The NarX-NarL two-component system regulates biofilm formation, natural product biosynthesis, and host-associated survival in Burkholderia pseudomallei.

Burkholderia pseudomallei is a saprophytic bacterium endemic throughout the tropics causing severe disease in humans and animals. Environmental signals such as the accumulation of inorganic ions mediates the biofilm forming capabilities and survival of B. pseudomallei. We have previously shown that B. pseudomallei responds to nitrate and nitrite by inhibiting biofilm formation and altering cyclic di-GMP signaling. To better understand the roles of nitrate-sensing in the biofilm inhibitory phenotype of B. pseudomallei, we created in-frame deletions of narX (Bp1026b_I1014) and narL (Bp1026b_I1013), which are adjacent components of a conserved nitrate-sensing two-component system. We observed transcriptional downregulation in key components of the biofilm matrix in response to nitrate and nitrite. Some of the most differentially expressed genes were nonribosomal peptide synthases (NRPS) and/or polyketide synthases (PKS) encoding the proteins for the biosynthesis of bactobolin, malleilactone, and syrbactin, and an uncharacterized cryptic NRPS biosynthetic cluster. RNA expression patterns were reversed in ∆narX and ∆narL mutants, suggesting that nitrate sensing is an important checkpoint for regulating the diverse metabolic changes occurring in the biofilm inhibitory phenotype. Moreover, in a macrophage model of infection, ∆narX and ∆narL mutants were attenuated in intracellular replication, suggesting that nitrate sensing contributes to survival in the host.

Evaluation of four sampling devices for Burkholderia pseudomallei laboratory aerosol studies.

Previous field and laboratory studies investigating airborne Burkholderia pseudomallei have used a variety of different aerosol samplers to detect and quantify concentrations of the bacteria in aerosols. However, the performance of aerosol samplers can vary in their ability to preserve the viability of collected microorganisms, depending on the resistance of the organisms to impaction, desiccation, or other stresses associated with the sampling process. Consequently, sampler selection is critical to maximizing the probability of detecting viable microorganisms in collected air samples in field studies and for accurate determination of aerosol concentrations in laboratory studies. To inform such decisions, the present study assessed the performance of four laboratory aerosol samplers, specifically the all-glass impinger (AGI), gelatin filter, midget impinger, and Mercer cascade impactor, for collecting aerosols containing B. pseudomallei generated from suspensions in two types of culture media. The results suggest that the relative performance of the sampling devices is dependent on the suspension medium utilized for aerosolization. Performance across the four samplers was similar for aerosols generated from suspensions supplemented with 4% glycerol. However, for aerosols generated from suspensions without glycerol, use of the filter sampler or an impactor resulted in significantly lower estimates of the viable aerosol concentration than those obtained with either the AGI or midget impinger. These results demonstrate that sampler selection has the potential to affect estimation of doses in inhalational animal models of melioidosis, as well as the likelihood of detection of viable B. pseudomallei in the environment, and will be useful to inform design of future laboratory and field studies.

Rhamnolipid enhances Burkholderia pseudomallei biofilm susceptibility, disassembly and production of virulence factors.

Aim: This study evaluated the effect of the biosurfactant rhamnolipid on the antimicrobial susceptibility, biofilm growth dynamics and production of virulence factors by Burkholderia pseudomallei. Materials & methods: The effects of rhamnolipid on planktonic and biofilm growth and its interaction with antibacterial drugs were evaluated. Then, its effects on growing and mature biofilms and on protease and siderophore production were assessed. Results: Rhamnolipid did not inhibit B. pseudomallei growth, but significantly enhanced the activity of meropenem and amoxicillin-clavulanate against mature biofilms. Rhamnolipid significantly reduced the biomass of mature biofilms, significantly increased protease production by growing and mature biofilms and siderophore release by growing biofilms. Conclusion: Rhamnolipid enhances the antimicrobial activity against B. pseudomallei, assists biofilm disassembly and alters protease and siderophore production by bacterial biofilms.

Survival of Burkholderia pseudomallei and Pathogenic Leptospira in Cola, Beer, Energy Drinks, and Sports Drinks.

Burkholderia pseudomallei and pathogenic Leptospira in contaminated drinking water can cause melioidosis and leptospirosis, respectively. Here, we evaluated their survival in beverages. We mixed six isolates (three isolates per organism) in four beverages (Coca-Cola®, Red Bull®, Singha® beer, and Gatorade®) and distilled water as the control at two final concentrations (1 × 107 colony-forming units [CFU]/mL and 1 × 103 CFU/mL). The solution was kept at two temperatures (37°C and 4°C). At 4°C and at the high concentration, pathogenic Leptospira survived in Coca-Cola® up to 3 minutes and in Singha, Red Bull®, and Gatorade up to 15 minutes, whereas B. pseudomallei survived in these beverages up to 8 hours, and 14, 14, and 28 days, respectively. The survival time of both organisms was shorter at 37°C (P = 0.01) and at the lower concentration (P = 0.001). In conclusion, Leptospira can survive in some beverages for up to 15 minutes, whereas B. pseudomallei can survive in some beverages for up to 4 weeks.

A comparative genomic analysis of small-colony variant and wild-type Burkholderia pseudomallei in a patient with bacterial liver abscess.

OBJECTIVE: To understand the genotypic variations of Burkholderia pseudomallei (B. pseudomallei) small-colony variant (SCV). METHODS: A pair of isogenic wild-type (WT) and SCV B. pseudomallei strains (CX1-1 and CX2-1, respectively) were isolated from a patient with a bacterial liver abscess. They were further identified by multilocus sequence typing (MLST) analysis. To compare their growth speed, the time to detection for the two strains was assessed by BacT/Alert 3D. Antibiotic susceptibility tests were performed by disc diffusion method and Etest assay according to the Clinical and Laboratory Standards Institute (CLSI) guidelines. The whole genomes of the two strains were sequenced. A comparative genome analysis was performed to determine the genotypic variations of the CX2-1 strain. RESULTS: The CX1-1 and CX2-1 strains were both identified as ST70 by MLST. The CX2-1 grew more slowly than the WT strain CX1-1 and was more resistant to imipenem, meropenem, doxycycline, trimethoprim-sulfamethoxazole, and ceftazidime. The comparative genome analysis revealed 38 variations in 30 genes associated with metabolism, drug resistance and virulence. The mutated genes encoded some cell membrane proteins, membrane transporters and synthetases, including: LolB, HisP, PchF, putative polyketide synthetases, probable non-ribosomal peptide synthetases, putative TonB-dependent outer-membrane receptor protein, and putative type III secretion protein. CONCLUSIONS: The reduced growth speed and increased drug resistance of B. pseudomallei SCV strain may be related to those variations in the genome. This provides some clues to their association between the morphotypic and phenotypic characteristics of colony variants, and the potential association of its colony morphotypes with metabolism, antibiotic resistance and virulence.

Erythritol as a single carbon source improves cultural isolation of Burkholderia pseudomallei from rice paddy soils.

BACKGROUND: Isolation of the soil bacterium Burkholderia pseudomallei from tropical environments is important to generate a global risk map for man and animals to acquire the infectious disease melioidosis. There is increasing evidence, that the currently recommended soil culture protocol using threonine-basal salt solution with colistin (TBSS-C50) for enrichment of B. pseudomallei and Ashdown agar for subsequent subculture lacks sensitivity. We therefore investigated, if the otherwise rarely encountered erythritol catabolism of B. pseudomallei might be exploited to improve isolation of this bacterium from soil. METHODOLOGY/PRINCIPAL FINDINGS: Based on TBSS-C50, we designed a new colistin-containing medium with erythritol as the single carbon source (EM). This medium was validated in various culture protocols by analyzing 80 soil samples from 16 different rice fields in Vietnam. B. pseudomallei enrichment was determined in all culture supernatants by a specific quantitative PCR (qPCR) targeting the type three secretion system 1. 51 out of 80 (63.8%) soil samples gave a positive qPCR signal in at least one of the culture conditions. We observed a significantly higher enrichment shown by lower median cycle threshold values for B. pseudomallei in a two-step culture with TBSS-C50 for 48 h followed by EM for 96h compared to single cultures in TBSS-C50 for either 48h or 144h (p<0.0001, respectively). Accordingly, B. pseudomallei could be isolated on Ashdown agar in 58.8% (30/51) of samples after subcultures from our novel two-step enrichment culture compared to only 9.8% (5/51) after standard enrichment with TBSS-C50 for 48h (p<0.0001) or 25.5% (13/51; p<0.01) after TBSS-C50 for 144h. CONCLUSIONS/SIGNIFICANCE: In the present study, we show that specific exploitation of B. pseudomallei metabolic capabilities in enrichment protocols leads to a significantly improved isolation rate of this pathogen from soil compared to established standard procedures. Our new culture method might help to facilitate the creation of environmental risk maps for melioidosis in the future.

The cost-effectiveness of the use of selective media for the diagnosis of melioidosis in different settings.

BACKGROUND: Melioidosis is a frequently fatal disease requiring specific treatment. The yield of Burkholderia pseudomallei from sites with a normal flora is increased by culture using selective, differential media such as Ashdown's agar and selective broth. However, since melioidosis mainly affects people in resource-poor countries, the cost effectiveness of selective culture has been questioned. We therefore retrospectively evaluated this in two laboratories in southeast Asia. METHODOLOGY/PRINCIPAL FINDINGS: The results of all cultures in the microbiology laboratories of Mahosot Hospital, Vientiane, Laos and Angkor Hospital for Children, Siem Reap, Cambodia, in 2017 were reviewed. We identified patients with melioidosis who were only diagnosed as a result of culture of non-sterile sites and established the total number of such samples cultured using selective media and the associated costs in each laboratory. We then conducted a rudimentary cost-effectiveness analysis by determining the incremental cost-effectiveness ratio (ICER) per DALY averted and compared this against the 2017 GDP per capita in each country. Overall, 29 patients in Vientiane and 9 in Siem Reap (20% and 16.9% of all culture-positive patients respectively) would not have been diagnosed without the use of selective media, the majority of whom (18 and 8 respectively) were diagnosed by throat swab culture. The cost per additional patient detected by selective culture was approximately $100 in Vientiane and $39 in Siem Reap. Despite the different patient populations (all ages in Vientiane vs. only children in Siem Reap) and testing strategies (all samples in Vientiane vs. based on clinical suspicion in Siem Reap), selective B. pseudomallei culture proved highly cost effective in both settings, with an ICER of ~$170 and ~$28 in Vientiane and Siem Reap, respectively. CONCLUSIONS/SIGNIFICANCE: Selective culture for B. pseudomallei should be considered by all laboratories in melioidosis-endemic areas. However, the appropriate strategy for implementation should be decided locally.

Slow growth of Burkholderia pseudomallei compared to other pathogens in an adapted blood culture system in Phnom Penh, Cambodia.

PURPOSE: Burkholderia pseudomallei is a key pathogen causing bloodstream infections at Sihanouk Hospital Center of Hope, Phnom Penh, Cambodia. Here, visual instead of automated detection of growth of commercial blood culture bottles is done. The present study assessed the performance of this system. METHODOLOGY: Blood culture sets, consisting of paired adult aerobic and anaerobic bottles (bioMérieux, FA FAN 259791 and FN FAN 252793) were incubated in a standard incubator for 7 days after reception. Each day, the bottle growth indicator was visually inspected for colour change indicating growth. Blind subculture was performed from the aerobic bottle at day 3. RESULTS: From 2010 to 2015, 11  671 sets representing 10  389 suspected bloodstream infection episodes were documented. In 1058 (10.2  %) episodes, pathogens grew; they comprised Escherichia coli (31.7 %), Salmonella Paratyphi A (13.9 %), B. pseudomallei (8.5 %), Staphylococcus aureus (7.8 %) and Klebsiella pneumoniae (7.0 %). Blind subculture yielded 72 (4.1  %) pathogens, mostly (55/72, 76.4 %) B. pseudomallei. Cumulative proportions of growth at day 2 were as follows: E. coli: 85.0 %, Salmonella Paratyphi A: 85.0 %, K. pneumoniae: 76.3  % and S. aureus: 52.2  %; for B. pseudomallei, this was only 4.0  %, which increased to 70.1  % (70/99) at day 4 mainly by detection on blind subculture (55/99). Compared to the anaerobic bottles, aerobic bottles had a higher yield and a shorter time-to-detection, particularly for B. pseudomallei. CONCLUSIONS: Visual inspection for growth of commercial blood culture bottles in a low-resource setting provided satisfactory yield and time-to-detection. However, B. pseudomallei grew slowly and was mainly detected by blind subculture. The aerobic bottle outperformed the anaerobic bottle.

Burkholderia thailandensis strain E555 is a surrogate for the investigation of Burkholderia pseudomallei replication and survival in macrophages.

BACKGROUND: Burkholderia pseudomallei is a human pathogen causing severe infections in tropical and subtropical regions and is classified as a bio-threat agent. B. thailandensis strain E264 has been proposed as less pathogenic surrogate for understanding the interactions of B. pseudomallei with host cells. RESULTS: We show that, unlike B. thailandensis strain E264, the pattern of growth of B. thailandensis strain E555 in macrophages is similar to that of B. pseudomallei. We have genome sequenced B. thailandensis strain E555 and using the annotated sequence identified genes and proteins up-regulated during infection. Changes in gene expression identified more of the known B. pseudomallei virulence factors than changes in protein levels and used together we identified 16% of the currently known B. pseudomallei virulence factors. These findings demonstrate the utility of B. thailandensis strain E555 to study virulence of B. pseudomallei. CONCLUSIONS: A weakness of studies using B. thailandensis as a surrogate for B. pseudomallei is that the strains used replicate at a slower rate in infected cells. We show that the pattern of growth of B. thailandensis strain E555 in macrophages closely mirrors that of B. pseudomallei. Using this infection model we have shown that virulence factors of B. pseudomallei can be identified as genes or proteins whose expression is elevated on the infection of macrophages. This finding confirms the utility of B. thailandensis strain E555 as a surrogate for B. pseudomallei and this strain should be used for future studies on virulence mechanisms.

Burkholderia pseudomallei BimC Is Required for Actin-Based Motility, Intracellular Survival, and Virulence.

The intracellular pathogen Burkholderia pseudomallei, the etiological agent of melioidosis in humans and various animals, is capable of survival and movement within the cytoplasm of host cells by a process known as actin-based motility. The bacterial factor BimA is required for actin-based motility through its direct interaction with actin, and by mediating actin polymerization at a single pole of the bacterium to promote movement both within and between cells. However, little is known about the other bacterial proteins required for this process. Here, we have investigated the role of the bimC gene (bpss1491) which lies immediately upstream of the bimA gene (bpss1492) on the B. pseudomallei chromosome 2. Conserved amongst all B. pseudomallei, B. mallei and B. thailandensis strains sequenced to date, this gene encodes an iron-binding protein with homology to a group of proteins known as the bacterial autotransporter heptosyltransferase (BAHT) family. We have constructed a B. pseudomallei bimC deletion mutant and demonstrate that it is defective in intracellular survival in HeLa cells, but not in J774.1 macrophage-like cells. The bimC mutant is defective in cell to cell spread as demonstrated by ablation of plaque formation in HeLa cells, and by the inability to form multi-nucleated giant cells in J774.1 cells. These phenotypes in intracellular survival and cell to cell spread are not due to the loss of expression and polar localization of the BimA protein on the surface of intracellular bacteria, however they do correlate with an inability of the bacteria to recruit and polymerize actin. Furthermore, we also establish a role for bimC in virulence of B. pseudomallei using a Galleria mellonella larvae model of infection. Taken together, our findings indicate that B. pseudomallei BimC plays an important role in intracellular behavior and virulence of this emerging pathogen.

Misidentification of Burkholderia pseudomallei as Acinetobacter species in northern Thailand.

Background: Burkholderia pseudomallei is the causative agent of melioidosis, a disease endemic throughout the tropics. Methods: A study of reported Acinetobacter spp. bacteraemia was performed at Chiang Rai provincial hospital from 2014 to 2015. Isolates were collected and tested for confirmation. Results: A total of 419 putative Acinetobacter spp. isolates from 412 patients were re-identified and 5/419 (1.2%) were identified as B. pseudomallei. Four of the five patients with melioidosis died. An estimated 88/419 (21%) isolates were correctly identified as Acinetobacter spp. Conclusions: Misidentification of Acinetobacter spp. as B. pseudomallei or other bacteria is not uncommon and programmes to address these shortfalls are urgently required.

Rapid antimicrobial susceptibility testing and ß-lactam-induced cell morphology changes of Gram-negative biological threat pathogens by optical screening.

BACKGROUND: For Yersinia pestis, Burkholderia pseudomallei, and Burkholderia mallei, conventional broth microdilution (BMD) is considered the gold standard for antimicrobial susceptibility testing (AST) and, depending on the species, requires an incubation period of 16-20 h, or 24-48 h according to the Clinical and Laboratory Standards Institute (CLSI) guidelines. After a diagnosis of plague, melioidosis or glanders during an outbreak or after an exposure event, the timely distribution of appropriate antibiotics for treatment or post-exposure prophylaxis of affected populations could reduce mortality rates. RESULTS: Herein, we developed and evaluated a rapid, automated susceptibility test for these Gram-negative bacterial pathogens based on time-lapse imaging of cells incubating in BMD microtitre drug panels using an optical screening instrument (oCelloScope). In real-time, the instrument screened each inoculated well containing broth with various concentrations of antibiotics published by CLSI for primary testing: ciprofloxacin (CIP), doxycycline (DOX) and gentamicin (GEN) for Y. pestis; imipenem (IPM), ceftazidime (CAZ) and DOX for B. mallei; and IPM, DOX, CAZ, amoxicillin-clavulanic acid (AMC) and trimethoprim-sulfamethoxazole (SXT) for B. pseudomallei. Based on automated growth kinetic data, the time required to accurately determine susceptibility decreased by ≥70% for Y. pestis and ≥ 50% for B. mallei and B. pseudomallei compared to the times required for conventional BMD testing. Susceptibility to GEN, IPM and DOX could be determined in as early as three to six hours. In the presence of CAZ, susceptibility based on instrument-derived growth values could not be determined for the majority of B. pseudomallei and B. mallei strains tested. Time-lapse video imaging of these cultures revealed that the formation of filaments in the presence of this cephalosporin at inhibitory concentrations was detected as growth. Other ß-lactam-induced cell morphology changes, such as the formation of spheroplasts and rapid cell lysis, were also observed and appear to be strain- and antibiotic concentration-dependent. CONCLUSIONS: A rapid, functional AST was developed and real-time video footage captured ß-lactam-induced morphologies of wild-type B. mallei and B. pseudomallei strains in broth. Optical screening reduced the time to results required for AST of three Gram-negative biothreat pathogens using clinically relevant, first-line antibiotics compared to conventional BMD.

Lipid A Remodeling Is a Pathoadaptive Mechanism That Impacts Lipopolysaccharide Recognition and Intracellular Survival of Burkholderia pseudomallei.

Burkholderia pseudomallei causes the severe disease melioidosis. The bacterium subverts the host immune system and replicates inside cells, and host mortality results primarily from sepsis-related complications. Lipopolysaccharide (LPS) is a major virulence factor and mediator of sepsis that many pathogens capable of intracellular growth modify to reduce their immunological "footprint." The binding strength of B. pseudomallei LPS for human LPS binding protein (hLBP) was measured using surface plasmon resonance. The structures of lipid A isolated from B. pseudomallei under different temperatures were analyzed by matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS), and the gene expression of two lipid A remodeling genes, lpxO and pagL, was investigated. The LPS was characterized for its ability to trigger tumor necrosis factor alpha (TNF-α) release and to activate caspase-11-triggered pyroptosis by introduction of LPS into the cytosol. Lipid A from long-term chronic-infection isolates was isolated and characterized by MALDI-TOF MS and also by the ability to trigger caspase-11-mediated cell death. Lipid A from B. pseudomallei 1026b lpxO and pagL mutants were characterized by positive- and negative-mode MALDI-TOF MS to ultimately identify their role in lipid A structural modifications. Replication of lpxO and pagL mutants and their complements within macrophages showed that lipid A remodeling can effect growth in host cells and activation of caspase-11-mediated cytotoxicity.

Diagnostic accuracy of the InBiOS AMD rapid diagnostic test for the detection of Burkholderia pseudomallei antigen in grown blood culture broth.

To assess the diagnostic and operational performance of the InBiOS AMD rapid diagnostic test (RDT) (Seattle, USA) for the detection of B. pseudomallei in grown blood culture broth. The InBiOS RDT is a lateral flow immunoassay in a strip format detecting B. pseudomallei capsular polysaccharide in culture fluids, marketed for research only. Broth of blood culture bottles (BacT/Alert, bioMérieux, Marcy L'Etoile, France) sampled in adult patients at the Sihanouk Hospital Center of HOPE, Phnom Penh, Cambodia, during 2010-2017 and stored at - 80 °C was tested. They included samples grown with B. pseudomallei (n = 114), samples with no growth (n = 12), and samples with growth of other pathogens (n = 139, among which Burkholderia cepacia (n = 5)). Diagnostic sensitivity and specificity were 96.5% [95% confidence interval (CI): 91.3-98.6%] and 100% [CI: 97.5-100%] respectively. Background clearance and line intensities were good and very good. The RDT's test strip, not housed in a cassette, caused difficulties in manipulation and biosafety. The centrifugation step prescribed by the procedure challenged biosafety, but processing of 19 B. pseudomallei samples without centrifugation showed similar results for line intensity and background clearance, compared to centrifugation. The InBiOS RDT showed excellent accuracy for detection of B. pseudomallei in grown blood culture broth. Provided operational adaptations such as cassette housing, it has the potential to reduce time to diagnosis of melioidosis.

Impact of nutritional stress on drug susceptibility and biofilm structures of Burkholderia pseudomallei and Burkholderia thailandensis grown in static and microfluidic systems.

Burkholderia pseudomallei is the causative agent of melioidosis and regarded as a bioterrorism threat. It can adapt to the nutrient-limited environment as the bacteria can survive in triple distilled water for 16 years. Moreover, B. pseudomallei exhibits intrinsic resistance to diverse groups of antibiotics in particular while growing in biofilms. Recently, nutrient-limited condition influenced both biofilm formation and ceftazidime (CAZ) tolerance of B. pseudomallei were found. However, there is no information about how nutrient-limitation together with antibiotics used in melioidosis treatment affects the structure of the biofilm produced by B. pseudomallei. Moreover, no comparative study to investigate the biofilm architectures of B. pseudomallei and the related B. thailandensis under different nutrient concentrations has been reported. Therefore, this study aims to provide new information on the effects of four antibiotics used in melioidosis treatment, viz. ceftazidime (CAZ), imipenem (IMI), meropenem (MEM) and doxycycline (DOX) on biofilm architecture of B. pseudomallei and B. thailandensis with different nutrient concentrations under static and flow conditions using confocal laser scanning microscopy. Impact of nutritional stress on drug susceptibility of B. pseudomallei and B. thailandensis grown planktonically or as biofilm was also evaluated. The findings of this study indicate that nutrient-limited environment enhanced survival of B. pseudomallei in biofilm after exposure to the tested antibiotics. The shedding planktonic B. pseudomallei and B. thailandensis were also found to have increased CAZ tolerance in nutrient-limited environment. However, killing activities of MEM and IMI were stronger than CAZ and DOX on B. pseudomallei and B. thailandensis both in planktonic cells and in 2-day old biofilm. In addition, MEM and IMI were able to inhibit B. pseudomallei and B. thailandensis biofilm formation to a larger extend compared to CAZ and DOX. Differences in biofilm architecture were observed for biofilms grown under static and flow conditions. Under static conditions, biofilms grown in full strength modified Vogel and Bonner's medium (MVBM) showed honeycomb-like architecture while a knitted-like structure was observed under limited nutrient condition (0.1×MVBM). Under flow conditions, biofilms grown in MVBM showed a multilayer structure while merely dispersed bacteria were found when grown in 0.1×MVBM. Altogether, this study provides more insight on the effect of four antibiotics against B. pseudomallei and B. thailandensis in biofilm under different nutrient and flow conditions. Since biofilm formation is believed to be involved in disease relapse, MEM and IMI may be better therapeutic options than CAZ for melioidosis treatment.

Physicochemical properties associated with the presence of Burkholderia pseudomallei in small ruminant farm water supplies in Peninsular Malaysia.

Burkholderia pseudomallei causes melioidosis, a life-threatening infection in both humans and animals. Water is an important reservoir of the bacteria and may serve as a source of environmental contamination leading to infection. B. pseudomallei has an unusual ability to survive in water for a long period. This paper investigates physicochemical properties of water associated with the presence of B. pseudomallei in water supply in small ruminant farms in Peninsular Malaysia. Physicochemical properties of water samples taken from small ruminant farms that included temperature, pH, dissolved oxygen (DO2), optical density (OD), and chemical oxygen demand (COD) were measured after which the samples were cultured for B. pseudomallei. Multivariable logistic regression model revealed that slightly acidic water pH and higher COD level were significantly associated with the likelihood of the B. pseudomallei presence in the water.

Comprehensive analysis of clinical Burkholderia pseudomallei isolates demonstrates conservation of unique lipid A structure and TLR4-dependent innate immune activation.

Burkholderia pseudomallei is an environmental bacterium that causes melioidosis, a major community-acquired infection in tropical regions. Melioidosis presents with a range of clinical symptoms, is often characterized by a robust inflammatory response, may relapse after treatment, and results in high mortality rates. Lipopolysaccharide (LPS) of B. pseudomallei is a potent immunostimulatory molecule comprised of lipid A, core, and O-polysaccharide (OPS) components. Four B. pseudomallei LPS types have been described based on SDS-PAGE patterns that represent the difference of OPS-type A, type B, type B2 and rough LPS. The majority of B. pseudomallei isolates are type A. We used matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) followed by electrospray ionization quadrupole time-of-flight mass spectrometry (ESI-QqTOF MS) and gas chromatography to characterize the lipid A of B. pseudomallei within LPS type A isolates. We determined that B. pseudomallei lipid A is represented by penta- and tetra-acylated species modified with 4-amino-4-deoxy-arabinose (Ara4N). The MALDI-TOF profiles from 171 clinical B. pseudomallei isolates, including 68 paired primary and relapse isolates and 35 within-host isolates were similar. We did not observe lipid A structural changes when the bacteria were cultured in different growth conditions. Dose-dependent NF-κB activation in HEK cells expressing TLR4 was observed using multiple heat-killed B. pseudomallei isolates and corresponding purified LPS. We demonstrated that TLR4-dependent NF-κB activation induced by heat-killed bacteria or LPS prepared from OPS deficient mutant was significantly greater than those induced by wild type B. pseudomallei. These findings suggest that the structure of B. pseudomallei lipid A is highly conserved in a wide variety of clinical and environmental circumstances but that the presence of OPS may modulate LPS-driven innate immune responses in melioidosis.

Case Report: Chorioamnionitis and Premature Delivery due to Burkholderia pseudomallei Infection in Pregnancy.

We report a case of placental infection leading to preterm delivery in a mother diagnosed with septicemia and pneumonia due to Burkholderia pseudomallei in pregnancy. Placental infection occurred despite prolonged ceftazidime therapy.

High-Throughput Flow Cytometry Screening of Multidrug Efflux Systems.

The resistance nodulation cell division (RND) family of proteins are inner membrane transporters that associate with periplasmic adaptor proteins and outer membrane porins to affect substrate transport from the cytosol and periplasm in Gram-negative bacteria. Various structurally diverse compounds are substrates of RND transporters. Along with their notable role in antibiotic resistance, these transporters are essential for niche colonization, quorum sensing, and virulence as well as for the removal of fatty acids and bile salts. As such, RNDs are an attractive target for antimicrobial development. However, while enhancing the utility of antibiotics with an RND inhibitor is an appealing concept, only a small core of chemotypes has been identified as efflux pump inhibitors (EPIs). Thus, our key objective is the development and validation of an efflux profiling and discovery strategy for RND model systems. Here we describe a flow cytometric dye accumulation assay that uses fluorescein diacetate (FDA) to interrogate the model Gram-negative pathogens Escherichia coli, Franscisella tularensis, and Burkholderia pseudomallei. Fluorochrome retention is increased in the presence of known efflux inhibitors and in RND deletion strains. The assay can be used in a high-throughput format to evaluate efflux of dye-substrate candidates and to screen chemical libraries for novel EPIs. Triaged compounds that inhibit efflux in pathogenic strains are tested for growth inhibition and antibiotic potentiation using microdilution culture plates in a select agent Biosafety Level-3 (BSL3) environment. This combined approach demonstrates the utility of flow cytometric analysis for efflux activity and provides a useful platform in which to characterize efflux in pathogenic Gram-negative bacteria. Screening small molecule libraries for novel EPI candidates offers the potential for the discovery of new classes of antibacterial compounds.

Identification of sRNA mediated responses to nutrient depletion in Burkholderia pseudomallei.

The Burkholderia genus includes many species that are known to survive in diverse environmental conditions including low nutrient environments. One species, Burkholderia pseudomallei is a versatile pathogen that can survive in a wide range of hosts and environmental conditions. In this study, we investigated how a nutrient depleted growth environment evokes sRNA mediated responses by B. pseudomallei. Computationally predicted B. pseudomallei D286 sRNAs were mapped to RNA-sequencing data for cultures grown under two conditions: (1) BHIB as a nutrient rich media reference environment and (2) M9 media as a nutrient depleted stress environment. The sRNAs were further selected to identify potentially cis-encoded systems by investigating their possible interactions with their flanking genes. The mappings of predicted sRNA genes and interactions analysis to their flanking genes identified 12 sRNA candidates that may possibly have cis-acting regulatory roles that are associated to a nutrient depleted growth environment. Our approach can be used for identifying novel sRNA genes and their possible role as cis-mediated regulatory systems.