Pseudomallei Clinical Isolates Research Articles

Multilocus sequence typing of clinical Burkholderia pseudomallei isolates from Cambodia.

Melioidosis is a neglected tropical disease caused by Burkholderia pseudomallei, endemic to Southeast Asia and Northern Australia. Despite its increasing global public health and clinical significance, the molecular epidemiology of melioidosis and genetic diversity of B. pseudomallei in Cambodia remains poorly understood. This study aims to elucidate the genetic diversity and antibiotic susceptibility profiles of B. pseudomallei isolates responsible for melioidosis in humans. For this purpose, 14 clinical isolates cryopreserved at the Medical Biology Laboratory at Institut Pasteur du Cambodge from 2016 to 2020 were subjected to antimicrobial susceptibility testing and Multilocus Sequence Typing (MLST). Phenotypic testing revealed that 92.86% (13/14) of the isolates were sensitive to all tested antibiotics, while one isolate exhibited resistance to trimethoprim-sulfamethoxazole. MLST analysis resolved our isolates into 14 unique Sequence Types (STs), including 10 previously documented in Southeast Asia. Notably, ST1858, ST2064, ST2065, and ST2066 were identified as novel STs, while ST54, ST99, ST211, and ST1359 were reported in Cambodia for the first time in this study. Comparing our MLST data with available sequences on PubMLST (n = 165), our study unveiled a high genetic diversity of B. pseudomallei in Cambodia. The identified STs were closely associated with isolates from other Southeast Asian countries, particularly Thailand, Vietnam, and Malaysia. In conclusion, this study provided insight into the genetic diversity among B. pseudomallei clinical isolates in Cambodia and their close genetic association with Southeast Asian isolates. To further our understanding, a One Health approach, incorporating human, environmental (mainly soil), and animal compartments, is essential to decipher the epidemiology of B. pseudomallei in Cambodia.

Melioidosis Queensland: An analysis of clinical outcomes and genomic factors.

The clinical and genomic epidemiology of melioidosis varies across regions. To describe the clinical and genetic diversity of B. pseudomallei across Queensland, Australia. Whole genome sequencing of clinical isolates stored at the melioidosis reference lab from 1996-2020 was performed and analysed in conjunction with available clinical data. Isolates from 292 patients were analysed. Bacteraemia was present in 71% and pneumonia in 65%. The case-fatality rate was 25%. Novel sequence types (ST) accounted for 51% of all isolates. No association was identified between the variable virulence factors assessed and patient outcome. Over time, the proportion of First Nation's patients declined from 59% to 26%, and the proportion of patients aged >70 years rose from 13% to 38%. This study describes a genomically diverse and comparatively distinct collection of B. pseudomallei clinical isolates from across Queensland, Australia. An increasing incidence of melioidosis in elderly patients may be an important factor in the persistently high case-fatality in this region and warrants further investigation and directed intervention.

In Vitro Susceptibility of Burkholderia pseudomallei Isolates to Cefiderocol and Ceftazidime/Avibactam from Odisha, India.

Introduction and Objectives The availability of a limited arsenal of antibacterial agents effective against Burkholderia pseudomallei, the causative agent of melioidosis, together with sporadic reports of emergence of resistance necessitates an evaluation of in vitro activity of new antimicrobials against clinical B. pseudomallei isolates. Cefiderocol (CFDC), a novel siderophore cephalosporin, and ceftazidime-avibactam (CZA), a new β lactam combination agent, have shown promising results for the treatment of difficult-to-treat Gram-negative bacilli infections with limited treatment options. This study was conducted to determine the in vitro activity of CFDC and CZA against a contemporary collection of 60 B. pseudomallei clinical isolates. Materials and Methods Minimum inhibitory concentrations (MIC) of CFDC and CZA were determined by broth microdilution and E-test, respectively. The performance of disk diffusion was also evaluated for CFDC. Results All B. pseudomallei isolates were susceptible to CFDC and CZA with MIC range of 0.125 to 2 mg/L and 0.19 to 1 mg/L, respectively. Zone diameters for CFDC ranged from 31 to 40 mm. Conclusion CFDC and CZA exhibited excellent in vitro activity against 60 B. pseudomallei isolates. Further pharmacokinetic-pharmacodynamics studies and clinical trials are needed to prove the clinical efficacy of CFDC and CZA in the treatment of melioidosis.

Phenotype and Virulence Assessment of a Burkholderia pseudomallei Soil Isolate from Malaysia

Burkholderia pseudomallei, a Gram-negative soil saprophyte, is the causative agent of life-threatening melioidosis. B. pseudomallei from soil and water remains a common source of human and animal infection via skin abrasions, ingestion or inhalation. Despite the reported sero-prevalence in healthy individuals among Malaysian rice farmers, there are limited reports on B. pseudomallei isolated from water or soil around the country. In this study, we characterized a B. pseudomallei soil isolate and compared it to local clinical isolates. 16s rRNA sequencing was adopted to confirm the identity of the soil isolate, NC20. B. pseudomallei NC20 colony morphology, in vitro growth rate and antibiotic sensitivity were examined and compared to two B. pseudomallei clinical isolates, UM6 and D286. Virulence properties such as biofilm formation and infection in a nematode host were also examined. The soil isolate NC20 exhibited distinguishable features of B. pseudomallei, comparable growth rate and similar antibiotic resistance profile to UM6 and D286. Additionally, NC20 is a medium-level biofilm producer with levels similar to D286, where the amount of biofilm produced was much less relative to UM6. Interestingly, NC20 exhibited weaker killing of the Caenorhabditis elegans infection model relative to the clinical isolates. The comparison between soil-derived and clinical isolates of B. pseudomallei demonstrated that both soil and clinical isolates shared certain phenotypic properties but the soil isolate was somewhat less virulent than the clinical isolates used in this study.

One-step, multiplex, dual-function oligonucleotide of loop-mediated isothermal amplification assay for the detection of pathogenic Burkholderia pseudomallei

This study highlights the development of a multiplex real-time loop-mediated isothermal amplification assay. The developed assay employed a dual-function oligonucleotide (DFO) which simultaneously monitors the emitted amplification signals and accelerates the amplification process. The DFO was a modification of loop primer (LP); the 5′-end and 3′-end of the LP was tagged with fluorophore and quencher, respectively. The DFO was quenched in its unbound state and fluoresces only when it anneals to the specific target during the amplification process. With the same working mechanism as LP, DFO allowed the detection of target genes in less than 1 h in a real time monitoring system. We demonstrated this detection platform with Burkholderia pseudomallei, the causative agent of melioidosis. An internal amplification control (IAC) was incorporated in the assay to rule out false negative result and to demonstrate that the assay was successfully developed in a multiplex system. The assay was 100% specific when it was evaluated against 96 B. pseudomallei clinical isolates and 48 other bacteria species. The detection limit (sensitivity) of the developed assay was 1 fg/μl of B. pseudomallei genomic DNA and 18.2 CFU/ml at the bacterial cell level. In spiked blood samples, the assay's detection limit was 14 CFU/ml. The assay's diagnostic evaluation showed 100% diagnostic sensitivity, diagnostic specificity, positive predictive value, and negative predictive value. An integrated multiplex LAMP and real-time monitoring system was successfully developed, simplifying the workflow for the rapid and specific nucleic acid diagnostic test.

Whole-genome comparative analysis of Malaysian Burkholderia pseudomallei clinical isolates.

Burkholderia pseudomallei , a soil-dwelling Gram-negative bacterium, is the causative agent of the endemic tropical disease melioidosis. Clinical manifestations of B. pseudomallei infection range from acute or chronic localized infection in a single organ to fulminant septicaemia in multiple organs. The diverse clinical manifestations are attributed to various factors, including the genome plasticity across B. pseudomallei strains. We previously characterized B. pseudomallei strains isolated in Malaysia and noted different levels of virulence in model hosts. We hypothesized that the difference in virulence might be a result of variance at the genome level. In this study, we sequenced and assembled four Malaysian clinical B. pseudomallei isolates, UKMR15, UKMPMC2000, UKMD286 and UKMH10. Phylogenomic analysis showed that Malaysian subclades emerged from the Asian subclade, suggesting that the Malaysian strains originated from the Asian region. Interestingly, the low-virulence strain, UKMH10, was the most distantly related compared to the other Malaysian isolates. Genomic island (GI) prediction analysis identified a new island of 23 kb, GI9c, which is present in B. pseudomallei and Burkholderia mallei , but not Burkholderia thailandensis . Genes encoding known B. pseudomallei virulence factors were present across all four genomes, but comparative analysis of the total gene content across the Malaysian strains identified 104 genes that are absent in UKMH10. We propose that these genes may encode novel virulence factors, which may explain the reduced virulence of this strain. Further investigation on the identity and role of these 104 proteins may aid in understanding B. pseudomallei pathogenicity to guide the design of new therapeutics for treating melioidosis.

Multi locus sequence typing of clinical Burkholderia pseudomallei isolates from Malaysia.

BackgroundMelioidosis is a neglected tropical disease with rising global public health and clinical importance. Melioidosis is endemic in Southeast Asia and Northern Australia and is of increasing concern in Malaysia. Despite a number of reported studies from Malaysia, these reports are limited to certain parts of the country and do not provide a cohesive link between epidemiology of melioidosis cases and the nation-wide distribution of the causative agent Burkholderia pseudomallei.Methodology/principle findingsHere we report on the distribution of B. pseudomallei sequence types (STs) in Malaysia and how the STs are related to STs globally. We obtained 84 culture-confirmed B. pseudomallei from confirmed septicaemic melioidosis patients from all over Malaysia. Prior to performing Multi Locus Sequence Typing, the isolates were subjected to antimicrobial susceptibility testing and detection of the YLF/BTFC genes and BimA allele. Up to 90.5% of the isolates were sensitive to all antimicrobials tested while resistance was observed for antimicrobials typically administered during the eradication stage of treatment. YLF gene cluster and bimABp allele variant were detected in all the isolates. The epidemiological distribution patterns of the Malaysian B. pseudomallei isolates were analysed in silico using phylogenetic tools and compared to Southeast Asian and world-wide isolates. Genotyping of the 84 Malaysian B. pseudomallei isolates revealed 29 different STs of which 6 (7.1%) were novel. ST50 was identified as the group founder followed by subgroup founders ST376, ST211 and ST84. A low-level diversity is noted for the B. pseudomallei isolates described in this study while phylogenetic analysis associated the Malaysian STs to Southeast Asian isolates especially isolates from Thailand. Further analysis also showed a strong association that implicates agriculture and domestication activities as high-risk routes of infection.Conclusions/significanceIn conclusion, MLST analysis of B. pseudomallei clinical isolates from all states in Malaysia revealed low diversity and a close association to Southeast Asian isolates.

Molecular analysis of clinical Burkholderia pseudomallei isolates from southwestern coastal region of India, using multi-locus sequence typing.

BackgroundThe Gram-negative soil dwelling bacterium Burkholderia pseudomallei is the etiological agent of melioidosis. The disease is endemic in most parts of Southeast Asia and northern Australia. Over last few years, there has been an increase in number of melioidosis cases from India; however the disease epidemiology is less clearly understood. Multi-locus sequence typing (MLST) is a powerful genotypic method used to characterize the genetic diversity of B. Pseudomallei both within and across the geographic regions.MethodsIn this study, MLST analysis was performed on 64 B. pseudomallei clinical isolates. These isolates were obtained between 2008–2014 from southwestern coastal region of India. Broad population patterns of Indian B. pseudomallei isolates in context with isolates of Southeast Asia or global collection was determined using in silico phylogenetic tools.ResultsA total of 32 Sequence types (STs) were reported among these isolates of which 17 STs (53%) were found to be novel. ST1368 was found as group founder and the most predominant genotype (n = 11, 17%). Most of the B. pseudomallei isolates reported in this study (or other Indian isolates available in MLST database) clustered in one major group suggesting clonality in Indian isolates; however, there were a few outliers. When analyzed by measure of genetic differentiation (FST) and other phylogenetic tools (e.g. PHYLOViZ), Indian STs were found closer to Southeast Asian isolates than Australian isolates. The phylogenetic analysis further revealed that within Asian clade, Indian isolates grouped more closely with isolates from Sri Lanka, Vietnam, Bangladesh and Thailand.ConclusionsOverall, the results of this study suggest that the Indian B. pseudomallei isolates are closely related with lesser heterogeneity among them and cluster in one major group suggesting clonality of the isolates. However, it appears that there are a few outliers which are distantly related to the majority of Indian STs. Phylogenetic analysis suggest that Indian isolates are closely related to isolates from Southeast Asia, particularly from South Asia.

Multilocus sequencing-based evolutionary analysis of 52 strains of Burkholderia pseudomallei in Hainan, China.

Previously, we reported a phylogenetic study of 98 Burkholderia pseudomallei clinical isolates from Hainan, China. Here, we update the B. pseudomallei strain library with 52 strains from newly identified cases dating from 2014 to 2017, analysed by multilocus sequence typing. Twenty-two sequence types (STs) were identified from the 52 cases, illustrating high genetic diversity; five of them (ST1480, ST1481, ST1482, ST1483 and ST1484) were novel. ST46, ST50 and ST58 predominated (34.6%) as was the case in the previous study (35.7%). An e-BURST map of the ST profiles of the two collections of isolates showed their genetic foundation to be largely unchanged. Neighbour-joining tree analysis was suggestive of a close phylogenetic relationship between the novel STs from this series and those first reported from Hainan (ST1105, ST1099, ST55 and ST1095). Moreover, the two novel STs (1481 and 1483) showed close similarity to ST58 which originated in Thailand indicating a close relationship between B. pseudomallei strains from both countries. The previously described allele profiles gmhD-36 and lepA-68 were found for the first time in our strain collections. Our study emphasises the importance of monitoring the epidemiological status and evolutionary trends of B. pseudomallei in China.

Multi locus sequence typing of Burkholderia pseudomallei isolates from India unveils molecular diversity and confers regional association in Southeast Asia.

ObjectivesBurkholderia pseudomallei, the causative agent for melioidosis, has become a public health problem in India and across the world. Melioidosis can be difficult to diagnose because of the inconsistent clinical presentations of the disease. This study aims to determine the genetic diversity among the clinical isolates of B. pseudomaelli from India in order to establish a molecular epidemiology and elucidate the Southeast Asian association.MethodsMolecular typing using multi locus sequence typing was performed on thirty one archived B. pseudomallei clinical isolates, previously characterised from specimens obtained from patients admitted to the Christian Medical College & Hospital, Vellore from 2015 to 2016. Further investigations into the genetic heterogeneity and evolution at a regional and global level were performed using insilico tools.ResultsMulti locus sequence typing (MLST) of the isolates from systemic and localized forms of melioidosis, including blood, pus, tissue, and urine specimens, revealed twenty isolates with novel sequence types and eleven with previously reported sequence types. High genetic diversity was observed using MLST with a strong association within the Southeast Asian region.ConclusionsMolecular typing of B. pseudomallei clinical isolates using MLST revealed high genetic diversity and provided a baseline molecular epidemiology of the disease in India with a strong Southeast Asian association of the strains. Future studies should focus on whole genome based Single-Nucleotide-Polymorphism (SNP) which has the advantage of a high discriminatory power, to further understand the novel sequence types reported in this study.

Virulence of the Melioidosis Pathogen Burkholderia pseudomallei Requires the Oxidoreductase Membrane Protein DsbB.

ABSTRACTThe naturally antibiotic-resistant bacterium Burkholderia pseudomallei is the causative agent of melioidosis, a disease with stubbornly high mortality and a complex, protracted treatment regimen. The worldwide incidence of melioidosis is likely grossly underreported, though it is known to be highly endemic in northern Australia and Southeast Asia. Bacterial disulfide bond (DSB) proteins catalyze the oxidative folding and isomerization of disulfide bonds in substrate proteins. In the present study, we demonstrate that B. pseudomallei membrane protein disulfide bond protein B (BpsDsbB) forms a functional redox relay with the previously characterized virulence mediator B. pseudomallei disulfide bond protein A (BpsDsbA). Genomic analysis of diverse B. pseudomallei clinical isolates demonstrated that dsbB is a highly conserved core gene. Critically, we show that DsbB is required for virulence in B. pseudomallei. A panel of B. pseudomallei dsbB deletion strains (K96243, 576, MSHR2511, MSHR0305b, and MSHR5858) were phenotypically diverse according to the results of in vitro assays that assess hallmarks of virulence. Irrespective of their in vitro virulence phenotypes, two deletion strains were attenuated in a BALB/c mouse model of infection. A crystal structure of a DsbB-derived peptide complexed with BpsDsbA provides the first molecular characterization of their interaction. This work contributes to our broader understanding of DSB redox biology and will support the design of antimicrobial drugs active against this important family of bacterial virulence targets.

Adhesion and invasion attributes of Burkholderia pseudomallei are dependent on airway surface liquid and glucose concentrations in lung epithelial cells.

Physiological constituents in airway surface liquids (ASL) appear to impact the adherence and invasion potentials of Burkholderia pseudomallei contributing to recrudescent melioidosis. Here, we investigated the factors present in ASL that is likely to influence bacterial adhesion and invasion leading to improved understanding of bacterial pathogenesis. Six B. pseudomallei clinical isolates from different origins were used to investigate the ability of the bacteria to adhere and invade A549 human lung epithelial cells using a system that mimics the physiological ASL with different pH, NaCl, KCl, CaCl2 and glucose concentrations. These parameters resulted in markedly differential adherence and invasion abilities of B. pseudomallei to the lung epithelial cells. The concentration of 20 mM glucose dramatically increased adherence and invasion by increasing the rate of pili formation in depiliated bacteria. Glucose significantly increased adherence and invasion of B. pseudomallei to A549 cells, and presence of NaCl, KCl and CaCl2 markedly ablated the effect despite the presence ofglucose. Our data established a link between glucose, enhanced adhesion and invasion potentials of B. pseudomallei, hinting increased susceptibility of individuals with diabetes mellitus to clinical melioidosis.

Susceptibility of Clinical Isolates of Burkholderia pseudomallei to a Lipid A Biosynthesis Inhibitor

.Burkholderia pseudomallei is the causative agent of melioidosis, a serious infection associated with high mortality and relapse. Current antimicrobial therapy using ceftazidime (CAZ) is often ineffective. Inhibitors of LpxC, the enzyme responsible for lipid A biosynthesis, have potential antimicrobial activity against several Gram-negative bacteria in vivo, but their activity against B. pseudomallei is unclear. Herein, we investigated the susceptibility of B. pseudomallei clinical isolates to LpxC-4, an LpxC inhibitor, and LpxC-4 in combination with CAZ. Time-kill assays for bactericidal activity were conducted for B. pseudomallei K96243, revealing growth inhibition and bactericidal effect at LpxC-4 concentrations of 2 μg/mL and 4 μg/mL, respectively. No significant synergistic effect was observed with the combination of LpxC-4 and CAZ. LpxC-4 susceptibility was tested on three groups of clinical isolates:1) CAZ- and trimethoprim–sulfamethoxazole (SXT)–susceptible (N = 71), 2) CAZ-resistant (N = 14), and 3) SXT-resistant (N = 23) isolates, by broth microdilution. The minimum concentration of LpxC-4 required to inhibit the growth of 90% of organisms was 2 μg/mL for all isolates. The median minimum inhibitory concentration of both the CAZ/SXT-susceptible and CAZ-resistant groups was 1 μg/mL (interquartile range [IQR] = 1–2 μg/mL), compared with 2 μg/mL (IQR = 2–4 μg/mL) for the SXT-resistant group. Cell morphology was observed after drug exposure by immunofluorescent staining, and a change from rod-shaped to cell wall–defective spherical cells was observed in surviving bacteria. LpxC-4 is a potent bactericidal agent against B. pseudomallei and warrants further testing as a new antibiotic to treat melioidosis.

Multilocus sequence typing of 102 Burkholderia pseudomallei strains isolated from China.

The phylogenetic and epidemiological relationships of 102 Burkholderia pseudomallei clinical isolates from different geographical and population sources in China were investigated by multilocus sequence typing (MLST). The MLST data were analysed using the e-BURST algorithm, and an unweighted pair-group method with arithmetic mean dendrogram was constructed based on the pair-wise differences in the allelic profiles of the strains. Forty-one sequence types (STs) were identified, of which eight were novel (ST1341, ST1345, ST1346, ST1347, ST1348, ST1349, ST1350, ST1351). No geographical-specific or host population-specific phylogenetic lineages were identified. ST46, ST50, ST55, ST58, ST70 and ST1095 predominated, but ~44% of isolates were assigned to 45 STs illustrating high genetic diversity in the strain collection. Additionally, the phylogenetic relationships of the dominant STs in China showed significant linkeage with B. pseudomallei isolates from Thailand. Analysis of the gmhD allele suggests high genetic variation in B. pseudomallei in China.

T Cell Immunity to the Alkyl Hydroperoxide Reductase of Burkholderia pseudomallei: A Correlate of Disease Outcome in Acute Melioidosis.

There is an urgent need for a better understanding of adaptive immunity to Burkholderia pseudomallei, the causative agent of melioidosis that is frequently associated with sepsis or death in patients in Southeast Asia and Northern Australia. The imperative to identify vaccine targets is driven both by the public health agenda in these regions and biological threat concerns. In several intracellular bacterial pathogens, alkyl hydroperoxidase reductases are upregulated as part of the response to host oxidative stress, and they can stimulate strong adaptive immunity. We show that alkyl hydroperoxidase reductase (AhpC) of B. pseudomallei is strongly immunogenic for T cells of ‘humanized’ HLA transgenic mice and seropositive human donors. Some T cell epitopes, such as p6, are able to bind diverse HLA class II heterodimers and stimulate strong T cell immunity in mice and humans. Importantly, patients with acute melioidosis who survive infection show stronger T cell responses to AhpC relative to those who do not. Although the sequence of AhpC is virtually invariant among global B. pseudomallei clinical isolates, a Cambodian isolate varies only in C-terminal truncation of the p6 T cell epitope, raising the possibility of selection by host immunity. This variant peptide is virtually unable to stimulate T cell immunity. For an infection in which there has been debate about centrality of T cell immunity in defense, these observations support a role for T cell immunity to AhpC in disease protection.

Burkholderia pseudomallei Isolates from Sarawak, Malaysian Borneo, Are Predominantly Susceptible to Aminoglycosides and Macrolides

Melioidosis is a potentially fatal disease caused by the saprophytic bacterium Burkholderia pseudomallei. Resistance to gentamicin is generally a hallmark of B. pseudomallei, and gentamicin is a selective agent in media used for diagnosis of melioidosis. In this study, we determined the prevalence and mechanism of gentamicin susceptibility found in B. pseudomallei isolates from Sarawak, Malaysian Borneo. We performed multilocus sequence typing and antibiotic susceptibility testing on 44 B. pseudomallei clinical isolates from melioidosis patients in Sarawak district hospitals. Whole-genome sequencing was used to identify the mechanism of gentamicin susceptibility. A novel allelic-specific PCR was designed to differentiate gentamicin-sensitive isolates from wild-type B. pseudomallei. A reversion assay was performed to confirm the involvement of this mechanism in gentamicin susceptibility. A substantial proportion (86%) of B. pseudomallei clinical isolates in Sarawak, Malaysian Borneo, were found to be susceptible to the aminoglycoside gentamicin, a rare occurrence in other regions where B. pseudomallei is endemic. Gentamicin sensitivity was restricted to genetically related strains belonging to sequence type 881 or its single-locus variant, sequence type 997. Whole-genome sequencing identified a novel nonsynonymous mutation within amrB, encoding an essential component of the AmrAB-OprA multidrug efflux pump. We confirmed the role of this mutation in conferring aminoglycoside and macrolide sensitivity by reversion of this mutation to the wild-type sequence. Our study demonstrates that alternative B. pseudomallei selective media without gentamicin are needed for accurate melioidosis laboratory diagnosis in Sarawak. This finding may also have implications for environmental sampling of other locations to test for B. pseudomallei endemicity.

The Effect of Environmental Conditions on Biofilm Formation of Burkholderia pseudomallei Clinical Isolates

Burkholderia pseudomallei, a Gram-negative saprophytic bacterium, is the causative agent of the potentially fatal melioidosis disease in humans. In this study, environmental parameters including temperature, nutrient content, pH and the presence of glucose were shown to play a role in in vitro biofilm formation by 28 B. pseudomallei clinical isolates, including four isolates with large colony variants (LCVs) and small colony variants (SCVs) morphotypes. Enhanced biofilm formation was observed when the isolates were tested in LB medium, at 30°C, at pH 7.2, and in the presence of as little as 2 mM glucose respectively. It was also shown that all SVCs displayed significantly greater capacity to form biofilms than the corresponding LCVs when cultured in LB at 37°C. In addition, octanoyl-homoserine lactone (C8-HSL), a quorum sensing molecule, was identified by mass spectrometry analysis in bacterial isolates referred to as LCV CTH, LCV VIT, SCV TOM, SCV CTH, 1 and 3, and the presence of other AHL's with higher masses; decanoyl-homoserine lactone (C10-HSL) and dodecanoyl-homoserine lactone (C12-HSL) were also found in all tested strain in this study. Last but not least, we had successfully acquired two Bacillus sp. soil isolates, termed KW and SA respectively, which possessed strong AHLs degradation activity. Biofilm formation of B. pseudomallei isolates was significantly decreased after treated with culture supernatants of KW and SA strains, demonstrating that AHLs may play a role in B. pseudomallei biofilm formation.

Burkholderia pseudomallei animal and human isolates from Malaysia exhibit different phenotypic characteristics

Burkholderia pseudomallei is a Gram-negative saprophytic soil bacterium, which is the etiologic agent of melioidosis, a severe and fatal infectious disease occurring in human and animals. Distinct clinical and animal isolates have been shown to exhibit differences in phenotypic trait such as growth rate, colony morphology, antimicrobial resistance, and virulence. This study was carried out to gain insight into the intrinsic differences between 4 clinical and 6 animal B. pseudomallei isolates from Malaysia. The 16S rRNA-encoding genes from these 10 isolates of B. pseudomallei were sequenced to confirm the identity of these isolates along with the avirulent Burkholderia thailandensis. The nucleotide sequences indicated that the 16S rRNA-encoding genes among the 10 B. pseudomallei isolates were identical to each other. However, the nucleotide sequence differences in the 16S rRNA-encoding genes appeared to be B. pseudomallei and B. thailandensis specific. The growth rate of all B. pseudomallei isolates was determined by generating growth curves at 37 °C for 72 h. The isolates were found to differ in growth rates with doubling time varying from 1.5 to 2.3 h. In addition, the B. pseudomallei isolates exhibited considerable variation in colony morphology when grown on Ashdown media, brain–heart infusion agar, and Luria–Bertani agar over 9 days of observation. Antimicrobial susceptibility tests indicated that 80% of the isolates examined were Amp R Cb R Kn R Gm R Chl S Te S. Virulence of the B. pseudomallei clinical and animal isolates was evaluated in B. pseudomallei-susceptible BALB/c mice. Most of the clinical isolates were highly virulent. However, virulence did not correlate with isolate origin since 2 of the animal isolates were also highly virulent.

A comparison of antibiotic susceptibility testing methods for cotrimoxazole with Burkholderia pseudomallei

Melioidosis is caused by the Gram-negative soil saprophyte, Burkholderia pseudomallei and is endemic in tropical and subtropical regions of southeast Asia and northern Australia. Cotrimoxazole has been traditionally used for the therapy of melioidosis despite results indicating resistance often produced in the disc diffusion test against B. pseudomallei. This inconsistency was addressed by comparing this method with the agar dilution, MicroScan and E -test methods. The results demonstrated that by disc diffusion, 41.3% of 80 B. pseudomallei clinical isolates tested were susceptible to cotrimoxazole, whereas the MicroScan, agar dilution and the E -test demonstrated 92.5, 90 and 97.5% of the isolates to be susceptible, respectively. These results indicate that an MIC based method is required to test the susceptibility of B. pseudomallei against cotrimoxazole.

Differentiation between non-virulent and virulent Burkholderia pseudomallei with monoclonal antibodies to the Ara+ or Ara- biotypes.

Burkholderia pseudomallei is the causative agent of melioidosis, a fatal tropical infectious disease endemic in Southeast Asia. Environmental isolates of B. pseudomallei have two distinctive biotypes. Some soil isolates are arabinose-assimilators (Ara+ biotype) and are non-virulent in experimental animals. The others cannot assimilate arabinose (Ara- biotype) and are virulent in experimental animals. The Ara- biotype is found in almost all B. pseudomallei clinical isolates. In the present study, a panel of eight monoclonal antibodies that agglutinate the bacteria were produced and tested. The first group, Bps-D2, -D3, -D5, -L1, and -L2 agglutinated 100% of Ara+ clinical and soil isolates of B. pseudomallei. Another group Bps-A1, -A2, and -D1 agglutinated 92.9% and 90.9% of Ara- clinical and soil isolates, respectively. This panel of monoclonal antibodies may be useful for rapid differentiation between non-virulent Ara+ and virulent Ara- B. pseudomallei.