How Genomics Is Changing What We Know About the Evolution and Genome of Bordetella pertussis

The evolution of Bordetella pertussis from a common ancestor similar to Bordetella bronchiseptica has occurred through large-scale gene loss, inactivation and rearrangements, largely driven by the spread of insertion sequence element repeats throughout the genome. B. pertussis is widely considered to be monomorphic, and recent evolution of the B. pertussis genome appears to, at least in part, be driven by vaccine-based selection. Given the recent global resurgence of whooping cough despite the wide-spread use of vaccination, a more thorough understanding of B. pertussis genomics could be highly informative. In this chapter we discuss the evolution of B. pertussis, including how vaccination is changing the circulating B. pertussis population at the gene-level, and how new sequencing technologies are revealing previously unknown levels of inter- and intra-strain variation at the genome-level

Comparative genomics of Rhodococcus equi virulence plasmids indicates host-driven evolution of the vap pathogenicity island

The conjugative virulence plasmid is a key component of the Rhodococcus equi accessory genome essential for pathogenesis. Three host-associated virulence plasmid types have been identified: the equine pVAPA and porcine pVAPB circular variants, and the linear pVAPN found in bovine (ruminant) isolates. We recently characterized the R. equi pangenome (Anastasi E, et al. 2016. Pangenome and phylogenomic analysis of the pathogenic actinobacterium Rhodococcus equi. Genome Biol Evol. 8:3140–3148.) and we report here the comparative analysis of the virulence plasmid genomes. Plasmids within each host-associated type were highly similar despite their diverse origins. Variation was accounted for by scattered single nucleotide polymorphisms and short nucleotide indels, while larger indels—mostly in the plasticity region near the vap pathogencity island (PAI)—defined plasmid genomic subtypes. Only one of the plasmids analyzed, of pVAPN type, was exceptionally divergent due to accumulation of indels in the housekeeping backbone. Each host-associated plasmid type carried a unique PAI differing in vap gene complement, suggesting animal host-specific evolution of the vap multigene family. Complete conservation of the vap PAI was observed within each host-associated plasmid type. Both diversity of host-associated plasmid types and clonality of specific chromosomal-plasmid genomic type combinations were observed within the same R. equi phylogenomic subclade. Our data indicate that the overall strong conservation of the R. equi host-associated virulence plasmids is the combined result of host-driven selection, lateral transfer between strains, and geographical spread due to international livestock exchanges

The evolution of Bordetella pertussis has selected for mutations of acr that lead to sensitivity to hydrophobic molecules and fatty acids

Whooping cough, or pertussis, is resurgent in numerous countries worldwide. This has renewed interest in Bordetella pertussis biology and vaccinology. The in vitro growth of B. pertussis has been a source of difficulty, both for the study of the organism and the production of pertussis vaccines. It is inhibited by fatty acids and other hydrophobic molecules. The AcrAB efflux system is present in many different bacteria and in combination with an outer membrane factor exports acriflavine and other small hydrophobic molecules from the cell. Here, we identify that the speciation of B. pertussis has selected for an Acr system that is naturally mutated and displays reduced activity compared to B. bronchiseptica, in which the system appears intact. Replacement of the B. pertussis locus with that of B. bronchiseptica conferred higher levels of resistance to growth inhibition by acriflavine and fatty acids. In addition, we identified that the transcription of the locus is repressed by a LysR-type transcriptional regulator. Palmitate de-represses the expression of the acr locus, dependent on the LysR regulator, strongly suggesting that it is a transcriptional repressor that is regulated by palmitate. It is intriguing that the speciation of B. pertussis has selected for a reduction in activity of the Acr efflux system that typically is regarded as protective to bacteria

A qPCR assay for Bordetella pertussis cells that enumerates both live and dead bacteria

Bordetella pertussis is the causative agent of whooping cough, commonly referred to as pertussis. Although the incidence of pertussis was reduced through vaccination, during the last thirty years it has returned to high levels in a number of countries. This resurgence has been linked to the switch from the use of whole-cell to acellular vaccines. Protection afforded by acellular vaccines appears to be short-lived compared to that afforded by whole cell vaccines. In order to inform future vaccine improvement by identifying immune correlates of protection, a human challenge model of B. pertussis colonisation has been developed. Accurate measurement of colonisation status in this model has required development of a qPCR-based assay to enumerate B. pertussis in samples that distinguishes between viable and dead bacteria. Here we report the development of this assay and its performance in the quantification of B. pertussis from human challenge model samples. This assay has future utility in diagnostic labs and in research where a quantitative measure of both B. pertussis number and viability is required

Pangenome and Phylogenomic Analysis of the Pathogenic Actinobacterium Rhodococcus equi

We report a comparative study of 29 representative genomes of the animal pathogen Rhodococcus equi. The analyses showed that R. equi is genetically homogeneous and clonal, with a large core genome accounting for ≈80% of an isolates’ gene content. An open pangenome, even distribution of accessory genes among the isolates, and absence of significant core–genome recombination, indicated that gene gain/loss is a main driver of R. equi genome evolution. Traits previously predicted to be important in R. equi physiology, virulence and niche adaptation were part of the core genome. This included the lack of a phosphoenolpyruvate:carbohydrate transport system (PTS), unique among the rhodococci except for the closely related Rhodococcus defluvii, reflecting selective PTS gene loss in the R. equi–R. defluvii sublineage. Thought to be asaccharolytic, rbsCB and glcP non-PTS sugar permease homologues were identified in the core genome and, albeit inefficiently, R. equi utilized their putative substrates, ribose and (irregularly) glucose. There was no correlation between R. equi whole-genome phylogeny and host or geographical source, with evidence of global spread of genomovars. The distribution of host-associated virulence plasmid types was consistent with the exchange of the plasmids (and corresponding host shifts) across the R. equi population, and human infection being zoonotically acquired. Phylogenomic analyses demonstrated that R. equi occupies a central position in the Rhodococcus phylogeny, not supporting the recently proposed transfer of the species to a new genus

Fundamental differences in physiology of Bordetella pertussis dependent on the two-component system Bvg revealed by gene essentiality studies.

The identification of genes essential for a bacterium's growth reveals much about its basic physiology under different conditions. Bordetella pertussis, the causative agent of whooping cough, adopts both virulent and avirulent states through the activity of the two-component system, Bvg. The genes essential for B. pertussis growth in vitro were defined using transposon sequencing, for different Bvg-determined growth states. In addition, comparison of the insertion indices of each gene between Bvg phases identified those genes whose mutation exerted a significantly different fitness cost between phases. As expected, many of the genes identified as essential for growth in other bacteria were also essential for B. pertussis. However, the essentiality of some genes was dependent on Bvg. In particular, a number of key cell wall biosynthesis genes, including the entire mre/mrd locus, were essential for growth of the avirulent (Bvg minus) phase but not the virulent (Bvg plus) phase. In addition, cell wall biosynthesis was identified as a fundamental process that when disrupted produced greater fitness costs for the Bvg minus phase compared to the Bvg plus phase. Bvg minus phase growth was more susceptible than Bvg plus phase growth to the cell wall-disrupting antibiotic ampicillin, demonstrating the increased susceptibility of the Bvg minus phase to disruption of cell wall synthesis. This Bvg-dependent conditional essentiality was not due to Bvg-regulation of expression of cell wall biosynthesis genes; suggesting that this fundamental process differs between the Bvg phases in B. pertussis and is more susceptible to disruption in the Bvg minus phase. The ability of a bacterium to modify its cell wall synthesis is important when considering the action of antibiotics, particularly if developing novel drugs targeting cell wall synthesis

An Invertron-Like Linear Plasmid Mediates Intracellular Survival and Virulence in Bovine Isolates of Rhodococcus equi.

We report a novel host-associated virulence plasmid in Rhodococcus equi, pVAPN, carried by bovine isolates of this facultative intracellular pathogenic actinomycete. Surprisingly, pVAPN is a 120-kb invertron-like linear replicon unrelated to the circular virulence plasmids associated with equine (pVAPA) and porcine (pVAPB variant) R. equi isolates. pVAPN is similar to the linear plasmid pNSL1 from Rhodococcus sp. NS1 and harbors six new vap multigene family members (vapN to vapS) in a vap pathogenicity locus presumably acquired via en bloc mobilization from a direct predecessor of equine pVAPA. Loss of pVAPN rendered R. equi avirulent in macrophages and mice. Mating experiments using an in vivo transconjugant selection strategy demonstrated that pVAPN transfer is sufficient to confer virulence to a plasmid-cured R. equi recipient. Phylogenetic analyses assigned the vap multigene family complement from pVAPN, pVAPA, and pVAPB to seven monophyletic clades, each containing plasmid type-specific allelic variants of a precursor vap gene carried by the nearest vap island ancestor. Deletion of vapN, the predicted “bovine-type” allelic counterpart of vapA, essential for virulence in pVAPA, abrogated pVAPN-mediated intramacrophage proliferation and virulence in mice. Our findings support a model in which R. equi virulence is conferred by host-adapted plasmids. Their central role is mediating intracellular proliferation in macrophages, promoted by a key vap determinant present in the common ancestor of the plasmid-specific vap islands, with host tropism as a secondary trait selected during coevolution with specific animal species

Leadership in Compassionate Care: Final Report 2012

This report reflects the initiation, planning, running and the important outcomes emerging from the Leadership in Compassionate Care Programme. The team worked in close partnership across the School of Nursing, Midwifery and Social Care, Edinburgh Napier University and NHS Lothian. This report also shares the highlights, challenges and solutions to embed compassionate care education and nursing practice.Additional co-authors: Fiona Smith, Stephen DM Smith, Ria Tocher, and Anne Waug