Early Career Scientist Research Symposium 2021

Professor Duncan Cameron

The University of Sheffield

Monday 22 March 12:00pm GMT - Decoding the secret language of soils: chemical communication in the rhizosphere and its implications for crop health

Duncan Cameron is Professor of Plant and Soil Biology in the Department of Animal and Plant Sciences at the University of Sheffield where his research group investigates the physiology and chemistry of plant-microbe interactions in the soil in the context of sustainable agriculture and global food security. Duncan is co-director of the University of Sheffield Flagship research institute, the Institute for Sustainable Food and is the University of Sheffield's lead academic for the N8 AgriFood program. Duncan's research is highly interdisciplinary, with collaborative projects linking between science, social science and the arts. He is actively engaged in public engagement, recent projects include the multi-media sci-art collaboration, Gaiamycota, the Sound of Science, and AquaKulture. Duncan actively supports equality, diversity and inclusion at The University of Sheffield as a gay role model.

: @dunkie_c

Professor Julian Marchesi

Cardiff University

Wednesday 24 March 1:00pm GMT: Denver Russel Memorial Lecture - FMT and going beyond “What did you say you are going to do!” and C. difficile infection

The human microbiome has recently been found to be an integral part of human biology.  For many years it was viewed as a source of infection, but now we know it is an essential component of the host and needed for normal development and function.  Professor Julian Marchesi's research since 2001 has focused on understanding the roles that the bacterial component of the microbiome plays in maintaining health and promoting diseases.

Professor Tania Dottorini

The University of Nottingham

Friday 26 March 12:00 GMT - Fighting Antimicrobial Resistance with machine learning, omics and big data analysis

Professor Tania Dottorini is an Associate Professor in Bioinformatics at the Nottingham School of Veterinary Medicine and Science (since June, 2016). Previously, a research fellow at the Imperial College London (2011 - 2016), Professor Dottorini's research themes include the development of novel bioinformatics and machine learning solutions to understand and diagnose infectious diseases in humans and animals.

Professor Dottorini's current interests are in the development of original methods and algorithms to gain deeper insight in biological problems related to human and animal health. To this purpose their research aims to merge different disciplines and knowledge/skills, including: bioinformatics and machine learning to develop predictive models and solve data mining tasks, in particular in scenarios involving large-scale data analysis from omics technologies (genomics, transcriptomics, proteomics, etc).

Judith Huygens

A one health perspective: The presence of antibiotic residues and antibiotic resistant bacteria in cattle manure intended for agricultural fields fertilization

Aims

In this study 25 cattle farmyard manure samples and 9 fattening calve slurry samples from Belgian farms were investigated on the presence of antibiotic resistant E. coli and 69 antibiotic residues. 

Methods and Results

Antimicrobial susceptibility testing was performed on preselected (for resistance to cefotaxime, ciprofloxacin, meropenem or colistin) and non-preselected E. coli isolates. In the fattening calve slurry samples, ciprofloxacin- and cefotaxime-resistant E. coli bacteria were both present in 7 of the 9 samples. In each fattening calve slurry sample, at least 8 different antibiotic residues were detected. Doxycycline, oxytetracycline, ciprofloxacin, enrofloxacin, flumequine and lincomycin were detected in all fattening calve slurry samples. Doxycycline and oxytetracycline were found in the highest mean concentrations; 2776 and 4078 µg/kg manure, respectively. Flumequine was found in a concentration of 536 µg/kg manure whereas the other residues were found in mean concentrations <100 µg/kg manure. Sulfadiazine was detected in 8 of the 9 samples in a mean concentration of 10895 µg/kg. In the cattle farmyard manure samples, ciprofloxacin- and cefotaxime-resistant E. coli were found in respectively 7 and 5 of the 25 samples. Antibiotic residues were found in only 4 of the 25 samples. Oxytetracycline was detected twice in a mean concentration of 250 µg/kg. Paromomycin, ciprofloxacin and enrofloxacin were detected in one sample in a concentration <100 µg/kg. Of the non-preselected E. coli isolates, 88% and 23% of the isolates from fattening calve slurry and cattle farmyard manure, respectively, were resistant to at least one of the antibiotics tested. A MDR up to 10 and 7 antibiotics, respectively, was observed.

Conclusions

The occurrence of antibiotic resistant E. coli and antibiotic residues seems to be higher in fattening calve slurry samples than in cattle farmyard manure samples used for agricultural field fertilization. 

Significance of Study

Antibiotic resistant bacteria and antibiotic residues enter the environment when using animal manure as fertilizer. 

Lewis Browett

How does seawater influx elicit compositional and functional changes in plant associated microbial communities? 

Aims

Storm surges, flooding, and the encroaching of seawater onto land that could potentially be used as farmland is predicted to increase with changes in our climate. It is, therefore, important to understand the knock-on effect seawater flooding may have on the health of the soil, and its ability to recover functioning.  The system chosen was a naturally occurring Saltmarsh-Pasture gradient, incorporating a legacy of differing levels of seawater ingress. We tested the hypotheses that (1) resistance to seawater flooding is a factor of pre-adaptation to the stress, and (2) this adaptation will resolve in different rates of recovery.

Methods and Results

This project was designed to simulate the flooding of terrestrial soil and elevations of Saltmarsh systems to monitor 1) changes in environmental parameters (pH, metal concentration, conductivity), 2) microbial functioning (metabolic activity and degradative enzymes), and 3) community composition (16S rRNA gene & ITS amplicon sequencing), over 0,1,96 and 192 hours submersion of soils from differing historical flooding exposure. This equates to 72 microcosms sampled across three timepoints: pre-flooding, post-flooding (immediately), and (14 days) post-recovery
Our data indicated that conductivity and pH levels within the pasture significantly increased with flooding duration, overall metal concentrations grouped dependent on site and concentrations decreased post recovery. Enzyme activity during flooding is similar between all sites, however post-flood pasture site and enzyme activity converges similar to Saltmarsh community activity as flooding treatment increased.

Conclusions

These results suggest that communities previously exposed to seawater flooding have increased resistance and resilience, with the addition of community composition (currently ongoing), we will be able to understand the community dynamics that underpin these data.  

Significance of Study

This research is important in understanding the impacts of seawater intrusion on potential farmland, potentially reducing community productivity and negatively impacting food security.

Alicia Showering

Elucidating the role of the skin microbiome in human attractiveness to mosquitoes

Aims

Some people are more attractive than others to mosquitoes, and consequently at higher risk of contracting vector borne diseases. The skin microbiome has a key role in producing volatiles which make up distinctive body odour profiles. Here, we aimed to identify differentially abundant bacteria between attractive and poorly attractive groups involved in production of repellent body odour.

Methods and Results

We used behavioural assays to measure attractiveness of participants to Anopheles coluzzii mosquitoes, 16S rRNA amplicon sequencing of the bacteria on human skin and gas chromatography of volatiles in body odour. We found differences in skin microbiome composition between the highly and poorly attractive groups. We identified eight differentially abundant amplicon sequence variants between poorly and highly attractive groups of participants. We found strong associations between the differentially abundant genera identified and known Anopheles attractants.

Conclusions

Bacterial genera we have identified explain some of the differences in attractiveness to mosquitoes between highly and poorly attractive people. 

Significance of Study

Understanding of the bacteria on human skin and associated volatile compounds within body odour offers potential for development of next generation vector control tools in the future. 

: @AliciaShowering

Gareth LuTheryn

Ultrasound-Mediated Therapy for the Treatment of Biofilms in Chronic Wounds

Aims

This research sought to develop an ultrasound-responsive agent capable of delivering nitric oxide (NO) contained in lipid-shelled microbubbles (MBs), which can co-administered with antimicrobial compounds for dispersal and elimination of biofilms.

Methods and Results

Pseudomonas aeruginosa biofilms were grown on Ibidi® dishes for 48 hours in wound constituent media (WCM), featuring key pathophysiological components of the in vivo wound environment. The anti-biofilm (dispersal) and bactericidal (killing) efficacy of ultrasound-responsive, neutral and cationic lipid-shelled MBs with an air or NO-filled core, was assessed in the presence and absence of sub-inhibitory (4 µg/mL) concentrations of gentamicin. Ultrasound stimulation was carried out for 40 seconds at 0.9 MHz, 20% duty cycle and 500 Hz PRF, achieving an acoustic pressure of 0.5 MPa. Insonified NO-loaded MBs demonstrated a significant 99.9% reduction in viable cells, by enhancing the efficacy of gentamicin. Fluorescence microscopy of treated biofilms showed that the combination of NO and ultrasound-mediated perturbations, achieved a 99.9% reduction in biofilm surface area. 

Conclusions

A major challenge in the delivery of NO in antimicrobial therapies is that it has a very limited half-life. By encapsulating it as the gaseous core of an ultrasound-responsive microbubble, NO is shielded from the external environment and allows successful control over its delivery and release. By inducing dispersal and perturbation of the biofilm with NO-loaded MBs, the physical barrier imposed by the extracellular matrix can be negated to improve treatment outcomes. 

Significance of Study

Biofilms are implicated in over 90% of chronic wounds, the management of which represents an annual cost of £5.6 billion to the NHS as well as significant patient morbidity and mortality. This research is the first of its kind to report the efficacy of lipid-shelled NO-loaded MBs, for the elimination of biofilms with a sub-inhibitory concentration of gentamicin. A proof-of-concept clinically viable translation of this research has been developed, which has the potential to alter the clinical outcome of these patients. 

References

LuTheryn, G., Glynne‐Jones, P., Webb, J. S. and Carugo, D. (2019) ‘Ultrasound‐mediated therapies for the treatment of biofilms in chronic wounds: a review of present knowledge’, Microbial Biotechnology. Wiley Online Library, pp. 1751-7915.13471. doi: 10.1111/1751-7915.13471.
Stride, E. P. and Coussios, C. C. (2009) ‘Cavitation and contrast: the use of bubbles in ultrasound imaging and therapy’, Proceedings of the Institution of Mechanical Engineers, Part H: Journal of Engineering in Medicine, 224. doi: 10.1243/09544119JEIM622.
Lafond, M., Shekhar, H., Panmanee, W., Collins, S. D., Palaniappan, A., McDaniel, C. T., Hassett, D. J. and Holland, C. K. (2020) ‘Bactericidal activity of lipid-shelled nitric oxide-loaded microbubbles’, Frontiers in Pharmacology. Frontiers Media S.A., 10. doi: 10.3389/fphar.2019.01540.
Howlin, R. P., Cathie, K., Hall-Stoodley, L., Cornelius, V., Duignan, C., Allan, R. N., Fernandez, B. O., Barraud, N., Bruce, K. D., Jefferies, J., Kelso, M., Kjelleberg, S., Rice, S. A., Rogers, G. B., Pink, S., Smith, C., Sukhtankar, P. S., Salib, R., Legg, J., Carroll, M., Daniels, T., Feelisch, M., Stoodley, P., Clarke, S. C., Connett, G., Faust, S. N. and Webb, J. S. (2017) ‘Low-Dose Nitric Oxide as Targeted Anti-biofilm Adjunctive Therapy to Treat Chronic Pseudomonas aeruginosa Infection in Cystic Fibrosis’, Molecular Therapy. Cell Press, 25(9), pp. 2104–2116. doi: 10.1016/J.YMTHE.2017.06.021

: @GarethLuTheryn

Cormac Rice

First phage-encoded depolymerase active against exopolysaccharides of Proteus mirabilis as a potential tool for prevention and treatment of CAUTIs.     

Aims

This study aimed to investigate the potential of phage depolymerases to prevent and treat catheter-associated urinary tract infections (CAUTIs) caused by Proteus mirabilis. The main objectives of the project were:
1.    Identification and sequencing of bacteriophages demonstrating strong ability to depolymerise exopolysaccharides (EPS) produced by P. mirabilis;
2.    Recombinant production of phage enzymes with such activities;
3.    Screening of expressed recombinant proteins for potential antimicrobial activity against P .mirabilis isolates and
4.    In vitro testing of the isolated depolymerases as anti-biofilm agents.

Methods and Results

A number of P. mirabilis bacteriophages were isolated using standard methods from environmental and agricultural sources. Subsequent screening for the presence of zones of matrix depolymerisation (halos) surrounding phage plaques formed on P. mirabilis lawns resulted in identification of several candidate phages. These were sequenced and their genomes annotated. Based on the results of sequence comparisons to experimentally verified depolymerases, a gene of a tail-spike protein of a new Proteus phage PM (isolated and characterised in this study), was selected for molecular cloning. The vector construct containing the tail-spike gene was successfully used for heterologous expression in E. coli and the resulting recombinant tail-spike protein was purified and concentrated. The ability of the protein to degrade EPS matrix of P. mirabilis B2000 (the host of PM phage), was confirmed by standard spot tests. In the next phase, in vitro tests were conducted with the recombinant tail spike protein, which demonstrated its ability to reduce the adherence of P. mirabilis to surfaces, thus hindering normal biofilm formation. Finally, the stability of the depolymerase against one of the major virulence factors of uropathogenic P. mirabilis, the 54-kDa extracellular metalloprotease ZapA, was investigated. Despite partial degradation, the tail-spike protein retained its depolymerase activity. 

Conclusions

With P. mirabilis being a common cause of difficult-to-treat CAUTIs, novel antimicrobials are required to treat such infections, especially in light of the increasing antibiotic resistance. We have shown the ability of Proteus bacteriophages isolated in this study to significantly reduce microbial load on indwelling catheters. In this study we have isolated and characterised the first phage-encoded depolymerase specific to P. mirabilis and demonstrated the ability of this phage-derived enzyme to delay the formation of biofilms by reducing the number of bacteria attaching to surfaces. This work highlights the potential for phages and their respective enzymes to act as effective antimicrobials, particularly in the treatment of P. mirabilis CAUTIs.

Significance of Study

With antimicrobial resistance at an all timeall-time high, the need for novel therapeutics is essential to tackle such issues. Bacteriophages have demonstrated remarkable potential as novel therapeutic agents against infections caused by various bacterial pathogens, including multi- and pan-drug-resistant ones, but our knowledge of the advantages, disadvantages and the range of applications of phage therapy is incomplete. As P. mirabilis via its urease production causes the build up of biofilm crystals and subsequent blockage of urinary catheters, this presents a serious problem, particularly for immunocompromised patients, as bacteraemia can occur, usually resulting in death. We for the first time isolated a depolymerase enzyme from a Proteus phage and demonstrated its ability to control biofilms formed by Proteus. As phage depolymerases do not kill bacteria, acting as anti-virulence agents instead, the use of phage depolymerases could become a promising therapeutic option in treatment of CAUTIs, helping to reduce the use of antibiotics and contributing to tackling the antibiotic resistance crisis. 

References

1.     Macleod SM, Stickler DJ. Species interactions in mixed-community crystalline biofilms on urinary catheters. J Med Microbiol. 2007;56(11):1549-1557. doi:10.1099/jmm.0.47395-0
2.     Alves DR, Nzakizwanayo J, Dedi C, et al. Genomic and Ecogenomic Characterization of Proteus mirabilis Bacteriophages. 2019;10(August):1-14. doi:10.3389/fmicb.2019.01783
3.     Milo S, Thet NT, Liu D, Nzakizwanayo J, Jones B V., Jenkins ATA. An in-situ infection detection sensor coating for urinary catheters. Biosens Bioelectron. 2016;81:166-172. doi:10.1016/j.bios.2016.02.059
4.     Nicolle LE. Infections associated with urinary catheters. Clin Infect Dis Second Ed. 2015:722-727. doi:10.1017/CBO9781139855952.122
5.     Clokie MRJ, Kropinski AM. Bacteriophages Methods and Protocols Volume 2: Molecular and Applied Aspects. Methods Mol Biol. 2009;502:xxii, 373 p. doi:10.1007/978-1-60327-565-1

Hayley Greenfield

Investigating RNA polymerase regulation in the antibiotic-producing Streptomyces bacteria.

Aims

To understand regulation of the key transcriptional enzyme, RNA polymerase, in Streptomyces. Also, to investigate the influence of RNA polymerase levels on antibiotic production and expression of silent gene clusters, that are not expressed in this organism under standard growth conditions and potentially encode novel antibiotics.

Methods and Results

Analysis of RNA -seq data from S. coelicolor and S. venezuelae revealed that the rpoBC operon, which encodes the large β and β’ subunits of RNA polymerase, is subjected to a largely unstudied form of gene regulation known as reiterative transcription (RT) at the transcriptional start site. Expression level from wildtype and mutant rpoBC promoter regions were tested using qRT-PCR before and after the induction of the stringent response to determine effects of RT on transcript level during both exponential growth and in response to stress. 

Conclusions

RpoBC is downregulated quickly after the induction of the stringent response in Streptomyces.  RT is confirmed to occur at the transcriptional start site of the rpoBC operon and is hypothesised to contribute to this drop in expression observed in response to stress. 

Significance of Study

The Streptomyces genus are well known to be prolific producers of useful secondary metabolites, including over half of all clinically relevant antibiotics. Enabling the production of previously uncharacterised compounds may allow the production of new antibiotics that can contribute to overcoming the current antimicrobial resistance crisis.

References

Bentley, S., Chater, K., Cerdeño-Tárraga, A.-M., Challis, G. L., Thomson, N. R., James, K. D., Harris, D. E., Quail, M. a, Kieser, H., Harper, D., Bateman, A., Brown, S., Chandra, G., Chen, C. W., Collins, M., Cronin, A., Fraser, A., Goble, A., Hidalgo, J., … Hopwood, D. a. (2002). Complete genome sequence of the model actinomycete Streptomyces coelicolor A3(2). Nature, 417(6885), 141–147. https://doi.org/10.1038/417141a

Hesketh, A., Chen, W. J., Ryding, J., Chang, S., & Bibb, M. (2007). The global role of ppGpp synthesis in morphological differentiation and antibiotic production in Streptomyces coelicolor A3(2). Genome Biology, 8(8). https://doi.org/10.1186/gb-2007-8-8-r161

Küster, C., Piepersberg, W., & Distler, J. (1998). Cloning and transcriptional analysis of the rplKA-or f31-rplJL gene cluster of Streptomyces griseus. Mol Gen Genet, 257(2), 219–229. http://www.ncbi.nlm.nih.gov/pubmed/9491081

Ochi, K., & Hosaka, T. (2013). New strategies for drug discovery: Activation of silent or weakly expressed microbial gene clusters. Applied Microbiology and Biotechnology, 97(1), 87–98. https://doi.org/10.1007/s00253-012-4551-9

Turnbough, C. L. (2011). Regulation of gene expression by reiterative transcription. Current Opinion in Microbiology, 14(2), 142–147. https://doi.org/10.1016/j.mib.2011.01.012