Sion Bayliss

Clonal differences in Staphylococcus aureus bacteraemia-associated mortality

The bacterium Staphylococcus aureus is a major human pathogen for which the emergence of antibiotic resistance is a global public health concern. Infection severity, and in particular bacteraemia-associated mortality, has been attributed to several host-related factors, such as age and the presence of comorbidities. The role of the bacterium in infection severity is less well understood, as it is complicated by the multifaceted nature of bacterial virulence, which has so far prevented a robust mapping between genotype, phenotype and infection outcome. To investigate the role of bacterial factors in contributing to bacteraemia-associated mortality, we phenotyped a collection of sequenced clinical S. aureus isolates from patients with bloodstream infections, representing two globally important clonal types, CC22 and CC30. By adopting a genome-wide association study approach we identified and functionally verified several genetic loci that affect the expression of cytolytic toxicity and biofilm formation. By analysing the pooled data comprising bacterial genotype and phenotype together with clinical metadata within a machine-learning framework, we found significant clonal differences in the determinants most predictive of poor infection outcome. Whereas elevated cytolytic toxicity in combination with low levels of biofilm formation was predictive of an increased risk of mortality in infections by strains of a CC22 background, these virulence-specific factors had little influence on mortality rates associated with CC30 infections. Our results therefore suggest that different clones may have adopted different strategies to overcome host responses and cause severe pathology. Our study further demonstrates the use of a combined genomics and data analytic approach to enhance our understanding of bacterial pathogenesis at the individual level, which will be an important step towards personalized medicine and infectious disease management.A genome-wide association approach identifies differential biofilm and virulence attributes associated with mortality in two Staphylococcus aureus clonal complexes.

Genomic analysis of 495 vancomycin-resistant Enterococcus faecium reveals broad dissemination of a vanA plasmid in more than 19 clones from Copenhagen, Denmark

Objectives From 2012 to 2014, there has been a huge increase in vancomycin-resistant (vanA) Enterococcus faecium (VREfm) in Copenhagen, Denmark, with 602 patients infected or colonized with VREfm in 2014 compared with just 22 in 2012. The objective of this study was to describe the genetic epidemiology of VREfm to assess the contribution of clonal spread and horizontal transfer of the vanA transposon (Tn1546) and plasmid in the dissemination of VREfm in hospitals. Methods VREfm from Copenhagen, Denmark (2012–14) were whole-genome sequenced. The clonal structure was determined and the structure of Tn1546-like transposons was characterized. One VREfm isolate belonging to the largest clonal group was sequenced using long-read technology to close a 37 kb vanA plasmid. Results Phylogeny revealed a polyclonal structure where 495 VREfm isolates were divided into 13 main groups and 7 small groups. The majority of the isolates were located in three groups (n = 44, 100 and 218) and clonal spread of VREfm between wards and hospitals was identified. Five Tn1546-like transposon types were identified. A dominant truncated transposon (type 4, 92%) was spread across all but one VREfm group. The closed vanA plasmid was highly covered by reads from isolates containing the type 4 transposon. Conclusions This study suggests that it was the dissemination of the type 4 Tn1546-like transposon and plasmid via horizontal transfer to multiple populations of E. faecium, followed by clonal spread of new VREfm clones, that contributed to the increase in and diversity of VREfm in Danish hospitals.

The promise of whole genome pathogen sequencing for the molecular epidemiology of emerging aquaculture pathogens

Aquaculture is the fastest growing food-producing sector, and the sustainability of this industry is critical both for global food security and economic welfare. The management of infectious disease represents a key challenge. Here, we discuss the opportunities afforded by whole genome sequencing of bacterial and viral pathogens of aquaculture to mitigate disease emergence and spread. We outline, by way of comparison, how sequencing technology is transforming the molecular epidemiology of pathogens of public health importance, emphasizing the importance of community-oriented databases and analysis tools.

Comparative analyses of selection operating on non-translated intergenic regions of diverse bacterial species

To date, the existing very large genome sequence datasets of many bacterial species have not been exploited to quantify the strength and direction of... Nontranslated intergenic regions (IGRs) compose 10–15% of bacterial genomes, and contain many regulatory elements with key functions. Despite this, there are few systematic studies on the strength and direction of selection operating on IGRs in bacteria using whole-genome sequence data sets. Here we exploit representative whole-genome data sets from six diverse bacterial species: Staphylococcus aureus, Streptococcus pneumoniae, Mycobacterium tuberculosis, Salmonella enterica, Klebsiella pneumoniae, and Escherichia coli. We compare patterns of selection operating on IGRs using two independent methods: the proportion of singleton mutations and the dI/dS ratio, where dI is the number of intergenic SNPs per intergenic site. We find that the strength of purifying selection operating over all intergenic sites is consistently intermediate between that operating on synonymous and nonsynonymous sites. Ribosome binding sites and noncoding RNAs tend to be under stronger selective constraint than promoters and Rho-independent terminators. Strikingly, a clear signal of purifying selection remains even when all these major categories of regulatory elements are excluded, and this constraint is highest immediately upstream of genes. While a paucity of variation means that the data for M. tuberculosis are more equivocal than for the other species, we find strong evidence for positive selection within promoters of this species. This points to a key adaptive role for regulatory changes in this important pathogen. Our study underlines the feasibility and utility of gauging the selective forces operating on bacterial IGRs from whole-genome sequence data, and suggests that our current understanding of the functionality of these sequences is far from complete.

A novel bifunctional histone protein in Streptomyces : a candidate for structural coupling between DNA conformation and transcription during development and stress?

Antibiotic-producing Streptomyces are complex bacteria that remodel global transcription patterns and their nucleoids during development. Here, we describe a novel developmentally regulated nucleoid-associated protein, DdbA, of the genus that consists of an N-terminal DNA-binding histone H1-like domain and a C-terminal DksA-like domain that can potentially modulate RNA polymerase activity in conjunction with ppGpp. Owing to its N-terminal domain, the protein can efficiently bind and condense DNA in vitro. Loss of function of this DNA-binding protein results in changes in both DNA condensation during development and the ability to adjust DNA supercoiling in response to osmotic stress. Initial analysis of the DksA-like activity of DdbA indicates that overexpression of the protein suppresses a conditional deficiency in antibiotic production of relA mutants that are unable to synthesise ppGpp, just as DksA overexpression in Escherichia coli can suppress ppGpp0 phenotypes. The null mutant is also sensitive to oxidative stress owing to impaired upregulation of transcription of sigR, encoding an alternative sigma factor. Consequently, we propose this bifunctional histone-like protein as a candidate that could structurally couple changes in DNA conformation and transcription during the streptomycete life-cycle and in response to stress.

Evidence of land-sea transfer of the zoonotic pathogen Campylobacter to a wildlife marine sentinel species

Environmental pollution often accompanies the expansion and urbanization of human populations where sewage and wastewaters commonly have an impact on the marine environments. Here, we explored the potential for faecal bacterial pathogens, of anthropic origin, to spread to marine wildlife in coastal areas. The common zoonotic bacterium Campylobacter was isolated from grey seals (Halichoerus grypus), an important sentinel species for environmental pollution, and compared to isolates from wild birds, agricultural sources and clinical samples to characterize possible transmission routes. Campylobacter jejuni was present in half of all grey seal pups sampled (24/50 dead and 46/90 live pups) in the breeding colony on the Isle of May (Scotland), where it was frequently associated with histological evidence of disease. Returning yearling animals (19/19) were negative for C. jejuni suggesting clearance of infection while away from the localized colony infection source. The genomes of 90 isolates from seals were sequenced and characterized using a whole‐genome multilocus sequence typing (MLST) approach and compared to 192 published genomes from multiple sources using population genetic approaches and a probabilistic genetic attribution model to infer the source of infection from MLST data. The strong genotype‐host association has enabled the application of source attribution models in epidemiological studies of human campylobacteriosis, and here assignment analyses consistently grouped seal isolates with those from human clinical samples. These findings are consistent with either a common infection source or direct transmission of human campylobacter to grey seals, raising concerns about the spread of human pathogens to wildlife marine sentinel species in coastal areas.

Piggy: a rapid, large-scale pan-genome analysis tool for intergenic regions in bacteria

Abstract Background The concept of the “pan-genome,” which refers to the total complement of genes within a given sample or species, is well established in bacterial genomics. Rapid and scalable pipelines are available for managing and interpreting pan-genomes from large batches of annotated assemblies. However, despite overwhelming evidence that variation in intergenic regions in bacteria can directly influence phenotypes, most current approaches for analyzing pan-genomes focus exclusively on protein-coding sequences. Findings To address this we present Piggy, a novel pipeline that emulates Roary except that it is based only on intergenic regions. A key utility provided by Piggy is the detection of highly divergent (“switched”) intergenic regions (IGRs) upstream of genes. We demonstrate the use of Piggy on large datasets of clinically important lineages of Staphylococcus aureus and Escherichia coli. Conclusions For S. aureus, we show that highly divergent (switched) IGRs are associated with differences in gene expression and we establish a multilocus reference database of IGR alleles (igMLST; implemented in BIGSdb).

Bayesian identification of bacterial strains from sequencing data

Rapidly assaying the diversity of a bacterial species present in a sample obtained from a hospital patient or an environmental source has become possible after recent technological advances in DNA sequencing. For several applications it is important to accurately identify the presence and estimate relative abundances of the target organisms from short sequence reads obtained from a sample. This task is particularly challenging when the set of interest includes very closely related organisms, such as different strains of pathogenic bacteria, which can vary considerably in terms of virulence, resistance and spread. Using advanced Bayesian statistical modelling and computation techniques we introduce a novel pipeline for bacterial identification that is shown to outperform the currently leading pipeline for this purpose. Our approach enables fast and accurate sequence-based identification of bacterial strains while using only modest computational resources. Hence it provides a useful tool for a wide spectrum of applications, including rapid clinical diagnostics to distinguish among closely related strains causing nosocomial infections. The software implementation is available at https://github.com/PROBIC/BIB.

The use of Oxford Nanopore native barcoding for complete genome assembly

Abstract Background The Oxford Nanopore Technologies MinION(TM) is a mobile DNA sequencer that can produce long read sequences with a short turn-around time. Here we report the first demonstration of single contig genome assembly using Oxford Nanopore native barcoding when applied to a multiplexed library of 12 samples and combined with existing Illumina short read data. This paves the way for the closure of multiple bacterial genomes from a single MinION(TM) sequencing run, given the availability of existing short read data. The strain we used, MHO_001, represents the important community-acquired methicillin-resistant Staphylococcus aureus lineage USA300. Findings Using a hybrid assembly of existing short read and barcoded long read sequences from multiplexed data, we completed a genome of the S. aureus USA300 strain MHO_001. The long read data represented only ∼5% to 10% of an average MinION(TM) run (∼7x genomic coverage), but, using standard tools, this was sufficient to complete the circular chromosome of S. aureus strain MHO_001 (2.86 Mb) and two complete plasmids (27 Kb and 3 Kb). Minor differences were noted when compared to USA300 reference genome, USA300_FPR3757, including the translocation, loss, and gain of mobile genetic elements. Conclusion Here we demonstrate that MinION(TM) reads, multiplexed using native barcoding, can be used in combination with short read data to fully complete a bacterial genome. The ability to complete multiple genomes, for which short read data is already available, from a single MinION(TM) run is set to impact our understanding of accessory genome content, plasmid diversity, and genome rearrangements.

The large majority of intergenic sites in bacteria are selectively constrained, even when known regulatory elements are excluded

There are currently no broad estimates of the overall strength and direction of selection operating on intergenic variation in bacteria. Here we address this using large whole genome sequence datasets representing six diverse bacterial species; Escherichia coli, Staphylococcus aureus, Salmonella enterica, Streptococcus pneumoniae, Klebsiella pneumoniae, and Mycobacterium tuberculosis. Excluding M. tuberculosis, we find that a high proportion (62%-79%; mean 70%) of intergenic sites are selectively constrained, relative to synonymous sites. Non-coding RNAs tend to be under stronger selective constraint than promoters, which in turn are typically more constrained than rho-independent terminators. Even when these regulatory elements are excluded, the mean proportion of constrained intergenic sites only falls to 69%; thus our current understanding of the functionality of intergenic regions (IGRs) in bacteria is severely limited. Consistent with a role for positive as well as negative selection on intergenic sites, we present evidence for strong positive selection in Mycobacterium tuberculosis promoters, underlining the key role of regulatory changes as an adaptive mechanism in this highly monomorphic pathogen.