The Genotypic and Phenotypic Comparison of Virulent Yersinia Enterocolitica from Humans and Animals

Final report summary: Yersinia enterocolitica is a Gram negative bacterium which can cause diarrhoea and related, more serious, diseases in humans. This infection is believed to be foodborne. The 1999-2000 Defra national abattoir survey indicate that 6% of cattle, 13% of sheep and 26% of pigs were colonised with this potential pathogen. However, the role of veterinary strains in human disease is currently unknown.
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Strains of Y. enterocolitica are defined on the basis of biotype (BT 1-5) and serotype. Biotypes 2-5 are generally considered pathogenic, on the basis of the presence of the major, well-characterised virulence determinants, including the plasmid pYV, and correlation with pathogenicity in a mouse infection model. Strains of BT 1a conversely do not possess these virulence determinants and have no pathogenicity in mice and are consequently considered non-pathogenic. Strains of BT1b are lethal in the mouse model and contain all the virulence determinants known but also have an additional high pathogenicity island. With the aim of determining the relationship between human and animal strains, phenotypic comparison using the standard bio-serotype approach was undertaken of isolates from the abattoir survey with a isolates from human disease cases provided by the Health Protection Agency (HPA) from the same time period.
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In collaboration with Dr T Chesty (HPA), initial characterisation of the strains by biotype and serotype showed that 58% of the animal isolates and 53% of the human isolates were Biotype (BT) 1a. The main recognised pathogenic Y. enterocolitica biotype isolated from livestock was BT3 (O:5,27) (35% of sheep, 22% of pigs and 4% of cattle) but this biotype was not detected in any of the human isolates investigated. The major pathogenic biotypes of strains isolated from humans were BT3 (O:9) (24%) and BT4 (O:3) (19%) whereas of the veterinary isolates investigated, only pigs (11%) carried BT3 (O:9) strains.
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To further investigate the relationship between human and animal isolates, 88 strains were selected and further characterised by genotyping. In collaboration with Dr S. On, DFVF, Denmark, the technique of fluorescent Amplified Fragment Length Polymorphism (AFLP), was successfully established and used to genotype other enteric pathogens. AFLP primarily distinguished Y. enterocolitica strains according to their biotype, with strains separated into two distinct clusters; cluster A comprising largely of the putatively pathogenic biotypes (BT 2-4) and cluster B comprising of the putatively non-pathogenic biotype 1A strains and a single BT 1B isolate. Within these two genotypic clusters, sub-clusters were observed and these largely were serotype-associated. Similarity of AFLP profiles in BT3 (O:9) strains from pigs and humans appeared to confirm that pigs are a major source of human infection with this putatively pathogenic biotype. Evidence was also obtained that sheep may be a reservoir for human infections with BT4 (O:3). However, the results also suggested that some strains causing human disease do not come from veterinary sources identifiable at this time.
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As part of a collaborative venture, a PhD student, under the supervison of Prof Brendan Wren, LSHTM, genotyped the 88 selected strains using a pan-Yersinia micro-array. The Pan-Yersinia species array included all sequences that were present in the sequenced strains Y. pestis CO92, Y. pestis KIM and Y. enterocolitica 8081 1b. In addition, unique sequences from Y. enterocolitica and Y. pseudotuberculosis strains, which were available from collaborators and had been deposited in Genbank, were also included (e.g. the O-antigen locus of Y. enterocolitica serotypes O: 3 and O: 9). From this data, the phylogeny revealed two distinct clades; a “pathogenic” clade primarily comprising pathogenic biotypes from humans, cattle, pigs and sheep and a “non-pathogenic” clade primarily comprising the non pathogenic biotype 1a human, cattle, sheep and pig isolates. Within the “pathogenic” clade the data split into two distinct groups, caused by the presence or absence of the virulence plasmid pYV, which was confirmed using GeneSpring. Loss of the virulence plasmid alone did not account for the clear divide of the pathogenic and non-pathogenic strains from each other and this strongly indicates that there are large chromosomal differences between the two groups. Interestingly, the basis for formation of subgroups via array analysis was different from the AFLP analysis. AFLP subgrouped isolates according to serotype, though there was a difference between human and porcine BT4 (O:3) isolates. Microarray analysis subgrouped the strains on an, as yet, unidentified genetic basis, with the human and porcine BT4 (O:3) isolates forming a statistically significant link. The ability of array analysis to compare the presence and absence of individual genes will allow the future identification of genetic markers responsible for the groupings.
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In order to compare the isolates on the basis of phenotypic properties related to virulence, the 88 selected strains were assayed for their ability to adhere to, and invade, cultured epithelial cells in vitro. All of the isolates were capable of high levels of adhesion to epithelial cells, regardless of biotype or source. All isolates tested, excepting 3 non-motile BT1a strains, were also capable of invading HEp-2 cells. In general, BT1a strains were approximately 100-fold less invasive than the pathogenic biotype strains, but there was no correlation between source and invasive ability. To further the phenotypic comparison, the ability was assessed of the strains to survive within U937 human macrophages cells in vitro, and the levels of cytokines secreted by infected cells. All of the strains tested were capable of surviving within macrophages to the same degree. There were differences in cytokine secretion induced by challenge with the strains. BT3 (O:5,27) strains displayed increased secretion of the pro-inflammatory cytokines IL-6 and IL-8. The BT1a isolates were capable of modulating cytokine secretion in that the levels of IL-6 and IL-8 expressed were similar to those observed for the pathogenic biotypes. However, challenge with BT1a strains resulted in a large increase in secretion of the pro-inflammatory cytokine TNF <P>
Investigation of the molecular basis for virulence requires the use of defined mutants but the production of such mutants in Y. enterocolitica has been problematic. Both the VLA and LSTHM laboratories developed successful methodologies to solve this. At the LSTHM, two genes were identified from the microarray analysis with potential roles in the pathogenicity of BT 1b strains. Using a Red recombinase system of mutagenesis these two genes ( a two component regulatory system gene and an Intimin like gene) were mutated and shown to play a role in motility and invasion in vitro. These mutants will now be tested in a mouse model. At the VLA the molecular basis of the non-invasiveness of the non-motile BT1a isolates was further investigated. Using the pKNG suicide vector method, the regulator of flagellin expression in strain 53/03 was successfully deleted. This is the first report of a mutant being constructed in a BT1a strain. The data obtained suggested that flagella are involved in invasion, but not adherence, of BT1a Y. enterocolitica, as well as survival within macrophages. The flagella were also immunomodulatory, and appeared to play a role in down-regulating TNF production and up-regulating the anti-inflammatory cytokine IL-10.
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Although not an initial objective of this project the role of serum antibodies in livestock infections was investigated using serum from cattle and pigs experimentally infected as part of a previous Brucella project. These results showed that antibodies directed against a number of antigens of Y. enterocolitica, including flagellin and Yop virulence factors, protected against invasion of epithelial cells in vitro.
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The findings of OZ0405 highlight a wide range of options for future work. The microarray data demonstrated that the presence of the pYV plasmid alone is not responsible for virulence. The data requires further analysis to determine whether there are any CDSs that are specific to human isolates. In addition, many new genetic markers have been identified which can distinguish Y. enterocolitica subgroups. Some of these may relate to phenotypic traits. Interestingly the results suggest that host response (immunopathology) is an important factor in the outcome of infection with the various biotypes. Clearly this requires further investigation in order to accurately determine the public health risk of Y. enterocolitica strains in the human food chain.