P a g e 1 Uncovering pathogens in the soil microbiome using the Galleria mellonella virulence model Submitted by Benedict Lawton to the University of Exeter as a dissertation for the degree of Master of Science by Research in Medical Sciences in December 2019 This dissertation is available for Library use on the understanding that it is copyright material and that no quotation from the dissertation may be published without proper acknowledgement. I certify that all material in this dissertation which is not my own work has been identified and that no material has previously been submitted and approved for the award of a degree by this or any other University. Signature: ………………………………………………… P a g e 1 P a g e 2 Abstract Environmental reservoirs of disease can harbour opportunistic pathogens that are harmful to human health. Anthropogenic effects such as climate change and antimicrobial resistance are likely to cause increase exposure to, and harm from, these pathogens. It is thus vital to study these environmental reservoirs in order to be prepared appropriately and prevent these diseases from emerging or re- emerging. Studies into the prevalence and diversity of opportunistic pathogens in the environment are largely focused on water sources. Microbial communities in soil are known to harbour a diverse range of species, yet are relatively poorly understood. Studies typically screen for specific ‘indicator pathogens’, or test the virulence of individual pathogens. These methods overlook a great deal of pathogenic species that can be present. Recent advances in the Galleria mellonella model system have shown that it can be used to screen for pathogens in microbial communities taken from environmental samples. We used the G. mellonella model to screen for potentially harmful pathogens in soil microbial communities. We frequently detected high levels of virulence, suggesting that soil often harbours pathogenic species. We were subsequently able to isolate pathogens from these soils and characterise their virulence, finding most of them to be highly virulent. 16s sequencing determined that the majority of pathogens found were either Serratia liquefaciens or Providencia alcalifaciens, both of which are known human pathogens. A highly virulent Aeromonas hydrophila was also identified. AMR profiling of these isolates found lower levels of antibiotic resistance than has typically been reported from clinical isolates. This study emphasises the importance of soil as a reservoir for pathogens, and provides a glimpse at the range of potentially harmful species that it contains. P a g e 2 P a g e 3 List of contents Abstract ............................................................................................................. 2 List of contents ................................................................................................. 3 List of figures .................................................................................................... 6 List of tables ..................................................................................................... 7 List of supplementary data .............................................................................. 7 Author’s declaration ........................................................................................ 8 Acknowledgements .......................................................................................... 9 List of abbreviations ...................................................................................... 10 Introduction .................................................................................................... 11 What is a pathogen? ..................................................................................... 11 The problem of opportunistic pathogens ....................................................... 12 Notable examples of bacterial diseases ....................................................... 12 Emerging infectious diseases ....................................................................... 14 The influence of anthropogenic effects on EIDs ........................................... 14 The rise of antimicrobial resistance .............................................................. 16 Which environments are typically disease reservoirs? ................................. 18 Traditional methods for detecting environmental pathogens ........................ 20 Limitations of traditional detection techniques .............................................. 21 The Galleria mellonella model system .......................................................... 22 Using the Galleria model to detect environmental pathogens ....................... 25 Chapter 2 Bacterial pathogens in soil ...................................................... 26 Pathogens in soil .......................................................................................... 26 The significance of rhizosphere bacteria ...................................................... 28 Aims of this study .......................................................................................... 28 Materials and Methods ................................................................................... 30 P a g e 3 P a g e 4 Soil sample collection ................................................................................... 30 Extraction of bacterial communities from environmental samples ................ 31 G. mellonella injection with whole soil communities ...................................... 32 Pathogen clone isolation ............................................................................... 32 Naming conventions used for isolated soil clones ........................................ 33 G. mellonella injection with clonal soil isolates .............................................. 34 Species identification of isolated clones using 16s rRNA ............................. 34 Phylogenetic analysis of 16s genes .............................................................. 35 Antimicrobial resistance profiling .................................................................. 36 Results ............................................................................................................ 37 1: Suspected pathogens in 29 soil communities ........................................... 38 2: High levels of virulence in 115 isolated clones .......................................... 40 3: Detailed virulence assays for selected clones .......................................... 44 4: Clone identification ................................................................................... 48 5: 14 AMR profiles found across 32 clones ................................................... 56 Discussion ...................................................................................................... 58 The effect of soil type on the frequency of pathogens .................................. 58 Community virulence compared to clone virulence ....................................... 59 On the prevalence and diversity of pathogenic soil species ......................... 60 Detailed virulence assays of detected pathogens ......................................... 61 The relevance of Serratia to human infection ............................................... 63 The relevance of Providencia to human infection ......................................... 64 The relevance of Aeromonas to human infection .......................................... 64 The relevance of Enterococcus to human infection ...................................... 65 The relevance of Bacillus to human infection................................................ 66 Potential risk factors for infection .................................................................. 66 Comments on the Galleria mellonella virulence model ................................. 67 P a g e 4 P a g e 5 Limitations of the project ............................................................................... 68 Concluding remarks ...................................................................................... 69 Supplementary material ................................................................................. 71 Bibliography ................................................................................................... 88 P a g e 5 P a g e 6 List of figures Figure 1: Predicted deaths due to AMR Figure 2: Workflow diagram of the whole project Figure 3: Soil community virulence Figure 4: Soil community virulence by soil type Figure 5: Soil community virulence of paired root-soil samples Figure 6: Virulence of isolated clones Figure 7: Number of clones isolated from each sampling site Figure 8: Galleria survival curves for 8 pathogenic clones Figure 9: Galleria survival curves for AMR-profiled clones Figure 10: Clone morphologies Figure 11: Species identified Figure 12: Evolutionary relationships of Bacillus clones Figure 13: Evolutionary relationships of Serratia clones Figure 14: Evolutionary relationships of Providencia clones Figure 15: Evolutionary relationships of Enterococcus clones Figure 16: Sites where each genus was identified P a g e 6 P a g e 7 List of tables Table 1: Examples of clinically relevant soil-borne pathogens Table 2: Sampling areas Table 3: Numbers of clones isolated from Galleria hemocoel Table 4: Species identification using 16s rRNA sequencing Table 5: AMR profiling List of supplementary data Table S1: Sampling Sites Figure S1: Maps of sampling sites Table S2: Morphology of