Co-evolutionary Signals Identify Burkholderia pseudomallei Survival Strategies in a Hostile Environment.

The 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 "co-selection"-that is a combination of genetic markers that have been repeatedly selected together through B. pseudomallei 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. pseudomallei 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. pseudomallei survival during harsh environmental conditions by facilitating bacterial adherence to different surfaces, cells, or living hosts.

Benchmarking CRISPR-BP34 for point-of-care melioidosis detection in low-income and middle-income countries: a molecular diagnostics study.

BACKGROUND: Melioidosis is a neglected but often fatal tropical disease. The disease has broad clinical manifestations, which makes diagnosis challenging and time consuming. To improve diagnosis, we aimed to evaluate the performance of the CRISPR-Cas12a system (CRISPR-BP34) to detect Burkholderia pseudomallei DNA across clinical specimens from patients suspected to have melioidosis. METHODS: We conducted a prospective, observational cohort study of adult patients (aged ≥18 years) with melioidosis at Sunpasitthiprasong Hospital, a tertiary care hospital in Thailand. Participants were eligible for inclusion if they had culture-confirmed B pseudomallei infection from any clinical samples. Data were collected from patient clinical records and follow-up telephone calls. Routine clinical samples (blood, urine, respiratory secretion, pus, and other body fluids) were collected for culture. We documented time taken for diagnosis, and mortality at day 28 of follow-up. We also performed CRISPR-BP34 detection on clinical specimens collected from 330 patients with suspected melioidosis and compared its performance with the current gold-standard culture-based method. Discordant results were validated by three independent qualitative PCR tests. This study is registered with the Thai Clinical Trial Registry, TCTR20190322003. FINDINGS: Between Oct 1, 2019, and Dec 31, 2022, 876 patients with culture-confirmed melioidosis were admitted or referred to Sunpasitthiprasong Hospital, 433 of whom were alive at diagnosis and were enrolled in this study. Median time from sample collection to diagnosis by culture was 4·0 days (IQR 3·0-5·0) among all patients with known survival status at day 28, which resulted in delayed treatment. 199 (23%) of 876 patients died before diagnosis and 114 (26%) of 433 patients in follow-up were treated, but died within 28 days of admission. To test the CRISPR-BP34 assay, we enrolled and collected clinical samples from 114 patients with melioidosis and 216 patients without melioidosis between May 26 and Dec 31, 2022. Application of CRISPR-BP34 reduced the median sample-to-diagnosis time to 1·1 days (IQR 0·7-1·5) for blood samples, 2·3 h (IQR 2·3-2·4) for urine, and 3·3 h (3·1-3·4) for respiratory secretion, pus, and other body fluids. The overall sensitivity of CRISPR-BP34 was 93·0% (106 of 114 samples [95% CI 86·6-96·9]) compared with 66·7% (76 of 114 samples [57·2-75·2]) for culture. The overall specificity of CRISPR-BP34 was 96·8% (209 of 216 samples [95% CI 93·4-98·7]), compared with 100% (216 of 216 samples [98·3-100·0]) for culture. INTERPRETATION: The sensitivity, specificity, speed, and window of clinical intervention offered by CRISPR-BP34 support its prospective use as a point-of-care diagnostic tool for melioidosis. Future development should be focused on scalability and cost reduction. FUNDING: Chiang Mai University Thailand and Wellcome Trust UK.

BurkHostGEN: a study protocol for evaluating variations in the Burkholderia pseudomallei and host genomes associated with melioidosis infection.

Background: 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 manifest diverse outcomes. We postulate that genetic factors, whether from the bacterium, the host or the combination of both, may influence disease outcomes. 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. We are 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.

Highly specific and sensitive detection of Burkholderia pseudomallei genomic DNA by CRISPR-Cas12a.

Detection of Burkholderia pseudomallei, a causative bacterium for melioidosis, remains a challenging undertaking due to long assay time, laboratory requirements, and the lack of specificity and sensitivity of many current assays. In this study, we are presenting a novel method that circumvents those issues by utilizing CRISPR-Cas12a coupled with isothermal amplification to identify B. pseudomallei DNA from clinical isolates. Through in silico search for conserved CRISPR-Cas12a target sites, we engineered the CRISPR-Cas12a to contain a highly specific spacer to B. pseudomallei, named crBP34. The crBP34-based detection assay can detect as few as 40 copies of B. pseudomallei genomic DNA while discriminating against other tested common pathogens. When coupled with a lateral flow dipstick, the assay readout can be simply performed without the loss of sensitivity and does not require expensive equipment. This crBP34-based detection assay provides high sensitivity, specificity and simple detection method for B. pseudomallei DNA. Direct use of this assay on clinical samples may require further optimization as these samples are complexed with high level of human DNA.

Corrigendum: An Evolutionary Arms Race Between Burkholderia pseudomallei and Host Immune System: What Do We Know?

[This corrects the article DOI: 10.3389/fmicb.2020.612568.].

An Evolutionary Arms Race Between Burkholderia pseudomallei and Host Immune System: What Do We Know?

A better understanding of co-evolution between pathogens and hosts holds promise for better prevention and control strategies. This review will explore the interactions between Burkholderia pseudomallei, an environmental and opportunistic pathogen, and the human host immune system. B. pseudomallei causes "Melioidosis," a rapidly fatal tropical infectious disease predicted to affect 165,000 cases annually worldwide, of which 89,000 are fatal. Genetic heterogeneities were reported in both B. pseudomallei and human host population, some of which may, at least in part, contribute to inter-individual differences in disease susceptibility. Here, we review (i) a multi-host-pathogen characteristic of the interaction; (ii) selection pressures acting on B. pseudomallei and human genomes with the former being driven by bacterial adaptation across ranges of ecological niches while the latter are driven by human encounter of broad ranges of pathogens; (iii) the mechanisms that generate genetic diversity in bacterial and host population particularly in sequences encoding proteins functioning in host-pathogen interaction; (iv) reported genetic and structural variations of proteins or molecules observed in B. pseudomallei-human host interactions and their implications in infection outcomes. Together, these predict bacterial and host evolutionary trajectory which continues to generate genetic diversity in bacterium and operates host immune selection at the molecular level.