Comparative phenotypic, proteomic and genomic approaches to assess lipopolysaccharide and outer membrane protein diversity among isolates of Yersinia ruckeri recovered from Atlantic salmon and rainbow trout

Yersinia ruckeri is the causative agent of enteric redmouth (ERM) disease in farmed salmonids. ERM disease is traditionally associated with rainbow trout (Oncorhynchus mykiss¸ Walbaum), but the incidence of the disease in Atlantic salmon (Salmo salar) has increased in recent years. Historically, motile (biotype 1), serotype O1 isolates of Y. ruckeri have been mostly responsible for ERM in rainbow trout worldwide but non-motile (biotype 2), serotype O1 isolates have become increasingly prevalent in this species over wide geographic areas since their first isolation in the UK in the 1980s. Yersinia ruckeri isolates responsible for infection of salmon have been less well characterised than those from rainbow trout and little is known about their diversity. The emergence of new pathogenic strains, together with vaccine breakdown in the field, has emphasised the need for greater knowledge about strain diversity which may lead to the development of improved vaccines for both species. In the present study, a unique and extensive strain collection encompassing 135 isolates of Y. ruckeri were characterised using complementary phenotypic, proteomic and genomic approaches. In the initial part of this thesis, 135 isolates recovered over a 14 year period in the UK from infected Atlantic salmon (109 isolates) and rainbow trout (26 isolates) were phenotypically characterised through biotype, serotype, and outer membrane protein (OMP) -type analysis. Atlantic salmon isolates represented a wider range of O-serotypes and associated lipopolysaccharide (LPS) types, and had more diverse OMP profiles than those from rainbow trout. Most significantly, a new O-serotype/LPS type was identified in 56 Atlantic salmon isolates; other Atlantic salmon isolates were represented by serotypes O1 (five isolates), O2 (34 isolates) and O5 (14 isolates). This new LPS type comprises a core polysaccharide region similar to that of serotype O1 but has a unique, previously unidentified O-antigen region. Atlantic salmon isolates could be assigned to one of four major OMP-types and to one of 11 OMP-sub types. Isolates recovered from rainbow trout were represented by the same non-motile clone that is responsible for the majority of ERM outbreaks in this species within the UK. This clone was not associated with any infected salmon. However, two isolates of the novel serotype O1/O5 were recovered from rainbow trout in 2010 and 2011. These data suggest that different Y. ruckeri strains are specifically adapted to cause disease in either Atlantic salmon or rainbow trout. The efficacy of current vaccine formulations against different clonal groups must be examined. Subsequently, an in-depth characterisation of the outer membrane (OM) proteome of isolates recovered from Atlantic salmon and rainbow trout was conducted. Outer membrane proteins are at the interface of host pathogen interactions, with important roles in adherence, evasion of host immune response, and transport. Using a bioinformatic prediction pipeline and four publicly available genomes, 141 proteins were confidently predicted to be OM associated. Subsequently, the OM proteomes of eight representative isolates (four from rainbow trout; four from Atlantic salmon) were analysed using a combination of gel-based and gel-free proteomic approaches. In total, 66 OMPs were identified through this combined approach, of which 28 were unique to the gel-free approach and 13 were unique to the gel-based approach. Further to this, the OM proteomes of these eight representative isolates were examined when cells were grown under conditions that aimed to mimic the in vivo and environmental conditions of Y. ruckeri. These included growing cells aerobically at 22°C and 28°C, anaerobically, under iron-depletion and in an artificial seawater medium at 22°C. In total, 76 OMPs were identified in all eight isolates under these growth conditions. Finally, a phylogenetic study was undertaken whereby the genomes of 16 representative isolates encompassing a range of biotypes, serotypes, host species (eight from rainbow trout, seven from Atlantic salmon and one from European eel), geographic locations and dates of isolation were considered. A phylogenetic species tree based on the concatenated sequences of 19 housekeeping genes revealed host specific lineages suggesting an earlier host-associated evolutionary split within Y. ruckeri. Subsequent analysis of the presence, absence and variation of the nucleotide and amino acid sequences of the 141 predicted OMPs revealed high levels of conservation (with 120 OMPs showing less than 1% nucleotide variation). One hundred and thirty proteins were identified in all 16 genomes examined. However, 11 proteins were not, and these included invasins, OmpE and proteins involved in pilus biogenesis. Further examination of the OMPs OmpA and OmpF, which were identified in the genomes of all 16 isolates, revealed variation in the surface exposed loop regions which may play a role in pathogenicity and/or host specificity. This study represents a comprehensive characterisation of Y. ruckeri isolates recovered from Atlantic salmon and rainbow trout using a range of molecular techniques, and reveals important adaptations that the bacteria may make in order to survive both inside and outside of the host. Importantly, this study provides comprehensive support for future work involving this fish pathogen

Yersinia ruckeri isolates recovered from diseased Atlantic Salmon (Salmo salar) in Scotland are more diverse than those from Rainbow Trout (Oncorhynchus mykiss) and represent distinct subpopulations

Yersinia ruckeri is the etiological agent of enteric redmouth (ERM) disease of farmed salmonids. Enteric redmouth disease is traditionally associated with rainbow trout (Oncorhynchus mykiss, Walbaum), but its incidence in Atlantic salmon (Salmo salar) is increasing. Yersinia ruckeri isolates recovered from diseased Atlantic salmon have been poorly characterized, and very little is known about the relationship of the isolates associated with these two species. Phenotypic approaches were used to characterize 109 Y. ruckeri isolates recovered over a 14-year period from infected Atlantic salmon in Scotland; 26 isolates from infected rainbow trout were also characterized. Biotyping, serotyping, and comparison of outer membrane protein profiles identified 19 Y. ruckeri clones associated with Atlantic salmon but only five associated with rainbow trout; none of the Atlantic salmon clones occurred in rainbow trout and vice versa. These findings suggest that distinct subpopulations of Y. ruckeri are associated with each species. A new O serotype (designated O8) was identified in 56 biotype 1 Atlantic salmon isolates and was the most common serotype identified from 2006 to 2011 and in 2014, suggesting an increased prevalence during the time period sampled. Rainbow trout isolates were represented almost exclusively by the same biotype 2, serotype O1 clone that has been responsible for the majority of ERM outbreaks in this species within the United Kingdom since the 1980s. However, the identification of two biotype 2, serotype O8 isolates in rainbow trout suggests that vaccines containing serotypes O1 and O8 should be evaluated in both rainbow trout and Atlantic salmon for application in Scotland

Diversification of OmpA and OmpF of Yersinia ruckeri is independent of the underlying species phylogeny and evidence of virulence-related selection

Yersinia ruckeri is the causative agent of enteric redmouth disease (ERM) which causes economically significant losses in farmed salmonids, especially Atlantic salmon (Salmo salar) and rainbow trout (Oncorhynchus mykiss, Walbaum). However, very little is known about the genetic relationships of disease-causing isolates in these two host species or about factors responsible for disease. Phylogenetic analyses of 16 representative isolates based on the nucleotide sequences of 19 housekeeping genes suggests that pathogenic Atlantic salmon and rainbow trout isolates represent distinct host-specific lineages. However, the apparent phylogenies of certain isolates has been influenced by horizontal gene transfer and recombinational exchange. Splits decomposition analysis demonstrated a net-like phylogeny based on the housekeeping genes, characteristic of recombination. Comparative analysis of the distribution of individual housekeeping gene alleles across the isolates demonstrated evidence of genomic mosaicism and recombinational exchange involving certain Atlantic salmon and rainbow trout isolates. Comparative nucleotide sequence analysis of the key outer membrane protein genes ompA and ompF revealed that the corresponding gene trees were both non-congruent with respect to the housekeeping gene phylogenies providing evidence that horizontal gene transfer has influenced the evolution of both these surface protein-encoding genes. Analysis of inferred amino acid sequence variation in OmpA identified a single variant, OmpA.1, that was present in serotype O1 and O8 isolates representing typical pathogenic strains in rainbow trout and Atlantic salmon, respectively. In particular, the sequence of surface-exposed loop 3 differed by seven amino acids to that of other Y. ruckeri isolates. These findings suggest that positive selection has likely influenced the presence of OmpA.1 in these isolates and that loop 3 may play an important role in virulence. Amino acid sequence variation of OmpF was greater than that of OmpA and was similarly restricted mainly to the surface-exposed loops. Two OmpF variants, OmpF.1 and OmpF.2, were associated with pathogenic rainbow trout and Atlantic salmon isolates, respectively. These OmpF proteins had very similar amino acid sequences suggesting that positive evolutionary pressure has also favoured the selection of these variants in pathogenic strains infecting both species

Plastic pollution and fungal, protozoan, and helminth pathogens – a neglected environmental and public health issue?

Plastic waste is ubiquitous in the environment and can become colonised by distinct microbial biofilm communities, known collectively as the ‘plastisphere.’ The plastisphere can facilitate the increased survival and dissemination of human pathogenic prokaryotes (e.g., bacteria); however, our understanding of the potential for plastics to harbour and disseminate eukaryotic pathogens is lacking. Eukaryotic microorganisms are abundant in natural environments and represent some of the most important disease-causing agents, collectively responsible for tens of millions of infections, and millions of deaths worldwide. While prokaryotic plastisphere communities in terrestrial, freshwater, and marine environments are relatively well characterised, such biofilms will also contain eukaryotic species. Here, we critically review the potential for fungal, protozoan, and helminth pathogens to associate with the plastisphere, and consider the regulation and mechanisms of this interaction. As the volume of plastics in the environment continues to rise there is an urgent need to understand the role of the plastisphere for the survival, virulence, dissemination, and transfer of eukaryotic pathogens, and the effect this can have on environmental and human health

Draft Genome Sequence of the Commensal Escherichia coli Strain F-18

Here, we report the draft genome sequence of Escherichia coli strain F-18, originally isolated from the feces of a healthy individual in 1977. The draft genome is 5,246,829 bp, with a G+C content of 50.50%, and it encodes 4,933 predicted coding sequences (CDSs), 10 rRNAs, and 84 tRNAs

Draft Genome Sequence of the Tumor-Targeting Salmonella enterica Serovar Typhimurium Strain SL7207

Salmonella enterica serovar Typhimurium strain SL7207 is a genetically modified derivative of strain SL1344, which preferentially accumulates in tumors and can be used as a vehicle for tis..

Comparative bioinformatic and proteomic approaches to evaluate the outer membrane proteome of the fish pathogen Yersinia ruckeri

Yersinia ruckeri is the aetiological agent of enteric redmouth (ERM) disease and is responsible for significant economic losses in farmed salmonids. Enteric redmouth disease is associated primarily with rainbow trout (Oncorhynchus mykiss, Walbaum) but its incidence in Atlantic salmon (Salmo salar) is increasing. Outer membrane proteins (OMPs) of Gram-negative bacteria are located at the host-pathogen interface and play important roles in virulence. The outer membrane of Y. ruckeri is poorly characterised and little is known about its composition and the roles of individual OMPs in virulence. Here, we employed a bioinformatic pipeline to first predict the OMP composition of Y. ruckeri. Comparative proteomic approaches were subsequently used to identify those proteins expressed in vitro in eight representative isolates recovered from Atlantic salmon and rainbow trout. One hundred and forty-one OMPs were predicted from four Y. ruckeri genomes and 77 of these were identified in three or more genomes and were considered as “core” proteins. Gel-free and gel-based proteomic approaches together identified 65 OMPs in a single reference isolate and subsequent gel-free analysis identified 64 OMPs in the eight Atlantic salmon and rainbow trout isolates. Together, our gel-free and gel-based proteomic analyses identified 84 unique OMPs in Y. ruckeri. Significance: Yersinia ruckeri is an important pathogen of Atlantic salmon and rainbow trout and is of major economic significance to the aquaculture industry worldwide. Disease outbreaks are becoming more problematic in Atlantic salmon and there is an urgent need to investigate in further detail the cell-surface (outer membrane) composition of strains infecting each of these host species. Currently, the outer membrane of Y. ruckeri is poorly characterised and very little is known about the OMP composition of strains infecting each of these salmonid species. This study represents the most comprehensive comparative outer membrane proteomic analysis of Y. ruckeri to date, encompassing isolates of different biotypes, serotypes, OMP-types and hosts of origin and provides insights into the potential roles of these diverse proteins in host-pathogen interactions. The study has identified key OMPs likely to be involved in disease pathogenesis and makes a significant contribution to furthering our understanding of the cell-surface composition of this important fish pathogen that will be relevant to the development of improved vaccines and therapeutics

Apparatus and method for centrifugation and robotic manipulation of samples

A device for centrifugation and robotic manipulation of specimen samples, including incubating eggs, and uses thereof are provided. The device may advantageously be used for the incubation of avian, reptilian or any type of vertebrate eggs. The apparatus comprises a mechanism for holding samples individually, rotating them individually, rotating them on a centrifuge collectively, injecting them individually with a fixative or other chemical reagent, and maintaining them at controlled temperature, relative humidity and atmospheric composition. The device is applicable to experiments involving entities other than eggs, such as invertebrate specimens, plants, microorganisms and molecular systems

From wastewater discharge to the beach: Survival of human pathogens bound to microplastics during transfer through the freshwater-marine continuum

Large quantities of microplastics are regularly discharged from wastewater treatment plants (WWTPs) into the aquatic environment. Once released, these plastics can rapidly become colonised by microbial biofilm, forming distinct plastisphere communities which may include potential pathogens. We hypothesised that the protective environment afforded by the plastisphere would facilitate the survival of potential pathogens during transitions between downstream environmental matrices and thus increase persistence and the potential for environmental dissemination of pathogens. The survival of Escherichia coli, Enterococcus faecalis and Pseudomonas aeruginosa colonising polyethylene or glass particles has been quantified in mesocosm incubation experiments designed to simulate, (1) the direct release of microplastics from WWTPs into freshwater and seawater environments; and (2) the movement of microplastics downstream following discharge from the WWTP through the river-estuary-marine-beach continuum. Culturable E. coli, E. faecalis and P. aeruginosa were successfully able to survive and persist on particles whether they remained in one environmental matrix or transitioned between different environmental matrices. All three bacteria were still detectable on both microplastic and glass particles after 25 days, with higher concentrations on microplastic compared to glass particles; however, there were no differences in bacterial die-off rates between the two materials. This potential for environmental survival of pathogens in the plastisphere could facilitate their transition into places where human exposure is greater (e.g., bathing waters and beach environments). Therefore, risks associated with pathogen-microplastic co-pollutants in the environment, emphasises the urgency for updated regulations on wastewater discharge and the management of microplastic generation and release