Detection and diversity of a putative novel heterogeneous polymorphic proline-glycine repeat (Pgr) protein in the footrot pathogen Dichelobacter nodosus

Dichelobacter nodosus, a Gram-negative anaerobic bacterium, is the essential causative agent of footrot in sheep. Currently, depending on the clinical presentation in the field, footrot is described as benign or virulent; D. nodosus strains have also been classified as benign or virulent, but this designation is not always consistent with clinical disease. The aim of this study was to determine the diversity of the pgr gene, which encodes a putative proline-glycine repeat protein (Pgr). The pgr gene was present in all 100 isolates of D. nodosus that were examined and, based on sequence analysis had two variants, pgrA and pgrB. In pgrA, there were two coding tandem repeat regions, R1 and R2: different strains had variable numbers of repeats within these regions. The R1 and R2 were absent from pgrB. Both variants were present in strains from Australia, Sweden and the UK, however, only pgrB was detected in isolates from Western Australia. The pgrA gene was detected in D. nodosus from tissue samples from two flocks in the UK with virulent footrot and only pgrB from a flock with no virulent or benign footrot for >10 years. Bioinformatic analysis of the putative PgrA protein indicated that it contained a collagen-like cell surface anchor motif. These results suggest that the pgr gene may be a useful molecular marker for epidemiological studies

Whole-genome sequencing of a quarter-century melioidosis outbreak in temperate Australia uncovers a region of low-prevalence endemicity

This study was funded by the National Health and Medical Research Council via awards 1046812 and 1098337, and the Wellcome Trust Sanger Institute via award 098051. S.J.P. receives funding from the NIHR Cambridge Biomedical Research Centre.Melioidosis, caused by the highly recombinogenic bacterium Burkholderia pseudomallei, is a disease with high mortality. Tracing the origin of melioidosis outbreaks and understanding how the bacterium spreads and persists in the environment are essential to protecting public and veterinary health and reducing mortality associated with outbreaks. We used whole-genome sequencing to compare isolates from a historical quarter-century outbreak that occurred between 1966 and 1991 in the Avon Valley, Western Australia, a region far outside the known range of B. pseudomallei endemicity. All Avon Valley outbreak isolates shared the same multilocus sequence type (ST-284), which has not been identified outside this region. We found substantial genetic diversity among isolates based on a comparison of genome-wide variants, with no clear correlation between genotypes and temporal, geographical or source data. We observed little evidence of recombination in the outbreak strains, indicating that genetic diversity among these isolates has primarily accrued by mutation. Phylogenomic analysis demonstrated that the isolates confidently grouped within the Australian B. pseudomallei clade, thereby ruling out introduction from a melioidosis-endemic region outside Australia. Collectively, our results point to B. pseudomallei ST-284 being present in the Avon Valley for longer than previously recognized, with its persistence and genomic diversity suggesting long-term, low-prevalence endemicity in this temperate region. Our findings provide a concerning demonstration of the potential for environmental persistence of B. pseudomallei far outside the conventional endemic regions. An expected increase in extreme weather events may reactivate latent B. pseudomallei populations in this region.Publisher PDFPeer reviewe

Molecular epidemiology, clonality and virulence of Dichelobacter nodosus, the agent of ovine footrot

Dichelobacter nodosus, an anaerobic bacterium, is the major transmissible agent of ovine footrot. The disease expresses as a virulent or benign lesion in the hoof. Virulence is related to the production of serine proteases, particularly a thermostable protease. Isolates of D. nodosus are characterised according to the type of protease produced (either heat-stable or heat-labile) and the electrophoretogram (zymogram) of the protease. This study reports on the use of the DNA-based typing techniques Pulsed-Field Gel Electrophoresis (PFGE) and Infrequent-Restriction-Site-PCR (IRS-PCR) to investigate the molecular epidemiology of D. nodosus, including a consideration of the relationship between genetic type, zymogram patterns and whole cell protein profiles. The aim of the project was to obtain a better understanding of D. nodosus strain diversity and dissemination in Australia and its relationship to virulence within the population. The overall intention was to use this information to assist in the long-term control of virulent footrot. Field isolates of D. nodosus from Western Australia (n = 735), New South Wales (n = 16), Victoria (n = 24) and South Australia (n = 21) were obtained and analysed. Both typing techniques that were used offered good differentiation between isolates for epidemiological purposes, and the results were in general agreement. PFGE provided slightly better discrimination between isolates, with 214 PFGE types (181 from Western Australia) compared to 94 IrsT types (77 from Western Australia). Within this diverse range of molecular types clonality was observed - with clones being defined as clusters of isolates having closely related PFGE types. The strains were categorised as genetically diverse, genetically similar or identified as the same strain. This diversity of genetic types was found overall, within flocks of sheep on a farm and within a single hoof where, on a number of occasions, multiple molecular types and zymogram types were found colonising a single hoof. One isolate that was experimentally inoculated into a flock of sheep produced six different genetic types when tested 12 months after the initial infection. This indicates that D. nodosus undergoes rapid genetic change, which means that follow-up epidemiological investigation of disease outbreaks and trace-backs need to be done as soon after infection as possible. The genetic differences appeared to be due to large insertions or deletions of DNA. Amongst sheep on some properties, isolates that had a different protease expression and virulence expression were found to have the same molecular type. Investigation of these isolates by SDS-PAGE showed that they also had the same whole cell protein profiles. Isolates from the same clonal groups also had the same protein profile, whereas genetically diverse isolates had different protein profiles. The lack of protein differences between isolates of the same molecular type, or within a clonal group, suggests that the differences in protease thermostability may be due to conformational changes in the protein, rather than to overall detectable genetic change and/or expression of different proteins. These results demonstrate that PFGE typing can be useful in predicting likely phenotypic expression of whole cell proteins. Further work is required to elucidate differences between virulent and benign strains of D. nodosus

Phase variation in latB associated with a fatal Pasteurella multocida outbreak in captive squirrel gliders

A septicaemic disease outbreak caused by Pasteurella multocida at a zoo in Western Australia (Zoo A) occurred in a resident group of squirrel gliders (Petaurus norfolcensis) following the introduction of two squirrel gliders imported from another zoo (Zoo B). P. multocida isolates obtained from the affected animals and asymptomatic, cohabiting marsupials at both zoos were typed via lipopolysaccharide outer core biosynthesis locus (LPS) typing, repetitive extragenic palindromic PCR (Rep-PCR) typing, and multilocus sequence typing (ST). Investigation of isolate relatedness via whole genome sequencing (WGS) and phylogenomic analysis found that the outbreak isolates shared the same genetic profile as those obtained from the imported gliders and the positive marsupials at Zoo B. Phylogenomic analysis demonstrated that these isolates belonged to the same clone (named complex one), confirming that the outbreak strain originated at Zoo B. As well, the carriage of multiple different strains of this pathogen in a range of marsupials in a zoo setting has been demonstrated. Importantly, the genomic investigation identified a missense mutation in the latB, a structural LPS gene, resulting in introduction of an immediate stop codon in the isolates carried by asymptomatic squirrel gliders in Zoo B. The identified diversity in the latB gene of LPS outer core biosynthesis loci of these isolates is consistent with a novel phase variable mechanism for virulence in P. multocida. Our study demonstrates the benefit of WGS and bioinformatics analysis in epidemiological investigations of pasteurellosis and its potential to reveal unexpected insights into bacterial virulence

Data underpinning - Whole-genome sequencing of a quarter-century melioidosis outbreak in temperate Australia uncovers a region of low-prevalence endemicity

All Illumina data from this study were uploaded to public databases. MSHRs 0161, 0169 and 0173 were deposited in the European Nucleotide Archive database. MSHRs 0157, 0160, 0162, 0163, 0167, 0170, 0171 and 0172 were deposited in the NCBI Sequence Read Archive database.. The MSHR0169 assembly has been deposited in GenBank under accession number LGKL0000000

Data underpinning - Whole-genome sequencing of a quarter-century melioidosis outbreak in temperate Australia uncovers a region of low-prevalence endemicity

All Illumina data from this study were uploaded to public databases. MSHRs 0161, 0169 and 0173 were deposited in the European Nucleotide Archive database. MSHRs 0157, 0160, 0162, 0163, 0167, 0170, 0171 and 0172 were deposited in the NCBI Sequence Read Archive database.. The MSHR0169 assembly has been deposited in GenBank under accession number LGKL0000000