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The environmental bacterium Burkholderia pseudomallei causes melioidosis, an important endemic human disease in tropical and sub-tropical countries. This bacterium occupies broad ecological niches including soil, contaminated water, single-cell microbes, plants and infection in a range of animal species. Here, we performed genome-wide association studies for genetic determinants of environmental and human adaptation using a combined dataset of 1,010 whole genome sequences of B. pseudomallei from Northeast Thailand and Australia, representing two major disease hotspots. With these data, we identified 47 genes from 26 distinct loci associated with clinical or environmental isolates from Thailand and replicated 12 genes in an independent Australian cohort. We next outlined the selective pressures on the genetic loci (dN/dS) and the frequency at which they had been gained or lost throughout their evolutionary history, reflecting the bacterial adaptability to a wide range of ecological niches. Finally, we highlighted loci likely implicated in human disease.
\n \n\n \n \nRapid 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.
\n \n\n \n \nBackground: Melioidosis is a frequently fatal disease caused by an environmental bacterium Burkholderia pseudomallei. The disease is prevalent in northeast Thailand, particularly among rice field farmers who are at risk of bacterial exposure through contact with contaminated soil and water. However, not all exposure results in disease, and infection can have different infection outcomes. Our hypothesis is that the acquisition and outcomes of melioidosis may be influenced by genetic factors of the bacterium, the host, or a combination of both. To address this hypothesis, we aim to collect, sequence, and analyse genetic data from melioidosis patients and controls, along with isolates of B. pseudomallei obtained from patients. Additionally, we will study the metagenomics of the household water supply for both patients and controls, including the presence of B. pseudomallei. Methods: BurkHostGEN is an ongoing observational study being conducted at Sunpasitthiprasong Hospital, Ubon Ratchathani, Thailand. Weare obtaining consent from 600 melioidosis patients and 700 controls, spanning both sexes, to collect 1 mL of blood for host DNA analysis, 3 mL of blood for RNA analysis, as well as 5 L of household water supply for metagenomic analysis. Additionally, we are isolating B. pseudomallei from the melioidosis patients to obtain bacterial DNA. This comprehensive approach will allow us to identify B. pseudomallei and their paired host genetic factors associated with disease acquisition and severity. Ethical approvals have been obtained for BurkHostGEN. Host and bacterial genetic data will be uploaded to European Genome-Phenome Archive (EGA) and European Nucleotide Archive (ENA), respectively. Conclusions: BurkHostGEN holds the potential to discover bacterial and host genetic factors associated with melioidosis infection and severity of illness. It can also support various study designs, including biomarker validation, disease pathogenesis, and epidemiological analysis not only for melioidosis but also for other infectious diseases.
\n \n\n \n \nThe extent to which evolution is constrained by the rate at which horizontal gene transfer (HGT) allows DNA to move between genetic lineages is an open question, which we address in the context of antibiotic resistance in Streptococcus pneumoniae. We analyze microbiological, genomic and epidemiological data from the largest-to-date sequenced pneumococcal carriage study in 955 infants from a refugee camp on the Thailand-Myanmar border. Using a unified framework, we simultaneously test prior hypotheses on rates of HGT and a key evolutionary covariate (duration of carriage) as determinants of resistance frequencies. We conclude that in this setting, there is only weak evidence for the rate of HGT playing a role in the evolutionary dynamics of resistance. Instead, observed resistance frequencies are best explained as the outcome of selection acting on a pool of variants, irrespective of the rate at which resistance determinants move between genetic lineages.
\n \n\n \n \nAbstractThe soil bacterium Burkholderia pseudomallei is the causative agent of melioidosis and a significant cause of human morbidity and mortality in many tropical and subtropical countries. The species notoriously survives harsh environmental conditions but the genetic architecture for these adaptations remains unclear. Here we employed a powerful combination of genome-wide epistasis and co-selection studies (2,011 genomes), condition-wide transcriptome analyses (82 diverse conditions), and a gene knockout assay to uncover signals of \u201cco-selection\u201d\u2014that is a combination of genetic markers that have been repeatedly selected together through B.\u00a0pseudomallei evolution. These enabled us to identify 13,061 mutation pairs under co-selection in distinct genes and noncoding RNA. Genes under co-selection displayed marked expression correlation when B.\u00a0pseudomallei was subjected to physical stress conditions, highlighting the conditions as one of the major evolutionary driving forces for this bacterium. We identified a putative adhesin (BPSL1661) as a hub of co-selection signals, experimentally confirmed a BPSL1661 role under nutrient deprivation, and explored the functional basis of co-selection gene network surrounding BPSL1661 in facilitating the bacterial survival under nutrient depletion. Our findings suggest that nutrient-limited conditions have been the common selection pressure acting on this species, and allelic variation of BPSL1661 may have promoted B.\u00a0pseudomallei survival during harsh environmental conditions by facilitating bacterial adherence to different surfaces, cells, or living hosts.
\n \n\n \n \nCovariance-based discovery of polymorphisms under co-selective pressure or epistasis has received considerable recent attention in population genomics. Both statistical modeling of the population level covariation of alleles across the chromosome and model-free testing of dependencies between pairs of polymorphisms have been shown to successfully uncover patterns of selection in bacterial populations. Here we introduce a model-free method, SpydrPick, whose computational efficiency enables analysis at the scale of pan-genomes of many bacteria. SpydrPick incorporates an efficient correction for population structure, which adjusts for the phylogenetic signal in the data without requiring an explicit phylogenetic tree. We also introduce a new type of visualization of the results similar to the Manhattan plots used in genome-wide association studies, which enables rapid exploration of the identified signals of co-evolution. Simulations demonstrate the usefulness of our method and give some insight to when this type of analysis is most likely to be successful. Application of the method to large population genomic datasets of two major human pathogens, Streptococcus pneumoniae and Neisseria meningitidis, revealed both previously identified and novel putative targets of co-selection related to virulence and antibiotic resistance, highlighting the potential of this approach to drive molecular discoveries, even in the absence of phenotypic data.
\n \n\n \n \nAbstractThe environmental bacterium Burkholderia pseudomallei causes melioidosis, an important endemic human disease in tropical and sub-tropical countries. This bacterium occupies broad ecological niches including soil, contaminated water, single-cell microbes, plants and infection in a range of animal species. Here, we performed genome-wide association studies for genetic determinants of environmental and human adaptation using a combined dataset of 1,010 whole genome sequences of B. pseudomallei from Northeast Thailand and Australia, representing two major disease hotspots. With these data, we identified 47 genes from 26 distinct loci associated with clinical or environmental isolates from Thailand and replicated 12 genes in an independent Australian cohort. We next outlined the selective pressures on the genetic loci (dN/dS) and the frequency at which they had been gained or lost throughout their evolutionary history, reflecting the bacterial adaptability to a wide range of ecological niches. Finally, we highlighted loci likely implicated in human disease.
\n \n\n \n \nAbstractStreptococcus pneumoniae is a significant human pathogen and a leading cause of infant mortality in developing countries. Considerable global variation in the pneumococcal carriage prevalence has been observed and the ecological factors contributing to it are not yet fully understood. We use data from a cohort of infants in Asia to study the effects of climatic conditions on both acquisition and clearance rates of the bacterium, finding significantly higher transmissibility during the cooler and drier months. Conversely, the length of a colonization period is unaffected by the season. Independent carriage data from studies conducted on the African and North American continents suggest similar effects of the climate on the prevalence of this bacterium, which further validates the obtained results. Further studies could be important to replicate the findings and explain the mechanistic role of cooler and dry air in the physiological response to nasopharyngeal acquisition of the pneumococcus.
\n \n\n \n \n\n There has been growing interest in the statistics community to develop methods for inferring transmission pathways of infectious pathogens from molecular sequence data. For many datasets, the computational challenge lies in the huge dimension of the missing data. Here, we introduce an importance sampling scheme\u00a0in which the transmission trees and phylogenies of pathogens are both sampled from reasonable importance distributions, alleviating the inference. Using this approach, arbitrary models of transmission could be considered, contrary to many earlier proposed methods. We illustrate the scheme by analysing transmissions of\n Streptococcus pneumoniae\n from household to household within a refugee camp, using data in which only a fraction of hosts is observed, but which is still rich enough to unravel the within-household transmission dynamics and pairs of households between whom transmission is plausible. We observe that while probability of direct transmission is low even for the most prominent cases of transmission, still those pairs of households are geographically much closer to each other than expected under random proximity.\n
\n \n\n \n \nScientific Reports 5: Article number: 11344; published online: 12 June 2015; updated: 29 March 2016 The Acknowledgements section in this Article is incomplete. \u201cWe thank Ben Cooper for helpful discussions, Ritva Syrj\u00e4nen for pointing out useful references on the seasonality of colonization, Marc Lipsitch for advice in parametrizing the effects of previous exposures in the model and Dan Weinberger on sharing the point estimates on the Navajo colonization study, visualized in Fig.
\n \n\n \n \nABSTRACT\n\n\n\nBurkholderia pseudomallei\n and\n Burkholderia cepacia\n are Gram-negative, soil-dwelling bacteria that are found in a wide variety of environmental niches. While\n B. pseudomallei\n is the causative agent of melioidosis in humans and animals, members of the\n B. cepacia\n complex typically only cause disease in immunocompromised hosts. In this study, we report the identification of\n B. cepacia\n strains isolated from either patients or soil in Laos and Thailand that express a\n B. pseudomallei\n -like 6-deoxyheptan capsular polysaccharide (CPS). These\n B. cepacia\n strains were initially identified based on their positive reactivity in a latex agglutination assay that uses the CPS-specific monoclonal antibody (mAb) 4B11. Mass spectrometry and\n recA\n sequencing confirmed the identity of these isolates as\n B. cepacia\n (formerly genomovar I). Total carbohydrates extracted from\n B. cepacia\n cell pellets reacted with\n B. pseudomallei\n CPS-specific mAbs MCA147, 3C5, and 4C4, but did not react with the\n B. pseudomallei\n lipopolysaccharide-specific mAb Pp-PS-W. Whole genome sequencing of the\n B. cepacia\n isolates revealed the presence of genes demonstrating significant homology to those comprising the\n B. pseudomallei\n CPS biosynthetic gene cluster. Collectively, our results provide compelling evidence that\n B. cepacia\n strains expressing the same CPS as\n B. pseudomallei\n co-exist in the environment alongside\n B. pseudomallei\n . Since CPS is a target that is often used for presumptive identification of\n B. pseudomallei\n , it is possible that the occurrence of these unique\n B. cepacia\n strains may complicate the diagnosis of melioidosis.\n \n\nIMPORTANCE\n\nBurkholderia pseudomallei\n , the etiologic agent of melioidosis, is an important cause of morbidity and mortality in tropical and subtropical regions worldwide. The 6-deoxyheptan capsular polysaccharide (CPS) expressed by this bacterial pathogen is a promising target antigen that is useful for rapidly diagnosing melioidosis. Using assays incorporating CPS-specific monoclonal antibodies, we identified both clinical and environmental isolates of\n Burkholderia cepacia\n that express the same CPS antigen as\n B. pseudomallei\n . Because of this, it is important that staff working in melioidosis-endemic areas are aware that these strains co-exist in the same niches as\n B. pseudomallei\n and do not solely rely on CPS-based assays such as latex-agglutination, AMD Plus Rapid Tests, or immunofluorescence tests for the definitive identification of\n B. pseudomallei\n isolates.\n \n\n
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