great to have some of your feedback to see if it is actually worth pursuing. I am starting with the premise that a) bacteria cannot be identified in the field and b) a guide on bacteria cannot be in any way, shape or from be representative of actual diversity (in contrast to say most regional bird guides). More positively, I also think that c) bacteria can be beautifully illustrated and d) that they have very diverse and interesting life-styles that would make for some good reading. Applying a ‘traditional’ natural history format focusing on species diversity could be a very good way of bringing the sophistication of bacteria to the attention of the general public.

The pitch: a guide book where 100 bacterial species are represented by a page of text and an opposing page with a Scanning Electron Microscopy (SEM) picture. The format will be like any other guide book, utilizing pictograms to signify key characteristics (e.g. genome size, metabolism and habitat) and a short text segment on general biology as well as a segment on how the species impacts on us humans. Using 100 species would allow covering a lot of bacterial variation: many different taxonomic groups, many different habitats and niches and many different types of metabolism. Examples would include oceanic species, soil species, gut species, phyllosphere species, pathogenic species, hotspring species, food fermenting species, endosymbiotic species, photosynthesizing species, lithotrophic species, multicellular species, tiny species, ‘giant’ species, magnetic species, halophilic species, Bacteroidetes, Actinobacteria, Chloroflexi, etc etc. Each species would serve to highlight one specific, interesting behaviour.

The illustrations: beautiful SEM colour illustrations will be essential. Hands down the most beautiful SEM pictures of bacteria I have seen so far are those of award-winning ‘micronaut‘ Martin Oeggerli. Martin uses highly sophisticated post-processing colouring for an ‘acrylic finish’. See below for a fantastic image of a consortium of gut bacteria (including a plant fibre and the eukaryotic parasite Giardia on the far right):06-intestinal-bacteria-670

The format: there is some precedent: MicrobeWiki features illustrated descriptions of selected bacterial species (example page here). Although this is a great resource, it is not comparable to the coffee table book I have in mind. There is a case to be made to not produce a book, but build an app instead. This might be cheaper to do and also allow reaching a wider audience. Also, it will be relatively easy to add many more species to the app when the project is succesful. And it will allow linking to relevant websites, such as those hosting genome sequences etc (the downside to this is that the app needs curating to prevent dead links and just having a multi-FASTA file with millions of A’s and T’s and C’s and G’s pop up by itself is not that helpful to the average reader and would need a lot of extra context).  Of course, the two formats are not mutually exclusive. I still think a book is nicer though.

An example page: because a picture is worth a 1000 words, my sister Leonie helped me to produce an example page using my favourite bacterium Myxococcus xanthus. The SEM is by Juergen Berger at the MPI and was used a cover for Current Biology for one of my papers with Gregory Velicer (although beautiful, I think the colours are a bit artificial, soil is not blue!). I am not sure about the pictograms yet. Most conventional guides have a little map with a distribution range coloured in. I do not think that will be very helpful for most bacterial species, as they are generally very widely distributed and they definitely do not migrate (my thoughts on bacterial biogeography are summarized in this paper and this other paper). A pictogram of the habitat would be very useful however. Likewise, a pictogram summarizing genome information (genome size, chromosome and plasmid number and shape (circular or lineair) and number of genes) would be essential. More tricky but absolutely necessary would be a clear pictogram summarizing metabolism (and whether the species is aerobic or anaerobic). There are many other small interesting pieces of information that could be summarized in such a standard way. BOOK

The contributors: this will have to be a team effort. Here’s who would be needed:

  • the scientific community. PI’s that work on an interesting model system would be asked to a) write two pages on what is cool about their model system (+ provide a list of basic information needed to produce the pictograms) and b) send a sample to an SEM facility. In return they will have an outlet to educate the public about their favourite organism by being featured in a book and they will have permission to use a beautiful picture of their bug in their future talks. I know (of) many labs that I could ask to contribute and I do not see any problems there.
  • an SEM wizard. The production of SEMs and, as importantly, the post-processing of images would need to be done by a single person/facility to guarantee continuity and consistency. The quality of the images would be vital to the project.
  • an editor. That would be me. As with the images, the text needs to have a consistent format and style. Editing text provided by other scientists would be much less work than writing copy on 100 species myself from  scratch. I like writing; this should be doable.
  • a graphic designer. The book needs to look slick. The pictograms need to be informative and beautiful at the same time.
  • a  couple of expert microbiologists. Guides usually have a background section at the start featuring more general information about habitats, morphology and life history. This will be a must for this guide, as most readers will know comparatively little about the featured organisms. For instance, having an expert on metabolism on board to briefly explain this complicated topic to the lay person would make the guide much more valuable. The same goes for genome sequencing. Hopefully Jonathan Eisen would want to write the foreword.

How to get there: I am not sure! I guess there are three ways of securing money to make this happen: approaching publishers, crowdfunding and outreach grants. I do not have a good idea about the costs involved and the sales needed to cover them. I would greatly appreciate your comments on the book idea itself and about financing!


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SETAC Nantes 2016

Another guest post, this time by Isobel Stanton, a PhD student who started work with Will Gaze October last year, co-funded by BBSRC and the pharmaceutical company AstraZeneca Global Environment (co-supervised by Prof Jason Snape). (See here for an overview of some of the work that the University of Exeter does in partnership with AstraZeneca.) She is currently investigating selection for antibiotic resistance in complex microbial communities at very low (environmentally relevant) concentrations of antibiotics. Due to the regulatory interest under the Water Framework Directive, macrolides are being used as a case study. Here goes:

Last month Will, Aimee and I visited Nantes, France for the STEAC Europe 26th Annual Meeting: Environmental contaminants from land to sea: continuities and interface in environmental toxicology and chemistry. We arrived in Nantes on Sunday 22nd May and had some time to explore the city before the opening ceremony that evening. We visited the Cathedral Saint-Peter-and-Saint-Paul, the Château des ducs de Bretagne and Les Machines De L’île Nantes. Unfortunately, we arrived too late and missed Le grand éléphant moving (

We all presented in the session titled “Antibiotics and Antibiotic Resistance in the Environment: Ecological Fate and Effects, Resistance Development and Implications for Human Health.” Will and Aimee gave platform presentations; Will talking about co-selection for AMR by biocides and Aimee spoke about her work looking into selection for AMR in the environment. I presented the work I’ve undertaken since starting my PhD, also on selection for AMR in the environment, as a poster presentation. The other sessions had a much more regulatory focus than our work but it was still a good insight into the other side of the industry.abcOur trip was unfortunately cut short and we had to miss the last day of the conference to make sure we were able to get out of the city (French strikes!). We managed to arrive at the airport hours early, to a scene which looked like the beginning of an apocalypse movie (standstill traffic, cars parked on verges, people trekking with suitcases and the odd casual fire), and ended up being delayed by four hours due to air traffic control strikes). Twelve hours later we arrived back in Cornwall – much more peaceful!nantes2

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Microbial water quality All-Party Parliamentary Group meeting at Westminster

A guest post Will Gaze’s student Anne Leonard:

Last Monday, 23rd May 2016, I was invited to attend an All-Party Parliamentary Group (APPG) at the Houses of Parliament in Westminster, London. The purpose of this meeting was to discuss the Environment Agency ‘Spill Frequency Trigger Permitting’ consultation, which questions the need to implement a limit on the number of times water companies are allowed to discharge untreated sewage into sensitive waters, such as bathing beaches and shellfish waters.

The majority of the UK’s sewers are combined, collecting sewage and grey water from people’s homes and industry as well as storm water from roads and roofs. During periods of wet weather, large volumes of wastewater enter these combined sewers and exceed their capacity. Combined sewer overflows (CSOs) – are used to release this mixture of untreated sewage and storm water directly into the environment, so that raw sewage does not back up into homes and streets. Raw sewage contains numerous disease-causing microorganisms (bacteria, viruses, protozoans, etc.), which can make people ill when they come into contact with them.

In accordance with the European Bathing Water Directive and the Water Framework Directive, bathing and shellfish waters are regularly tested for the presence of sewage during the bathing season (mid-May to the end of September). However, evidence suggests that the sampling regime used only detects 11% of CSO spills, and bathers may still be exposed to bathing waters contaminated by raw sewage.

Member of Parliament, Steve Double, chaired the meeting, which was attended by MPs, representatives from NGOs like Surfers Against Sewage and the Marine Conservation Society, water companies, the Environment Agency, the shellfish industry, and the watersports sector. Speaking to this room of experts, I presented the results of three studies that I have been working on with Dr Will Gaze and Dr Ruth Garside on the threat that bacteria in coastal waters pose to human health:

  1. Participants needed for new health survey ( )
  2. Are we exposed to antibiotic resistance in coastal waters? (
  3. Beach bum survey (

Since the meeting last week, MPs have confirmed that they will be writing to the Environment Agency, urging them to enforce tighter legal thresholds on CSOs from systems that have already been designed or recently updated to reduce the frequency of spills to just 3 per bathing season.large group westminster

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Visit to the Netherlands Institute of Ecology, Wageningen

NIOO2Last week I visited past colleague and collaborator Paolina Garbeva at the Netherlands Institute of Ecology NIOO in Wageningen where I worked as a postdoc between 2009 and 2011. Paolina is an expert in the secondary metabolites (especially the volatile ones) of soil bacteria and fungi. It was great to hear about the research in her group, sat in the typical blistering Dutch sun on the roof terrace of the groundbreaking eco-building. I also gave a talk about our seaweed antimicrobial work. Paolina and I plan to write papers and proposals in the near future, hopefully more on that soon on the blog!


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Paper out: A barrier to homologous recombination between sympatric strains of the cooperative soil bacterium Myxococcus xanthus

MyxofruitingbodyJust out in the ISME journal, a paper on my favourite bacterium, Myxococcus xanthus. I did my PhD with Greg Velicer on this fascinating species which builds beautiful multicellular fruiting bodies that can be seen with the naked eye (click on my personal page underneath the page header for a publication list with pdf downloads, including a review paper). I thought it would be nice to return to the strains I isolated back then and do some population genomics. It turned out to be an interesting project although not without significant obstacles; papers are a bit like sausages, they can be very enjoyable but you do not want to necessarily know how they were made. Anyway, from the Abstract:

The bacterium Myxococcus xanthus glides through soil in search of prey microbes, but when food sources run out, cells cooperatively construct and sporulate within multicellular fruiting bodies. M. xanthus strains isolated from a 16×16 centimetre-scale patch of soil were previously shown to have diversified into many distinct compatibility types that are distinguished by the failure of swarming colonies to merge upon encounter.  We sequenced the genomes of 22 isolates from this population belonging to the two most frequently occurring MultiLocus Sequence Type (MLST) clades in order to trace patterns of incipient genomic divergence, specifically related to social divergence. Although homologous recombination occurs frequently within the two MLST clades, we find an almost complete absence of recombination events between them. As the two clades are very closely related and live in sympatry, either ecological or genetic barriers must reduce genetic exchange between them. We find that the rate of change in the accessory genome is greater than the rate of amino acid substitution in the core genome. We identify a large genomic tract that consistently differs between isolates that do not freely merge and therefore is a candidate region for harbouring gene(s) responsible for self/non-self discrimination.

Fig_1-page-001Above a nice Figure made by first author Seb Wielgoss that shows the location in the centimeter-scale plot the representatives of the two clades (genotypes) were isolated from (A) and a core genome phylogenetic tree clearly showing that the two clades are distinct, with limited within-clade diversity (B). For instance, strains A31 and A34 do not have a single SNP difference across their aligned conserved sequence of 7.6 Mb! For the homologous recombination analysis, co-author Xavier Didelot employed the FineStructure software (for a nice explanation on how this works see here), which has also been used in a high profile study on the genetic ancestry of the British population. The figure below shows a co-ancestry matrix on the left, with warmer colours representing higher percentages of recombinational copying from one genome to another. Genomes belonging to the same FineStructure population are connected by a vertical branch in the tree on the right, with the rest of the tree indicating inferred relationships between seven identified populations. The dark blue colours indicate negligible (<0.01%), homologous recombination between Clades I (populations 5-7) and V (populations 1-4). To my knowledge, this is only the third time a barrier to recombination between bacteria from the same population has been shown, and the first for a soil bacterium.

Fig_2-page-001Actually the main reason I wanted to sequence these genomes is to find out how different strains are able to recognize kin from non-kin. It is likely that Myxococcus meets other distinct strains, as many types can be found on a scale of even centimeters. In another paper, it could be shown that genetically distinct swarms were generally very bad at building fruiting bodies together. That scenario might actually not even be very relevant, as mixing of distinct cells is most likely when they are actively swarming. In the same paper however, it could be shown that swarms did not mix upon encounter, and that there thus must be some genetic mechanism whereby cells can discriminate self from non-self:Presentation1

By sequencing a set of genomes of strains that can either mix or not, genetic differences that consistently differ between such sets can be identified. Indeed, a large genomic tract was located that consistently differed with swarming compatibility. Again Seb made a really nice Figure to visualize these differences in gene content. First a phylogeny based on gene content variation in the variable region (A). Clusters match compatibility type (CT) groupings derived from social swarm merger assays. On the right a visual representation of the absence (black) and presence of single (light red) and multicopy (dark red) genes in the focal region. The proof in the pudding was a strain (A92) that could not mix with strains that it was otherwise genomically closely related to that was identical in this genomic tract with other strains it could mix with.Fig_4-page-001This study was explorative in nature; you never know what you are going to find when sequencing new genomes. So there were a number of other significant findings (having multiple interesting findings perhaps paradoxically made the manuscript a harder sell…). For instance, we could show that changes in gene content (in the ‘accessory genome’) were much more prevalent than mutational changes (in the ‘core genome’). This finding actually was the inspiration of a recent Opinion paper on accessory genome evolution, see this recent post. I had hoped that differences on the DNA level would shed light on ecological differences between strains that could explain their coexistence but that proved very difficult as most genes were of unknown function. We did find massive differences in CRISPR-Cas systems though (these results ended up mainly in the supplemental material). The advance online publication is freely available from the journal’s website:

Wielgoss, Sébastien, et al. “A barrier to homologous recombination between sympatric strains of the cooperative soil bacterium Myxococcus xanthus.” The ISME Journal (2016).


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