Environmental exposure to Burkholderia pseudomallei, the causative agent of melioidosis, remains poorly characterised due to the low sensitivity of conventional detection methods. Here, we develop CRISPR-BEEPs, a sensitive and resource-efficient CRISPR-based assay, and evaluate its performance against conventional culture-based plate inspection using double-qPCR as the reference standard. CRISPR-BEEPs demonstrated higher sensitivity (93.5% vs 19.4%) and high specificity (100% vs 98.0%). We apply the assay to water samples from natural and piped sources across 15,118 km² in northeast Thailand, collected from or near the households of 439 participants with melioidosis. We compared these with households of 190 participants with other bacterial infections and 506 healthy control participants living in the same endemic region who had never developed melioidosis. CRISPR-BEEPs detects B. pseudomallei in 73.3% of groundwater, 32.9% of surface water, and 26.2% of piped water samples, with results comparable to double-qPCR. The improved sensitivity reveals a significant association between environmental detection within 10 km of households and melioidosis risk (OR 2.74; 95% CI:1.38-5.48), an association undetectable using conventional methods. These findings expose critical sanitation gaps and highlight the value of high-resolution environmental surveillance for disease prevention.

SUMMARY Burkholderia pseudomallei and Burkholderia mallei present significant biosafety challenges due to their high pathogenicity, environmental resilience, intrinsic antimicrobial resistance, and potential use as bioterrorism agents. This review examines key aspects of laboratory management and infection control for these organisms, focusing on inconsistencies in biosafety protocols and risk classifications across regions. We synthesize current evidence on biocontainment requirements, disinfection strategies, and personal protective equipment (PPE), with particular emphasis on sustainable practices for laboratories in low-resource settings. Although laboratory-acquired infections are rare, their potential severity underscores the importance of stringent safety measures. Critical gaps remain in our understanding of infectious dose, the effectiveness of post-exposure prophylaxis (PEP), and the development of risk assessment frameworks. We advocate for harmonized global biosafety standards and targeted research on transmission dynamics and inactivation protocols. These priorities are essential to enhance laboratory safety, especially in endemic areas, and to inform coherent international policy on B. pseudomallei and B. mallei containment and management.

Most studies on bacteriophages (phages) of the Gram-negative bacterium Burkholderia pseudomallei rely on in silico predictions and thus underestimate the true diversity of phages. Analysis of the whole genome sequences of culturable prophages induced from B. pseudomallei and B. thailandensis, along with their free Burkholderia phages isolated from soils in Thailand, identified six novel groups of Burkholderia phages, surpassing in silico expectations. The analysis also indicated that soil-dwelling phages may have originated from lysogenic B. pseudomallei strains. Free phages isolated from soil showed high nucleotide similarity to prophage sequences in B. pseudomallei, including phages previously cultured from melioidosis patients' hemocultures, indicating that similar phage types occur in both environmental and clinical sources. Phylogenomic analysis also revealed close genomic relatedness between prophages from B. thailandensis and B. pseudomallei, although the biological significance remains unknown. Together, these findings refine our understanding of the genomic diversity and ecological patterns of Burkholderia phages.

Environmental persistence allows opportunistic pathogens to survive a range of harsh conditions, increasing the likelihood of eventual infection. Burkholderia pseudomallei , the causative agent of melioidosis, can endure long-term nutrient-depletion in the environment, but its adaptive mechanisms remain poorly understood. Here, we investigated the evolutionary trajectory of a clinical B. pseudomallei strain maintained in sterile water since 1994. The strain was inoculated into nine individual tubes at nine initial concentrations (102 - 1010 CFU/mL) and has remained viable to date. Liquid chromatography-mass spectrometry analysis of the water identified potential carbon sources, including phthalic acid - a plastic degradation product likely leached from inoculation tubes - which the strain can metabolise via an intact catabolic operon. Genomic variations accumulated between 1994 and 2022 were characterised using both single-colony and plate-sweep sequencing which provided complementary insights. Across all tubes, we identified 249 single-nucleotide polymorphisms (SNPs), 73 indels, and a large-scale deletion. Cultures from each tube displayed a consistently low mutation rate (3.18 × 10-8 SNPs per site per year), suggesting that cells entered a dormant or slow-growth state. Of 393 genes with mutations, 193 were independently mutated in more than one tube, particularly those involved in signal transduction, cell wall and membrane biogenesis, and secondary metabolite synthesis. These patterns indicate parallel adaptation to long-term nutrient deprivation through modulation of cell-density-related functions and loss of metabolically costly pathways. Remarkably, B. pseudomallei from this experiment remain viable after nearly three decades, providing a rare natural model for understanding how environmental bacteria endure and adapt in extremely nutrient-depleted conditions. ### Competing Interest Statement The authors have declared no competing interest. Wellcome Trust, https://ror.org/029chgv08, 216457/Z/19/Z, 220211, 206194 Prince Mahidol foundation

Melioidosis, caused by the Gram-negative bacterium Burkholderia pseudomallei, is an infectious disease with high rates of morbidity and mortality, which primarily affects low- and middle-income countries in South and Southeast Asia and Australia. The clinical manifestations of this disease are nonspecific and, therefore, rapid laboratory diagnosis is especially critical as appropriate management requires specific antimicrobials. This article aims to provide an overview of the current diagnostic methodologies, emerging technologies, susceptibility testing, and future perspectives for laboratory diagnosis of melioidosis. By examining conventional culture methods, mass spectrometry, antimicrobial susceptibility testing, antigen detection, molecular diagnostics, and serological assays, this article highlights the current challenges in accurately and cost-effectively diagnosing melioidosis in diverse clinical and resource-limited settings. A detailed analysis of current and future diagnostic methodologies will offer valuable insights for clinicians, researchers, and public health professionals. This review aims to influence clinical and laboratory guidelines for diagnosing melioidosis and future research directions.

IntroductionClinical microbiology laboratories are essential for diagnosing and monitoring antimicrobial resistance (AMR). Here, we assessed the systems involved in generating, managing and analyzing blood culture data in these laboratories in an upper-middle-income country.MethodsFrom October 2023 to February 2024, we conducted a survey on the utilization of automated systems and laboratory information management systems (LIMS) for blood culture specimens in 2022 across 127 clinical microbiology laboratories (one each from 127 public referral hospitals) in Thailand. We categorized automated systems for blood culture processing into three steps: incubation, bacterial identification, and antimicrobial susceptibility testing (AST).ResultsOf the 81 laboratories that completed the questionnaires, the median hospital bed count was 450 (range, 150-1,387), and the median number of blood culture bottles processed was 17,351 (range, 2,900-80,330). All laboratories (100%) had an automated blood culture incubation system. Three-quarters of the laboratories (75%, n = 61) had at least one automated system for both bacterial identification and AST, about a quarter (22%, n = 18) had no automated systems for either step, and two laboratories (3%) outsourced both steps. The systems varied and were associated with the hospital level. Many laboratories utilized both automated systems and conventional methods for bacterial identification (n = 54) and AST (n = 61). For daily data management, 71 laboratories (88%) used commercial microbiology LIMS, three (4%) WHONET, three (4%) an in-house database software and four (5%) did not use any software. Many laboratories manually entered data of incubation (73%, n = 59), bacterial identification (27%, n = 22) and AST results (25%, n = 20) from their automated systems into their commercial microbiology LIMS. The most common barrier to data analysis was 'lack of time', followed by 'lack of staff with statistical skills' and 'difficulty in using analytical software'.ConclusionIn Thailand, various automated systems for blood culture and LIMS are utilized. However, barriers to data management and analysis are common. These challenges are likely present in other upper-middle-income countries. We propose that guidance and technical support for automated systems, LIMS and data analysis are needed.

Ceftazidime (CAZ) resistance in Burkholderia pseudomallei, the causative agent of melioidosis, complicates treatment in endemic regions. This study identified a novel A172T mutation and other known penA mutations as critical contributors to CAZ resistance in a large Thai strain collection. Frequent gene duplication and amplification of penA, likely driven by Palindromic GC-Rich Repeat Sequences, highlights the urgent need for rapid diagnostics and optimized treatment strategies to manage this life-threatening disease effectively.

BACKGROUND:The presence of antimicrobial-resistant (AMR) bacteria in edible ice in tropical countries is largely unknown. METHODS:We evaluate the presence of extended-spectrum β-lactamase (ESBL)-producing Enterobacterales in 100 edible ice samples from drink carts in 20 markets in four provinces (five markets/province) in Thailand. Ten samples of commercially sold edible ice in sealed packages were tested as controls. RESULTS:Of 100 samples, 29 (29%) were culture positive for ESBL-producing Enterobacterales, with a median quantitative count of 2 colony-forming units (CFU)/100 mL (range, 1 to 40 CFU/100 mL). All control samples were culture negative for ESBL-producing Enterobacterales. CONCLUSIONS:AMR bacteria is commonly found in edible ice from drink carts.

Burkholderia pseudomallei and Burkholderia cepacia are Gram-negative, soil-dwelling bacteria that are found in a wide variety of environmental niches. While B. pseudomallei is the causative agent of melioidosis in humans and animals, members of the B. cepacia complex typically only cause disease in immunocompromised hosts. In this study, we report the identification of B. cepacia strains isolated from either patients or soil in Laos and Thailand that express a B. pseudomallei-like 6-deoxyheptan capsular polysaccharide (CPS). These B. cepacia 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 recA sequencing confirmed the identity of these isolates as B. cepacia (formerly genomovar I). Total carbohydrates extracted from B. cepacia cell pellets reacted with B. pseudomallei CPS-specific mAbs MCA147, 3C5, and 4C4, but did not react with the B. pseudomallei lipopolysaccharide-specific mAb Pp-PS-W. Whole genome sequencing of the B. cepacia isolates revealed the presence of genes demonstrating significant homology to those comprising the B. pseudomallei CPS biosynthetic gene cluster. Collectively, our results provide compelling evidence that B. cepacia strains expressing the same CPS as B. pseudomallei co-exist in the environment alongside B. pseudomallei. Since CPS is a target that is often used for presumptive identification of B. pseudomallei, it is possible that the occurrence of these unique B. cepacia strains may complicate the diagnosis of melioidosis. IMPORTANCE Burkholderia pseudomallei, 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 Burkholderia cepacia that express the same CPS antigen as B. pseudomallei. 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 B. pseudomallei 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 B. pseudomallei isolates.

Summary Background Environmental acquisition of Burkholderia pseudomallei can cause melioidosis, a life-threatening yet underreported disease. Understanding environmental exposure is essential for effective public health interventions, yet existing tools are limited in their ability to quantify exposure risks. Methods We conducted two complementary studies across a 15,118 km2 area of northeast Thailand to improve detection methods and investigate risk factors for melioidosis. In the first study, we compared a newly developed, equipment-light CRISPR-based assay (CRISPR-BP34) with conventional culture methods using both spiked samples and real water samples from household and community sources (November 2020 - November 2021). The second study involved a case-control analysis of 1,135 participants (October 2019 - January 2023) to evaluate the association between environmental exposure to B. pseudomallei (detected in Study 1) and melioidosis risk. Findings The CRISPR-BP34 assay demonstrated improved sensitivity (93.52% vs 19.44% for conventional methods) and specificity (100% vs 97.98%), allowing for more accurate detection of B. pseudomallei and exposure risk quantification. Environmental exposure to B. pseudomallei in water sources within a 10 km radius of participant households was significantly associated with increased melioidosis risk (OR: 2.74 [95% CI 1.38-5.48]). This risk was also heightened by known factors: occupational exposure among agricultural workers (4.46 [2.91-6.91]), and health factors like elevated hemoglobin A1c, indicating diabetes (1.35 [1.19-1.31]). Interpretation Our findings underscore the impact of environmental contamination on melioidosis risk. The robust association between contaminated water sources, including piped water systems, and clinical cases highlights the urgent need for improved water sanitation to mitigate melioidosis risk. Funding Wellcome Trust ### Competing Interest Statement The authors have declared no competing interest. ### Funding Statement CChe was funded by the Wellcome International Intermediate Fellowship (216457/Z/19/Z), the Sanger International Fellowship, and the University of Oxford Nuffield Department of Medicine Career Development Scheme. This research was funded in part by the Wellcome Trust [220211 and 206194]. For the purpose of Open Access, the author has applied a CC BY public copyright license to any Author Accepted Manuscript version arising from this submission. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. ### Author Declarations I confirm all relevant ethical guidelines have been followed, and any necessary IRB and/or ethics committee approvals have been obtained. Yes The details of the IRB/oversight body that provided approval or exemption for the research described are given below: This study received ethical approval from the Sunpasitthiprasong Hospital Ethical Review Board (015/62C) and the Oxford Tropical Research Ethics Committee (OxTREC 25-19). I confirm that all necessary patient/participant consent has been obtained and the appropriate institutional forms have been archived, and that any patient/participant/sample identifiers included were not known to anyone (e.g., hospital staff, patients or participants themselves) outside the research group so cannot be used to identify individuals. Yes I understand that all clinical trials and any other prospective interventional studies must be registered with an ICMJE-approved registry, such as ClinicalTrials.gov. I confirm that any such study reported in the manuscript has been registered and the trial registration ID is provided (note: if posting a prospective study registered retrospectively, please provide a statement in the trial ID field explaining why the study was not registered in advance). Yes I have followed all appropriate research reporting guidelines, such as any relevant EQUATOR Network research reporting checklist(s) and other pertinent material, if applicable. Yes All data produced in the present study are available upon reasonable request to the authors

In September 2021, a total of 25 patients diagnosed with COVID-19 developed acute melioidosis after (median 7 days) admission to a COVID-19 field hospital in Thailand. Eight nonpotable tap water samples and 6 soil samples were culture-positive for Burkholderia pseudomallei. Genomic analysis suggested contaminated tap water as the likely cause of illness.

Bacteriophages (phages), viruses that infect bacteria, are found in abundance not only in the environment but also in the human body. The use of phages for the diagnosis of melioidosis, a tropical infectious disease caused by Burkholderia pseudomallei, is emerging as a promising novel approach, but our understanding of conditions under which Burkholderia prophages can be induced remains limited. Here, we first demonstrated the isolation of Burkholderia phages from the hemocultures of melioidosis patients. The B. pseudomallei-positive hemoculture bottles were filtered to remove bacteria, and then phages were isolated and purified by spot and double agar overlay plaque assays. Forty blood samples (hemoculture-confirmed melioidosis) were tested, and phages were found in 30% of the samples. Transmission electron microscopy and genome analysis of the isolated phages, vB_HM387 and vB_HM795, showed that both phages are Myoviruses. These two phages were stable at a pH of 5–7 and temperatures of 25–37°C, suggesting their ability to survive in human blood. The genome sizes of vB_HM387 and vB_HM795 are 36.3 and 44.0 kb, respectively. A phylogenetic analysis indicated that vB_HM387 has homologs, but vB_HM795 is a novel Myovirus, suggesting the heterogeneity of Burkholderia phages in melioidosis patients. The key finding that Burkholderia phages could be isolated from the blood of melioidosis patients highlights the potential application of phage-based assays by detecting phages in blood as a pathogen-derived biomarker of infection.

Summary Background Melioidosis is a grossly neglected but often-fatal tropical disease. The disease is named “a great mimicker” after its broad clinical manifestations, which makes disease diagnosis challenging and time-consuming. To improve diagnosis, we developed and evaluated the performance of the CRISPR-Cas12a system called “CRISPR-BP34” to detect Burkholderia pseudomallei DNA across clinical specimens from patients suspected to have melioidosis. Methods We documented time taken for diagnosis, antibiotics prescribed during the waiting period, and infection outcomes in 875 melioidosis patients treated in a hospital in northeast Thailand between October 2019 and December 2022. In the last six months, we performed CRISPR-BP34 detection on clinical specimens (blood, urine, respiratory secretion, pus and other body fluids) collected from 330 patients with suspected melioidosis and compared its performance to the current gold-standard culture-based method. Discordant results were validated by three independent qPCR tests. Findings A window of 3-4 days was required for gold-standard culture diagnosis, which resulted in delayed treatment. 199 [22·7%] of 875 patients died prior to diagnosis results while 114 [26·3%] of 433 follow-up cases had been diagnosed, treated, but died within 28 days of admission. A shorter sample-to-diagnosis time of less than 4 hours offered by CRISPR-BP34 technology could lead to faster administration of correct treatment. We demonstrated an improved sensitivity of CRISPR-BP34 (106 [93·0%] of 114 positive cases, 95% CI 86·6 - 96·9) compared to the culture approach (76 [66·7%] of 114 positive cases, 95% CI 57·2 - 75·2); while maintaining similar specificity (209 [96·8%] of 216 negative cases, 95% CI 93·4-98·7) to the culture (216 [100 %] of 216 negative cases, 95% CI 98·3-100·0). Interpretation The sensitivity, specificity, speed, window of clinical intervention, and ease of operation offered by the CRISPR-BP34 support its use as a point-of-care diagnostic for melioidosis. Funding Chiang Mai University Thailand and Wellcome Trust UK Research in context Evidence before this study Melioidosis is an often-severe infectious disease caused by the bacterium Burkholderia pseudomallei . It is estimated to affect 165,000 individuals annually worldwide, of which 89,000 cases are fatal. The disease diagnosis is challenging due to diverse clinical presentations, low awareness, limited diagnostic options, or even a lack of diagnostic tests. A PubMed search conducted from the database inception to 6 May 2023, using the terms “melioidosis” AND “diagnosis test,” yielded 207 results, 40 of which presented clinical evaluations of rapid melioidosis diagnostic tests. Antigen-based diagnostic tests, which detect the presence of B. pseudomallei , reported high specificity (median = 98·6%, IQR 94·0 - 100·0), but low sensitivity (median = 57·1%, IQR = 44·3 - 82·5). The test sensitivity suffers from the often-low concentration of the bacterial antigens in patients’ samples, which can vary by specimen type and stage of infection. Antibody-based diagnostic tests that detect host antibodies against B. pseudomallei typically exhibit satisfactory specificity (median = 94·5%, IQR = 88·6 - 96·2) but poor sensitivity (median = 80·2%, IQR = 71·0 - 88·1). These tests are often impacted by variations in antibody responses to B. pseudomallei and the duration required for antibody production. Furthermore, standardisation remains challenging due to the influence of different serum titres on sensitivity and background of the tests. Likewise, quantitative PCR exhibits a high degree of specificity (median = 99·8%, IQR = 91·6-100·0), but an observed low sensitivity for melioidosis (median = 77·1%, IQR = 20·8-97·8), which is likely attributed to the genetic heterogeneity of B. pseudomallei genomes. Additionally, these studies consistently reported a demand for improved speed and ease of implementation in resource-limited settings where melioidosis is endemic. With the limitations of current diagnostic methods, a culture-confirmed approach with 60% sensitivity, 100% specificity, and a diagnosis time of 2-7 days still stands as the gold standard for melioidosis diagnosis. Added value of this study To date, no study has measured the impact of delayed diagnosis on melioidosis. We assessed the number of deaths occurring prior to culture-confirmed diagnosis (22·7%) and those after diagnosis but within 28 days post-admission (26·3%), highlighting the urgent need for prompt action. To address this, we developed the CRISPR-BP34 test, which utilises isothermal amplification of a nucleic acid target followed by site-specific detection using a CRISPR-Cas12a enzyme. We successfully implemented this assay in a resource-limited setting in northeast Thailand, where the disease prevalence is among the highest in the world. The assay achieved a diagnostic sensitivity and specificity of 93·0% and 96·8%, respectively, with a limit of detection ranging from 50-250 cfu/mL. Early diagnosis can be achieved within four hours of patient admission, which is significantly faster than the gold-standard test that typically takes several days. Moreover, the ultrasensitivity of the CRISPR-BP34 assay enabled the detection of low levels of B. pseudomallei in hemoculture bottles, which could be missed due to mixed infections, poor aseptic technique, or other causes, leading to undiagnosed melioidosis. Implications of all available evidence The CRISPR-BP34 assay holds great promise for the management and control of melioidosis. Its minimal setup and shallow learning curve make it well-suited for resource-limited settings. Additionally, its speed and high sensitivity enable early diagnosis and treatment, which are crucial for saving patients’ lives.

Aims We investigated the antibacterial efficacy of Umonium 38 and Virkon ® against Burkholderia pseudomallei, Escherichia coli, Pseudomonas aeruginosa and Methicillin-Resistant Staphylococcus aureus (MRSA) up to 14 days following treatment. Methods and results Umonium 38 was diluted to 0.5%, 1.0%, 1.5%, 2.0%, 2.5% and 3%, tested against the bacterial strains at various contact times (15 min to 24 h), and incubated for up to 14 days. A minimum concentration of 0.5% Umonium 38 with a contact time of 15 min effectively killed approximately 10 8 CFU/ml of all four bacterial species. No growth was observed on agar plates from day 0 until day 14 for all six concentrations. The bacteria were also inactivated by a 30-minute treatment time using Virkon ® 1% solution. Conclusions Umonium 38 effectively inactivates B. pseudomallei, E. coli, P. aeruginosa and MRSA at a concentration of ≥ 0.5% with a contact time of at least 15 min. The antimicrobial effect of Umonium 38 remained for 14 days.

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.

Burkholderia pseudomallei is a soil-dwelling bacterium endemic to Southeast Asia and northern Australia that causes the disease, melioidosis. Although the global genomic diversity of clinical B. pseudomallei isolates has been investigated, there is limited understanding of its genomic diversity across small geographic scales, especially in soil. In this study, we obtained 288 B. pseudomallei isolates from a single soil sample (~100g; intensive site 2, INT2) collected at a depth of 30cm from a site in Ubon Ratchathani Province, Thailand. We sequenced the genomes of 169 of these isolates that represent 7 distinct sequence types (STs), including a new ST (ST1820), based on multi-locus sequence typing (MLST) analysis. A core genome SNP phylogeny demonstrated that all identified STs share a recent common ancestor that diverged an estimated 796-1260 years ago. A pan-genomics analysis demonstrated recombination between clades and intra-MLST phylogenetic and gene differences. To identify potential differential virulence between STs, groups of BALB/c mice (5 mice/isolate) were challenged via subcutaneous injection (500 CFUs) with 30 INT2 isolates representing 5 different STs; over the 21-day experiment, eight isolates killed all mice, 2 isolates killed an intermediate number of mice (1-2), and 20 isolates killed no mice. Although the virulence results were largely stratified by ST, one virulent isolate and six attenuated isolates were from the same ST (ST1005), suggesting that variably conserved genomic regions may contribute to virulence. Genomes from the animal-challenged isolates were subjected to a bacterial genome-wide association study to identify genomic regions associated with differential virulence. One associated region is a unique variant of Hcp1, a component of the type VI secretion system, which may result in attenuation. The results of this study have implications for comprehensive sampling strategies, environmental exposure risk assessment, and understanding recombination and differential virulence in B. pseudomallei.

Background Burkholderia pseudomallei is the bacterial causative agent of melioidosis, a difficult disease to diagnose clinically with high mortality if not appropriately treated. Definitive diagnosis requires isolation and identification of the organism. With the increased adoption of MALDI-TOF MS for the identification of bacteria, we established a method for rapid identification of B. pseudomallei using the Vitek MS, a system that does not currently have B. pseudomallei in its in-vitro diagnostic database. Results A routine direct spotting method was employed to create spectra and SuperSpectra. An initial B. pseudomallei SuperSpectrum was created at Shoklo Malaria Research Unit (SMRU) from 17 reference isolates (46 spectra). When tested, this initial SMRU SuperSpectrum was able to identify 98.2 % (54/55) of Asian isolates, but just 46.7 % (35/75) of Australian isolates. Using spectra (430) from different reference and clinical isolates, two additional SMRU SuperSpectra were created. Using the combination of all SMRU SuperSpectra with seven existing SuperSpectra from Townsville, Australia 119 (100 %) Asian isolates and 31 (100 %) Australian isolates were correctly identified. In addition, no misidentifications were obtained when using these 11 SuperSpectra when tested with 34 isolates of other bacteria including the closely related species Burkholderia thailandensis and Burkholderia cepacia. Conclusions This study has established a method for identification of B. pseudomallei using Vitek MS, and highlights the impact of geographical differences between strains for identification using this technique.

Background: Communicating about antimicrobial resistance (AMR) to the public is challenging. Methods: We developed a dictionary of terms commonly used to communicate about AMR. For each term, we developed learning points to explain AMR and related concepts in plain language. We conducted a pilot evaluation in 374 high school students in Ubon Ratchathani, Thailand. In three 50-minute sessions, students were asked to answer five true/false questions using a paper-based questionnaire. The first session assessed their understanding of AMR at baseline, the second after searching the internet, and the third after the provision of the printed AMR dictionary and its web address. Results: We developed the AMR dictionary as a web-based application (www.amrdictionary.net). The Thai version of the AMR dictionary included 35 terms and associated learning points, seven figures displaying posters promoting AMR awareness in Thailand, and 66 recommended online videos. In the pretest, the proportion of correct responses to each question ranged from 10% to 57%; 10% of the students correctly answered that antibiotics cannot kill viruses and 57% correctly answered that unnecessary use of antibiotics makes them ineffective. After the internet searches, the proportions of correct answers increased, ranging from 62% to 89% (all p<0.001). After providing the AMR dictionary, the proportions of correct answers increased further, ranging from 79% to 89% for three questions (p<0.001), and did not change for one question (p=0.15). Correct responses as to whether taking antibiotics often has side-effects such as diarrhoea reduced from 85% to 74% (p<0.001). The dictionary was revised based on the findings and comments received. Conclusions: Understanding of AMR among Thai high school students is limited. The AMR dictionary can be a useful supportive tool to increase awareness and improve understanding of AMR. Our findings support the need to evaluate the effectiveness of communication tools in the real-world setting.

The Burkholderia pseudomallei phylogenetic cluster includes B. pseudomallei, B. mallei, B. thailandensis, B. oklahomensis, B. humptydooensis and B. singularis. Regarded as the only pathogenic members of this group, B. pseudomallei and B. mallei cause the diseases melioidosis and glanders, respectively. Additionally, variant strains of B. pseudomallei and B. thailandensis exist that include the geographically restricted B. pseudomallei that express a B. mallei-like BimA protein (BPBM), and B. thailandensis that express a B. pseudomallei-like capsular polysaccharide (BTCV). To establish a PCR-based assay for the detection of pathogenic Burkholderia species or their variants, five PCR primers were designed to amplify species-specific sequences within the bimA (Burkholderia intracellular motility A) gene. Our multiplex PCR assay could distinguish pathogenic B. pseudomallei and BPBM from the non-pathogenic B. thailandensis and the BTCV strains. A second singleplex PCR successfully discriminated the BTCV from B. thailandensis. Apart from B. humptydooensis, specificity testing against other Burkholderia spp., as well as other Gram-negative and Gram-positive bacteria produced a negative result. The detection limit of the multiplex PCR in soil samples artificially spiked with known quantities of B. pseudomallei and B. thailandensis were 5 and 6 CFU/g soil, respectively. Furthermore, comparison between standard bacterial culture and the multiplex PCR to detect B. pseudomallei from 34 soil samples, collected from an endemic area of melioidosis, showed high sensitivity and specificity. This robust, sensitive, and specific PCR assay will be a useful tool for epidemiological study of B. pseudomallei and closely related members with pathogenic potential in soil.

Background Melioidosis, an infectious disease caused by Burkholderia pseudomallei , is endemic in many tropical developing countries and has a high mortality. Here we evaluated combinations of a lateral flow immunoassay (LFI) detecting B . pseudomallei capsular polysaccharide (CPS) and enzyme-linked immunosorbent assays (ELISA) detecting antibodies against hemolysin co-regulated protein (Hcp1) or O-polysaccharide (OPS) for diagnosing melioidosis. Methodology/Principal findings We conducted a cohort-based case-control study. Both cases and controls were derived from a prospective observational study of patients presenting with community-acquired infections and sepsis in northeast Thailand (Ubon-sepsis). Cases included 192 patients with a clinical specimen culture positive for B . pseudomallei . Controls included 502 patients who were blood culture positive for Staphylococcus aureus , Escherichia coli or Klebsiella pneumoniae or were polymerase chain reaction assay positive for malaria or dengue. Serum samples collected within 24 hours of admission were stored and tested using a CPS-LFI, Hcp1-ELISA and OPS-ELISA. When assessing diagnostic tests in combination, results were considered positive if either test was positive. We selected ELISA cut-offs corresponding to a specificity of 95%. Using a positive cut-off OD of 2.912 for Hcp1-ELISA, the combination of the CPS-LFI and Hcp1-ELISA had a sensitivity of 67.7% (130/192 case patients) and a specificity of 95.0% (477/502 control patients). The sensitivity of the combination (67.7%) was higher than that of the CPS-LFI alone (31.3%, p<0.001) and that of Hcp1-ELISA alone (53.6%, p<0.001). A similar phenomenon was also observed for the combination of CPS-LFI and OPS-ELISA. In case patients, positivity of the CPS-LFI was associated with a short duration of symptoms, high modified Sequential (sepsis-related) Organ Failure Assessment (SOFA) score, bacteraemia and mortality outcome, while positivity of Hcp1-ELISA was associated with a longer duration of symptoms, low modified SOFA score, non-bacteraemia and survival outcome. Conclusions/Significance A combination of antigen-antibody diagnostic tests increased the sensitivity of melioidosis diagnosis over individual tests while preserving high specificity. Point-of-care tests for melioidosis based on the use of combination assays should be further developed and evaluated.