Biology subjects

Parkhill, J.

Publications and source records attributed to Parkhill, J..

At least 37 records · Page 2Linked to original sources

bioRxiv2021

Inferring Mycobacterium bovis transmission between cattle and badgers using isolates from the Randomised Badger Culling Trial

Mycobacterium bovis (M. bovis) is a causative agent of bovine tuberculosis, a significant source of morbidity and mortality in the global cattle industry. The Randomised Badger Culling Trial was a field experiment carried out between 1998 and 2005 in the South West of England. As part of this trial, M. bovis isolates were collected from contemporaneous and overlapping populations of badgers and cattle within ten defined trial areas. We combined whole genome sequences from 1,442 isolates with location and cattle movement data, identifying transmission clusters and inferred rates and routes of transmission of M. bovis. Most trial areas contained a single transmission cluster that had been established shortly before sampling, often contemporaneous with the expansion of bovine tuberculosis in the 1980s. The estimated rate of transmission from badger to cattle was approximately two times higher than from cattle to badger, and the rate of within-species transmission considerably exceeded these for both species. We identified long distance transmission events linked to cattle movement, recurrence of herd breakdown by infection within the same transmission clusters and superspreader events driven by cattle but not badgers. Overall, our data suggests that the transmission clusters in different parts of South West England that are still evident today were established by long-distance seeding events involving cattle movement, not by recrudescence from a long-established wildlife reservoir. Clusters are maintained primarily by within-species transmission, with less frequent spill-over both from badger to cattle and cattle to badger.

genomics↗

bioRxiv2021

Cross-species evolutionary rewiring in the enteric bacterium Campylobacter

Significance StatementGenetic exchange among bacteria shapes the microbial world. From the acquisition of antimicrobial resistance genes to fundamental questions about the nature of bacterial species, this powerful evolutionary force has preoccupied scientists for decades. However, the mixing of genes between species rests on a paradox. On one hand, promoting adaptation by conferring novel functionality, on the other potentially introducing disharmonious gene combinations (negative epistasis) that will be selected against. Taking an interdisciplinary approach to analyse natural populations of the enteric bacteria Campylobacter, an ideal example of long-range admixture, we demonstrate that genes can independently transfer across species boundaries and rejoin in epistasis in a recipient genome. This challenges conventional ideas and highlights the possibility of single step evolution by saltation. AbstractRecombination of short DNA fragments via horizontal gene transfer (HGT) can both introduce beneficial alleles and create genomic disharmony through negative epistasis. For non-core (accessory) genes, the negative epistatic cost is likely to be minimal because the incoming genes have not co-evolved with the recipient genome. By contrast, for the core genome, interspecific recombination is expected to be rare because disruptive allelic replacement is likely to introduce negative epistasis. Why then is homologous recombination common in the core of bacterial genomes? To understand this enigma we take advantage of an exceptional model system, the common enteric pathogens Campylobacter jejuni and Campylobacter coli, that are known for very high magnitude interspecies gene flow in the core genome. As expected, HGT does indeed disrupt co-adapted allele pairings (negative epistasis). However, multiple HGT events enable recovery of the genomes co-adaption between introgressing alleles, even in core metabolism genes (e.g., formate dehydrogenase). These findings demonstrate that, even for complex traits, genetic coalitions can be decoupled, transferred and independently reinstated in a new genetic background - facilitating transition between fitness peaks. In this example, the two-step recombinational process is associated with C. coli that are adapted to the agricultural niche.

microbiology↗

bioRxiv2020

Population structure and transmission of Mycobacterium bovis in Ethiopia

Bovine tuberculosis (bTB) is endemic in cattle in Ethiopia, a country that hosts the largest national cattle herd in Africa. The intensive dairy sector, most of which is peri-urban, has the highest prevalence of disease. Previous studies in Ethiopia have demonstrated that the main cause is Mycobacterium bovis (M. bovis), which has been investigated using conventional molecular tools including deletion typing, spoligotyping and Mycobacterial interspersed repetitive unit-variable number tandem repeat (MIRU-VNTR). Here we use whole genome sequencing (WGS) to examine the population structure of M. bovis in Ethiopia. A total of 134 M. bovis isolates were sequenced including 128 genomes from 85 mainly dairy cattle and six genomes isolated from humans, originating from 12 study sites across Ethiopia. These genomes provided a good representation of the previously described population structure of M. bovis, based on spoligotyping and demonstrated that the population is dominated by the clonal complexes African 2 (Af2) and European 3 (Eu3). A range of within-host diversity was observed amongst the isolates and evidence was found for both short- and long-distance transmission. Detailed analysis of available genomes from the Eu3 clonal complex combined with previously published genomes revealed two distinct introductions of this clonal complex into Ethiopia between 1950 and 1987, likely from Europe. This work is important to help better understand bTB transmission in cattle in Ethiopia and can potentially inform national strategies for bTB control in Ethiopia and beyond.

genomics↗

bioRxiv2020

Identifying virulence determinants of multidrug-resistant Klebsiella pneumoniae in Galleria mellonella

Infections caused by Klebsiella pneumoniae are a major public health threat. Extensively drug-resistant and even pan-resistant strains have been reported. Understanding K. pneumoniae pathogenesis is hampered by the fact that murine models of infection offer limited resolution for the non-hypervirulent strains which cause the majority of infections. We have performed genome-scale fitness profiling of a multidrug-resistant K. pneumoniae ST258 strain during infection of the insect Galleria mellonella, with the aim to determine if this model is suitable for large-scale virulence factor discovery in this pathogen. Our results demonstrated a dominant role for surface polysaccharides in infection, with contributions from siderophores, cell envelope proteins, purine biosynthesis genes and additional genes of unknown function. Comparison with a hypervirulent strain, ATCC 43816, revealed substantial overlap in important infection-related genes, as well as additional putative virulence factors that may be specific to ST258. Our analysis also identified a role for the metalloregulatory protein NfeR (also called YqjI) in virulence. Overall, this study offers new insight into the infection fitness landscape of K. pneumoniae ST258, and provides a framework for using the highly flexible, scalable G. mellonella infection model to dissect the molecular virulence mechanisms of K. pneumoniae and other bacterial pathogens.

microbiology↗

bioRxiv2020

Screening for highly transduced genes in Staphylococcus aureus reveals both lateral and specialized transduction

Bacteriophage-mediated transduction of bacterial DNA is a major route of horizontal gene transfer in the human pathogen, Staphylococcus aureus. Transduction involves packaging of bacterial DNA by viruses and enables transmission of virulence and resistance genes between cells. To learn more about transduction in S. aureus, we searched a transposon mutant library for genes and mutations that enhanced transfer mediated by the temperate phage, {varphi}11. Using a novel screening strategy, we performed multiple rounds of transduction of transposon mutant pools selecting for an antibiotic resistance marker within the transposon element. When determining the locations of transferred mutations, we found that, within each pool of 96 mutants the screen had selected for just 1 or 2 transposon mutant(s). Subsequent analysis showed that the position of the transposon, rather than inactivation of bacterial genes, was responsible for the phenotype. Interestingly, from multiple rounds we identified a pattern of transduction that encompassed mobile genetic elements, as well as chromosomal regions both upstream and downstream of the phage integration site. The latter was confirmed by DNA sequencing of purified phage lysates. Importantly, transduction frequencies were lower for phage lysates obtained by phage infection rather than induction. Our results confirm previous reports of lateral transduction of bacterial DNA downstream of the integrated phage, but also indicate specialized transduction of DNA upstream of the phage, likely involving imprecise excision of the phage from the bacterial genome. These findings illustrate the complexity of transduction processes and increase our understanding of the mechanisms by which phages transfer bacterial DNA. ImportanceHorizontal transfer of DNA between bacterial cells contributes to the spread of virulence and antibiotic resistance genes in human pathogens. For Staphylococcus aureus, bacterial viruses are particularly important. These viruses, termed bacteriophages, can transfer bacterial DNA between cells by a process known as transduction, which despite of its importance is only poorly characterized. Here, we employed a transposon mutant library to investigate transduction in S. aureus. We show that the location of bacterial DNA in relation to bacteriophages integrated in the bacterial genome is a key decider of how frequently that DNA is transduced. Based on serial transduction of transposon mutant pools and direct sequencing of bacterial DNA in bacteriophage particles, we demonstrate both lateral and specialized transduction. The use of mutant libraries to investigate the patterns of bacterial DNA transfer between cells could help understand how bacteria evolve virulence and resistance and may ultimately lead to new intervention strategies.

microbiology↗

bioRxiv2020

Co-evolutionary signals from Burkholderia pseudomallei population genomics highlight its survival strategy in a hostile environment

BackgroundThe soil bacterium Burkholderia pseudomallei is the causative agent of melioidosis. It kills up to 40% of cases and contributes to human morbidity and mortality in many tropical and sub-tropical countries. As no vaccines are currently available, prevention is the key health policy and is achieved by avoiding direct contact with soil and standing water. The pathogen notoriously persists in ranges of environmental conditions which make disease prevention difficult. We aimed to scan B. pseudomallei genomes for signals of evolutionary adaptations that allow it to thrive across environmental conditions, which should ultimately inform prevention policy. MethodsWe conducted three layers of analyses: a genome-wide epistasis and co-selection study (GWES) on 2,011 B. pseudomallei genomes to detect signals of co-selection; gene expression analyses across 82 diverse physical, chemical, biological and infectious conditions to identify specific conditions in which such selection might have acted; and gene knockout assays to confirm the function of the co-selection hotspot. FindingsWe uncovered 13,061 mutation pairs in distinct genes and non-coding RNA that have been repeatedly co-selected through B. pseudomallei evolution. Genes under co-selection displayed marked expression correlation when B. pseudomallei was subjected to physical stress conditions including temperature stress, osmotic stress, UV radiation, and nutrient deprivation; highlighting these conditions as the major evolutionary driving forces for this bacterium. We identified a putative adhesin (BPSL1661) as a hub of co-selection signals, experimentally confirmed the role of BPSL1661 under nutrient deprivation, and explored the functional basis of the co-selection gene network surrounding BPSL1661 in facilitating bacterial survival under nutrient depletion. InterpretationOur findings suggest that B. pseudomallei has a selective advantage to survive nutrient-limited conditions. Anthropogenic activities such as shifting cultivation systems with more frequent rotations of cropping and shortened fallow periods or continuous cultivation of cash crops could directly or indirectly contribute to loss of soil nutrient; these may lead to the preferential survival of B. pseudomallei and a subsequent rise of melioidosis. Successful disease control for melioidosis needs to consider improving environmental health in addition to current preventive efforts. FundingWellcome Trust, European Research Council, UK Department of Health, Thailand Research Fund and Khon Kaen University Research in contextO_ST_ABSEvidence before this studyC_ST_ABSWe searched PubMed with terms (co-selection AND bacteria AND population) with no date or language restrictions from database inception until April 11, 2021. We identified 44 publications of which four were conducted at a genome-wide scale. These four studies were performed on human-restricted pathogens, detected co-selection of antibiotic resistance gene networks which highlight the use of antibiotics as major selection pressures and further inform treatment options. However, none of these studies were performed on Burkholderia pseudomallei or other opportunistic pathogens that have been adapted to both natural and host environments. The selection pressures exerted on these pathogens and the genetic determinants allowed for their adaptations remain unclear, which limit our understanding on the bacterial biology and the information used for disease control. Added value of this studyBased on genomes of 2,011 B. pseudomallei collected from melioidosis endemic areas, we identified and confirmed genetic signals for co-selection. Using transcriptome profiling covering a broad spectrum of conditions and exposures, we showed that genes under co-selection displayed marked expression correlation under physical stress conditions with the gene at the co-selection hotspot conditionally expressed under nutrient starvation. Furthermore, we experimentally validated the function of the hotspot gene and demonstrated that unlike host-restricted pathogens, the B. pseudomallei co-selection network does not facilitate host infection but is focused on bacterial survival in a harsh environment, particularly under nutrient depletion. Aside from providing a data resource, the study also showcases the power of combined genetics, transcriptomics and functional analysis as a tool for biology discovery. Implications of all available evidenceOur findings provide evolutionary and biological evidence for preferential survival of B. pseudomallei under nutrient starvation. Agricultural practice that induces soil loss, which is not uncommon in melioidosis endemic areas has been linked to soil nutrient depletion and may contribute to the prevalence of B. pseudomallei and a consequent rise of melioidosis in these regions. Successful melioidosis control has to consider environmental health in addition to existing prevention policy.

microbiology↗

bioRxiv2020

Mycobacterium tuberculosis complex lineage 5 exhibits high levels of within-lineage genomic diversity

Pathogens of the Mycobacterium tuberculosis complex (MTBC) are considered monomorphic, with little gene content variation between strains. Nevertheless, several genotypic and phenotypic factors separate the different MTBC lineages (L), especially L5 and L6 (traditionally termed Mycobacterium africanum), from each other. However, genome variability and gene content especially of L5 and L6 strains have not been fully explored and may be potentially important for pathobiology and current approaches for genomic analysis of MTBC isolates, including transmission studies. We compared the genomes of 358 L5 clinical isolates (including 3 completed genomes and 355 Illumina WGS (whole genome sequenced) isolates) to the L5 complete genomes and H37Rv, and identified multiple genes differentially present or absent between H37Rv and L5 strains. Additionally, considerable gene content variability was found across L5 strains, including a split in the L5.3 sublineage into L5.3.1 and L5.3.2. These gene content differences had a small knock on effect on transmission cluster estimation, with clustering rates influenced by the selection of reference genome, and with potential over-estimation of recent transmission when using H37Rv as the reference genome. Our data show that the use of H37Rv as reference genome results in missing SNPs in genes unique for L5 strains. This potentially leads to an underestimation of the diversity present in the genome of L5 strains and in turn affects the transmission clustering rates. As such, a full capture of the gene diversity, especially for high resolution outbreak analysis, requires a variation of the single H37Rv-centric reference genome mapping approach currently used in most WGS data analysis pipelines. Moreover, the high within-lineage gene content variability suggests that the pan-genome of M. tuberculosis is at least several kilobases larger than previously thought, implying a concatenated or reference-free genome assembly (de novo) approach may be needed for particular questions. Data summarySequence data for the Illumina dataset are available at European Genome-phenome Archive (EGA; https://www.ebi.ac.uk/ega/) under the study accession numbers PRJEB38317 and PRJEB38656. Individual runs accession numbers are indicated in Table S8. PacBio raw reads for the L5 Benin genome are available on the ENA accession SAME3170744. The assembled L5 Benin genome is available on NCBI with accession PRJNA641267. To ensure naming conventions of the genes in the three L5 genomes can be followed, we have uploaded these annotated GFF files to figshare at https://doi.org/10.6084/m9.figshare.12911849.v1. Custom python scripts used in this analysis can be found at https://github.com/conmeehan/pathophy.

genetics↗

bioRxiv2020

Phylogenomics of Mycobacterium africanum reveals a new lineage and a complex evolutionary history

Human tuberculosis is caused by members of the Mycobacterium tuberculosis Complex (MTBC). The MTBC comprises several human-adapted lineages known as M. tuberculosis sensu stricto as well as two lineages (L5 and L6) traditionally referred to as M. africanum. Strains of L5 and L6 are largely limited to West Africa for reasons unknown, and little is known on their genomic diversity, phylogeography and evolution. Here, we analyzed the genomes of 365 L5 and 326 L6 strains, plus five related genomes that had not been classified into any of the known MTBC lineages, isolated from patients from 21 African countries. Our population genomic and phylogeographical analyses show that the unclassified genomes belonged to a new group that we propose to name MTBC Lineage 9 (L9). While the most likely ancestral distribution of L9 was predicted to be East Africa, the most likely ancestral distribution for both L5 and L6 was the Eastern part of West Africa. Moreover, we found important differences between L5 and L6 strains with respect to their phylogeographical substructure, genetic diversity and association with drug resistance. In conclusion, our study sheds new light onto the genomic diversity and evolutionary history of M. africanum, and highlights the need to consider the particularities of each MTBC lineage for understanding the ecology and epidemiology of tuberculosis in Africa and globally.

genomics↗

bioRxiv2020

A global data-driven census of Salmonella small proteins and their potential functions in bacterial virulence

Small proteins are an emerging class of gene products with diverse roles in bacterial physiology. However, a full understanding of their importance has been hampered by insufficient genome annotations and a lack of comprehensive characterization in microbes other than Escherichia coli. We have taken an integrative approach to accelerate the discovery of small proteins and their putative virulence-associated functions in Salmonella Typhimurium. We merged the annotated small proteome of Salmonella with new small proteins predicted with in silico and experimental approaches. We then exploited existing and newly generated global datasets that provide information on small open reading frame expression during infection of epithelial cells (dual RNA-seq), contribution to bacterial fitness inside macrophages (TraDIS), and potential engagement in molecular interactions (Grad-seq). This integrative approach suggested a new role for the small protein MgrB beyond its known function in regulating PhoQ. We demonstrate a virulence and motility defect of a Salmonella {Delta}mgrB mutant and reveal an effect of MgrB in regulating the Salmonella transcriptome and proteome under infection-relevant conditions. Our study highlights the power of interpreting available "omics" datasets with a focus on small proteins, and may serve as a blueprint for a data integration-based survey of small proteins in diverse bacteria.

microbiology↗

bioRxiv2020

Linking phenotype, genotype and ecology: antimicrobial resistance in the zoonotic pathogen Streptococcus suis

Antimicrobial resistance (AMR) is among the gravest threats to human health and food security worldwide. Pigs receive more antimicrobials than most other livestock, and are a known source of zoonotic disease. We studied AMR in Streptococcus suis, a commensal found in most pigs, but which can also cause serious disease in both pigs and humans. We obtained replicated measures of Minimum Inhibitory Concentration (MIC) for 16 antibiotics, across a panel of 678 isolates, from the major pig-producing regions of the world. For several drugs, there was no natural separation into "resistant" and "susceptible", highlighting the need to treat MIC as a quantitative trait. We found differences in MICs between countries, consistent with their patterns of antimicrobial usage. AMR levels were high even for drugs not used to treat S. suis, with many multi-drug resistant isolates. And similar levels of resistance were found in pigs and humans from zoonotic regions. We next used whole genome sequences for each isolate to identify 43 candidate resistance determinants, 22 of which were novel in S. suis. The presence of these determinants explained most of the variation in MIC. But there were also complications, including epistatic interactions, where known resistance alleles had no effect in some genetic backgrounds. Beta-lactam resistance involved many variants of small effect, appearing in a characteristic order. Our results confirm the potential for genomic data to aid in the fight against AMR, but also demonstrate that it cannot be tackled one species or one drug at a time.

microbiology↗

bioRxiv2020

Kill and cure: genomic phylogeny and bioactivity of a diverse collection of Burkholderia gladioli bacteria capable of pathogenic and beneficial lifestyles

Burkholderia gladioli is one of few bacteria with a broad ecology spanning disease in humans, animals, and plants, and encompassing beneficial interactions with multiple eukaryotic hosts. It is a plant pathogen, a bongkrekic acid toxin producing food-poisoning agent, and a lung pathogen in people with cystic fibrosis (CF). Contrasting beneficial traits include antifungal production exploited by insects to protect their eggs, plant protective abilities and antibiotic biosynthesis. We explored the ecological diversity and specialized metabolite biosynthesis of 206 B. gladioli strains, phylogenomically defining 5 evolutionary clades. Historical disease pathovars (pv) B. gladioli pv. allicola and B. gladioli pv. cocovenenans were phylogenetically distinct, while B. gladioli pv. gladioli and B. gladioli pv. agaricicola were indistinguishable. Soft-rot disease and CF infection pathogenicity traits were conserved across all pathovars. Biosynthetic gene clusters for toxoflavin, caryoynencin and enacyloxin were dispersed across B. gladioli, but bongkrekic acid and gladiolin production were clade specific. Strikingly, 13% of CF-infection strains characterised (n=194) were bongkrekic acid toxin positive, uniquely linking this food-poisoning risk factor to chronic lung disease. Toxin production was suppressed by exposing strains to the antibiotic trimethoprim, providing a potential therapeutic strategy to minimise poisoning risk in CF.

microbiology↗

bioRxiv2020

Producing Polished Prokaryotic Pangenomes with the Panaroo Pipeline

Population-level comparisons of prokaryotic genomes must take into account the substantial differences in gene content, resulting from frequent horizontal gene transfer, gene duplication and gene loss. However, the automated annotation of prokaryotic genomes is imperfect, and errors due to fragmented assemblies, contamination, diverse gene families and mis-assemblies accumulate over the population, leading to profound consequences when analysing the set of all genes found in a species. Here we introduce Panaroo, a graph based pangenome clustering tool that is able to account for many of the sources of error introduced during the annotation of prokaryotic genome assemblies. We verified our approach through extensive simulations of de novo assemblies using the infinitely many genes model and by analysing a number of publicly available large bacterial genome datasets. Using a highly clonal Mycobacterium tuberculosis dataset as a negative control case, we show that failing to account for annotation errors can lead to pangenome estimates that are dominated by error. We additionally demonstrate the utility of the improved graphical output provided by Panaroo by performing a pan-genome wide association study in Neisseria gonorrhoeae and by analysing gene gain and loss rates across 51 of the major global pneumococcal sequence clusters. Panaroo is freely available under an open source MIT licence at https://github.com/gtonkinhill/panaroo.

genomics↗

bioRxiv2019

Genomic analysis of carbapenemase-encoding plasmids from Klebsiella pneumoniae across Europe highlights three major patterns of dissemination

The incidence of Klebsiella pneumoniae infections that are resistant to carbapenems, a last-line class of antibiotics, has been rapidly increasing. The primary mechanism of carbapenem resistance is production of carbapenemase enzymes, which are most frequently encoded on plasmids by blaOXA-48-like, blaVIM, blaNDM and blaKPC genes. Using short-read sequence data, we previously analysed genomes of 1717 isolates from the K. pneumoniae species complex submitted during the European survey of carbapenemase-producing Enterobacteriaceae (EuSCAPE). Here, we investigated the diversity, prevalence and transmission dynamics of carbapenemase-encoding plasmids using long-read sequencing of representative isolates (n=79) from this collection in combination with short-read data from all isolates. We highlight three major patterns by which carbapenemase genes have disseminated via plasmids. First, blaOXA-48-like genes have spread across diverse lineages primarily via a highly conserved, epidemic pOXA-48-like plasmid. Second, blaVIM and blaNDM genes have spread via transient associations of diverse plasmids with numerous lineages. Third, blaKPC genes have transmitted predominantly by stable association with one clonal lineage (ST258/512) despite frequent mobilisation between pre-existing yet diverse plasmids within the lineage. Despite contrasts in these three modes of carbapenemase gene spread, which can be summarised as using one plasmid/multiple lineages, multiple plasmids/multiple lineages, and multiple plasmids/one lineage, all are underpinned by significant propagation along high-risk clonal lineages.

microbiology↗

bioRxiv2019

Genomic surveillance of Escherichia coli ST131 identifies local expansion and serial replacement of subclones

Escherichia coli sequence type 131 (ST131) is a pandemic clone that is evolving rapidly with increasing levels of antimicrobial resistance. Here, we investigated an outbreak of E. coli ST131 producing extended spectrum {beta}-lactamases (ESBLs) in a long-term care facility (LTCF) in Ireland by combining data from this LTCF (n=69) with other Irish (n=35) and global (n=690) ST131 genomes to reconstruct the evolutionary history and understand changes in population structure and genome architecture over time. This required a combination of short and long-read genome sequencing, de novo assembly, read mapping, ESBL gene screening, plasmid alignment and temporal phylogenetics. We found that clade C was the most prevalent (686 out of 794 isolates, 86%) of the three major ST131 clades circulating worldwide (A, B, C), and was associated with the presence of different ESBL alleles, diverse plasmids and transposable elements. Clade C was estimated to have emerged in [~]1985 and subsequently acquired different ESBL gene variants (blaCTX-M-14 vs blaCTX-M-15). An ISEcp1- mediated transposition of the blaCTX-M-15 gene further increased the diversity within Clade C. We discovered a local clonal expansion of a rare C2 lineage (C2_8) with a chromosomal insertion of blaCTX-M-15 at the mppA gene. This was acquired from an IncFIA plasmid. The C2_8 lineage clonally expanded in the Irish LTCF from 2006, displacing the existing C1 strain (C1_10) and highlighting the potential for novel ESBL-producing ST131 with a distinct genetic profile to cause outbreaks strongly associated with specific healthcare environments.\n\nImportanceExtraintestinal pathogenic E. coli (ExPEC) ST131 is adapting in the context of antibiotic exposure, resulting in a pandemic with distinct genetic subtypes. Here, we track the evolution of antibiotic-resistance gene variants originally discovered in an ExPEC ST131 outbreak that was identified in a LTCF in Ireland. Analysis of 794 global ST131 genomes show that subclade C1 was associated with the initial infection outbreak, but that a new lineage from subclade C2 successfully displaced C1. This genetically distinct C2 subclade with a chromosomal insertion of a key antibiotic-resistance gene had clonally expanded within the LTCF. We provide new insights into the timing of genetic events driving the diversification of C2 subclades to show that that outbreak C2 strain likely evolved elsewhere before spreading to the LTCF. This study highlights the scope of antibiotic-resistance gene rearrangement within ST131, reinforcing the need to integrate genomic, epidemiological and microbiological approaches to understand ST131 transmission.

genomics↗

bioRxiv2019

Contrasting approaches to genome-wide association studies impact the detection of resistance mechanisms in Staphylococcus aureus

Rapid detection of antibiotic resistance using whole-genome sequencing (WGS) could improve clinical outcomes and limit the spread of resistance. For this to succeed, we need an accurate way of linking genotype to phenotype, that identifies new resistance mechanisms as they appear. To assess how close we are to this goal, we characterized antimicrobial resistance determinants in >4,000 Staphylococcus aureus genomes of isolates associated with bloodstream infection in the United Kingdom and Ireland. We sought to answer three questions: 1) how well did known resistance mechanisms explain phenotypic resistance in our collection, 2) how many previously identified resistance mechanisms appeared in our collection, and 3) how many of these were detectable using four contrasting genome-wide association study (GWAS) methods. Resistance prediction based on the detection of known resistance determinants was 98.8% accurate. We identified challenges in correcting for population structure, clustering orthologous genes, and identifying causal mechanisms in rare or common phenotypes, which reduced the recovery of known mechanisms. Limited sensitivity and specificity of these methods made prediction using GWAS-discovered hits alone less accurate than using literature-derived genetic determinants. However, GWAS methods identified novel mutations associated with resistance, including five mutations in rpsJ, which improved tetracycline resistance prediction for 28 isolates, and a T118I substitution in fusA which resulted in better fusidic acid resistance prediction for 5 isolates. Thus, GWAS approaches in conjunction with phenotypic testing data can support the development of comprehensive databases to enable real-time use of WGS for patient management.

bioinformatics↗

bioRxiv2019

Discovery of a Pseudomonas aeruginosa Type VI secretion system toxin targeting bacterial protein synthesis using a global genomics approach

The Type VI secretion system (T6SS) is a bacterial weapon which delivers toxic effectors to kill competitors or subvert some of their key functions. Here we use transposon directed insertion-site sequencing (TraDIS) to identify T6SS toxins associated with the H1-T6SS, one of the three T6SS machines found in Pseudomonas aeruginosa. This approach identified several putative toxin-immunity pairs, including Tse8-Tsi8. Full characterization of this protein pair demonstrated that Tse8 is delivered by the VgrG1a spike complex into prey cells where it targets the transamidosome, a multiprotein complex involved in protein synthesis in bacteria lacking either one or both of the asparagine or glutamine tRNA synthases. Our data suggests that Tse8 combines as a non-cognate component of the transamidosome complex, reducing fitness by limiting the ability of the cell to synthesize proteins. This is the first demonstration of a T6SS toxin affecting protein synthesis, expanding the range of cellular components targeted by this bacterial weapon. The success of the current study validates the use of our TraDIS approach as a tool to drastically expand the repertoire of T6SS toxins in any T6SS-encoding bacterium.

microbiology↗

bioRxiv2019

Engineering bacteriocin-mediated resistance against plant pathogenic bacteria in plants

Pseudomonas syringae (Ps) and related plant pathogenic bacteria are responsible for losses in diverse crops such as tomato, kiwifruit, pepper, olive and soybean. Current solutions, involving the use of chemicals and the introduction of resistance genes, have enjoyed only limited success and may have adverse environmental impacts. Consequently, there is a pressing need to develop alternative technologies to address the problem of bacterial disease in crops. An alternative strategy is to utilise the narrow spectrum protein antibiotics (bacteriocins) used by diverse bacteria for competition against closely related species. Here, we demonstrate that active putidacin L1 (PL1) can be expressed at high levels in planta and expression of PL1 provides effective resistance against diverse pathovars of Ps. Furthermore, we found that strains which evolve to become insensitive to PL1; lose their O-antigen, exhibit reduced motility and are less virulent in PL1 transgenic plants. Our results provide proof-of-principle that transgene-mediated expression of a bacteriocin in planta is an effective strategy for providing disease resistance against bacterial pathogens. Genetically modified (GM) crops expressing insecticidal proteins have proved extremely successful as a strategy for pest management; expressing bacteriocins to control bacterial disease may have a similar potential. Crucially, nearly all genera of bacteria, including many plant pathogenic species, produce bacteriocins, providing an extensive source of these antimicrobial agents.\n\nSIGNIFICANCEWith the global population to surpass 9 billion by 2050 there is a huge demand to make industrial farming as efficient as possible. A disadvantage of industrial farming is the lack of genetic diversity within crop monocultures, which make them highly susceptible to diseases caused by plant pathogenic bacteria like Pseudomonas syringae. Bacteriocins are narrow spectrum protein antibiotics which are produced by all major bacterial lineages. Their main purpose is to eliminate competitor strains to establish dominance within a niche. By arming plants with bacteriocins we can increase the genetic toolbox used to engineer crops to be resistant to specific bacterial plant pathogens.

plant biology↗

bioRxiv2019

Genetic variation regulates the activation and specificity of Restriction-Modification systems in Neisseria gonorrhoeae

Restriction-Modification systems (RMS) are one of the main mechanisms of defence against foreign DNA invasion and can have an important role in the regulation of gene expression. The obligate human pathogen Neisseria gonorrhoeae carries one of the highest loads of RMS in its genome; between 13 to 15 of the three main types. Previous work has described their organization in the reference genome FA1090 and has experimentally inferred the associated methylated motifs. Here, we studied the structure of RMS and target methylated motifs in 25 gonococcal strains sequenced with Single Molecule Real-Time (SMRT) technology, which provides data on DNA modification. The results showed a variable picture of active RMS in different strains, with phase variation switching the activity of Type III RMS, and both the activity and specificity of a Type I RMS. Interestingly, the Dam methylase was found in place of the NgoAXI endonuclease in two of the strains, despite being previously thought to be absent in the gonococcus. We also identified the real methylation target of NgoAX as 5-GCAGA-3, different from that previously described. Results from this work give further insights into the diversity and dynamics of RMS and methylation patterns in N. gonorrhoeae.

microbiology↗