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 gene.mt-labThe 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.

mt-data

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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