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Fulltext Duplex TaqMan Hydrolysis Probe-Based Molecular Assay for Simultaneous Detection and Differentiation of Burkholderia pseudomallei and Leptospira spp. DNA

Mohd Ali MR, Lih Huey L, Foo PC, Goay YX, Ismail AS, Mustaffa KMF, et al.

Biomed Res Int, 2019;2019:9451791.
PMID: 31355287 DOI: 10.1155/2019/9451791

Melioidosis and leptospirosis, caused by two different bacteria, Burkholderia pseudomallei and Leptospira spp., are potentially fatal infections that share a very similar spectrum of clinical features and cause significant mortality and morbidity in humans and livestock. Early detection is important for better clinical consequences. To our knowledge, there is no diagnostic tool available to simultaneously detect and differentiate melioidosis and leptospirosis in humans and animals. In this study, we described a duplex TaqMan probe-based qPCR for the detection of B. pseudomallei and Leptospira spp. DNA. The performance of the assay was evaluated on 20 B. pseudomallei isolates, 23 Leptospira strains, and 39 other microorganisms, as well as two sets of serially diluted reference strains. The duplex qPCR assay was able to detect 0.02 pg (~ 4 copies) Leptospira spp. DNA and 0.2 pg (~ 25.6 copies) B. pseudomallei DNA. No undesired amplification was observed in other microorganisms. In conclusion, the duplex qPCR assay was sensitive and specific for the detection of B. pseudomallei & Leptospira spp. DNA and is suitable for further analytical and clinical evaluation.

Matched MeSH terms: Burkholderia pseudomallei/genetics*
Adhesion and invasion attributes of Burkholderia pseudomallei are dependent on airway surface liquid and glucose concentrations in lung epithelial cells

Mariappan V, Thimma J, Vellasamy KM, Shankar EM, Vadivelu J

Environ Microbiol Rep, 2018 04;10(2):217-225.
PMID: 29393577 DOI: 10.1111/1758-2229.12624

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 of glucose. 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.

Matched MeSH terms: Burkholderia pseudomallei/genetics
Fulltext Multi locus sequence typing of clinical Burkholderia pseudomallei isolates from Malaysia

Arushothy R, Amran F, Samsuddin N, Ahmad N, Nathan S

PLoS Negl Trop Dis, 2020 12;14(12):e0008979.
PMID: 33370273 DOI: 10.1371/journal.pntd.0008979

BACKGROUND: Melioidosis 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 FINDINGS: Here 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/SIGNIFICANCE: In conclusion, MLST analysis of B. pseudomallei clinical isolates from all states in Malaysia revealed low diversity and a close association to Southeast Asian isolates.

Matched MeSH terms: Burkholderia pseudomallei/genetics*
Ribotyping and DNA macrorestriction analysis of isolates of Burkholderia pseudomallei from cases of melioidosis in Malaysia

Vadivelu J, Puthucheary SD, Mifsud A, Drasar BS, Dance DA, Pitt TI

Trans R Soc Trop Med Hyg, 1997 5 1;91(3):358-60.
PMID: 9231217

Forty-nine isolates of Burkholderia pseudomallei from sporadic cases of melioidosis in Malaysia over the past 18 years were examined by BamHI ribotyping and pulsed-field gel electrophoresis (PFGE) of XbaI digests of total deoxyribonucleic acid (DNA). Twenty-four patients had septicaemic melioidosis with a mortality of 70%; mortality in the non-septicaemic disease was 16%. Five ribotype patterns were identified, 2 of which accounted for 90% of all isolates. PFGE revealed a number of different strains within these ribotypes, but some pairs of isolates from unrelated cases gave closely similar DNA profiles. These results are in agreement with Australian studies which showed a high prevalence of a few ribotypes of B. pseudomallei which are further divisible by genotyping, in areas where melioidosis is endemic.

Matched MeSH terms: Burkholderia pseudomallei/genetics*
Stability of strain genotypes of Burkholderia pseudomallei from patients with single and recurrent episodes of melioidosis

Vadivelu J, Puthucheary SD, Drasar BS, Dance DA, Pitt TL

Trop Med Int Health, 1998 Jul;3(7):518-21.
PMID: 9705184

The constancy of strain genotypes of multiple isolates of Burkholderia pseudomallei from 13 patients with melioidosis was examined by BamHI ribotyping and pulsed-field gel electrophoresis (PFGE) of XbaI digests of DNA. Seven of 8 patients with single episodes of melioidosis each yielded genetically identical isolates and only one of five patients with recurrent episodes was infected with a new strain clearly distinct from the original primary strain. Variation was observed in PFGE patterns of primary and relapse isolates of another patient but this was insufficient to define genetically distinct strains. We conclude that most patients with single or multiple episodes of melioidosis retain a single strain.

Matched MeSH terms: Burkholderia pseudomallei/genetics*
Molecular investigation of virulence determinants between a virulent clinical strain and an attenuated strain of Burkholderia pseudomallei

Puthucheary SD, Puah SM, Chai HC, Thong KL, Chua KH

J. Mol. Microbiol. Biotechnol., 2012;22(3):198-204.
PMID: 22846664 DOI: 10.1159/000338985

Burkholderia pseudomallei is the causative agent of melioidosis. We initiated this investigation with a virulent and an attenuated strain of B. pseudomallei. Pulsed-field gel electrophoresis was carried out initially for macrogenomic comparison of both strains of B. pseudomallei. However, the pulsotypes obtained were identical and therefore we applied a subtractive hybridization technique to distinguish and determine the possible differences between the two strains. Six virulence strain-specific DNA fragments were obtained and the encoding homolog proteins were identified as a xenobiotic-responsive element family of transcriptional regulator, a hypothetical protein, an unknown protein, a plasmid recombination enzyme, a regulatory protein and a putative hemolysin activator protein. A combination of at least three of these determinants was identified in 45 clinical isolates when screening was carried out with self-designed multiplex PCR targeting the six putative virulent determinants. Our data demonstrated that different combinations of the six putative virulence genes were present in the clinical isolates indicating their probable role in the pathogenesis of B. pseudomallei infections.

Matched MeSH terms: Burkholderia pseudomallei/genetics*
Fulltext Molecular investigations of a locally acquired case of melioidosis in Southern AZ, USA

Engelthaler DM, Bowers J, Schupp JA, Pearson T, Ginther J, Hornstra HM, et al.

PLoS Negl Trop Dis, 2011 Oct;5(10):e1347.
PMID: 22028940 DOI: 10.1371/journal.pntd.0001347

Melioidosis is caused by Burkholderia pseudomallei, a Gram-negative bacillus, primarily found in soils in Southeast Asia and northern Australia. A recent case of melioidosis in non-endemic Arizona was determined to be the result of locally acquired infection, as the patient had no travel history to endemic regions and no previous history of disease. Diagnosis of the case was confirmed through multiple microbiologic and molecular techniques. To enhance the epidemiological analysis, we conducted several molecular genotyping procedures, including multi-locus sequence typing, SNP-profiling, and whole genome sequence typing. Each technique has different molecular epidemiologic advantages, all of which provided evidence that the infecting strain was most similar to those found in Southeast Asia, possibly originating in, or around, Malaysia. Advancements in new typing technologies provide genotyping resolution not previously available to public health investigators, allowing for more accurate source identification.

Matched MeSH terms: Burkholderia pseudomallei/genetics*
Development of a multiplex PCR assay for rapid identification of Burkholderia pseudomallei, Burkholderia thailandensis, Burkholderia mallei and Burkholderia cepacia complex

Koh SF, Tay ST, Sermswan R, Wongratanacheewin S, Chua KH, Puthucheary SD

J Microbiol Methods, 2012 Sep;90(3):305-8.
PMID: 22705921 DOI: 10.1016/j.mimet.2012.06.002

We have developed a multiplex PCR assay for rapid identification and differentiation of cultures for Burkholderia pseudomallei, Burkholderia thailandensis, Burkholderia mallei and Burkholderia cepacia complex. The assay is valuable for use in clinical and veterinary laboratories, and in a deployable laboratory during outbreaks.

Matched MeSH terms: Burkholderia pseudomallei/genetics*
Fulltext Computational discovery and RT-PCR validation of novel Burkholderia conserved and Burkholderia pseudomallei unique sRNAs

Khoo JS, Chai SF, Mohamed R, Nathan S, Firdaus-Raih M

BMC Genomics, 2012;13 Suppl 7:S13.
PMID: 23282220 DOI: 10.1186/1471-2164-13-S7-S13

The sRNAs of bacterial pathogens are known to be involved in various cellular roles including environmental adaptation as well as regulation of virulence and pathogenicity. It is expected that sRNAs may also have similar functions for Burkholderia pseudomallei, a soil bacterium that can adapt to diverse environmental conditions, which causes the disease melioidosis and is also able to infect a wide variety of hosts.

Matched MeSH terms: Burkholderia pseudomallei/genetics*
SpeI restriction enzyme displays greater discriminatory power than XbaI enzyme does in a pulsed-field gel electrophoresis study on 146 clinical Burkholderia pseudomallei isolates

Chua KH, See KH, Thong KL, Puthucheary SD

Jpn J Infect Dis, 2011;64(3):228-33.
PMID: 21617308

Restriction enzymes SpeI and XbaI were used in a pulsed-field gel electrophoresis (PFGE) study for molecular characterization of 146 clinical Burkholderia pseudomallei isolates. The PFGE parameters were optimized to enable comparable, reproducible, and robust results. The optimized parameters for both SpeI and XbaI restriction enzymes used in this study were 200 V and a pulse time of 5 to 65 s for a 28-h runtime. Using SpeI, 9 different clusters were identified, whereas 6 clusters were identified by XbaI digestion, which exhibited 85% similarity to SpeI. SpeI (discrimination index [D]=0.854) showed higher discriminatory power than XbaI did (D=0.464).

Matched MeSH terms: Burkholderia pseudomallei/genetics*
In silico analysis of Burkholderia pseudomallei genome sequence for potential drug targets

Chong CE, Lim BS, Nathan S, Mohamed R

In Silico Biol. (Gedrukt), 2006;6(4):341-6.
PMID: 16922696

Recent advances in DNA sequencing technology have enabled elucidation of whole genome information from a plethora of organisms. In parallel with this technology, various bioinformatics tools have driven the comparative analysis of the genome sequences between species and within isolates. While drawing meaningful conclusions from a large amount of raw material, computer-aided identification of suitable targets for further experimental analysis and characterization, has also led to the prediction of non-human homologous essential genes in bacteria as promising candidates for novel drug discovery. Here, we present a comparative genomic analysis to identify essential genes in Burkholderia pseudomallei. Our in silico prediction has identified 312 essential genes which could also be potential drug candidates. These genes encode essential proteins to support the survival of B. pseudomallei including outer-inner membrane and surface structures, regulators, proteins involved in pathogenenicity, adaptation, chaperones as well as degradation of small and macromolecules, energy metabolism, information transfer, central/intermediate/miscellaneous metabolism pathways and some conserved hypothetical proteins of unknown function. Therefore, our in silico approach has enabled rapid screening and identification of potential drug targets for further characterization in the laboratory.

Matched MeSH terms: Burkholderia pseudomallei/genetics*
DNA fingerprinting of septicemic and localized Burkholderia pseudomallei isolates from Malaysian patients

Azura MN, Norazah A, Kamel AG, Zorin SA

Southeast Asian J Trop Med Public Health, 2011 Jan;42(1):114-21.
PMID: 21323173

We have analysed DNA fingerprinting patterns by pulsed-field gel electrophoresis (PFGE) of 52 unrelated Burkholderia pseudomallei strains isolated from septicemic and localized infections from Malaysian subjects. A total of 38 PFGE types were observed among 36 septicemic and 16 localized strains with no predominant pattern. Type 25 was seen in 2 epidemiologically related strains, suggesting human to human transmission. Twelve PFGE types were shared among 26 strains (21 septicemic and 5 localized) showing close genetic relatedness with coefficient of similarity of 0.81 to 1.0. The other 26 strains (15 septicemic and 11 localized) were unrelated as shown by the similarity coefficient of < 0.8. This study showed that our B. pseudomallei strains in Malaysia were mainly heterogenous with no predominant type both in septicemic or localized strains.

Matched MeSH terms: Burkholderia pseudomallei/genetics*
Fulltext Sequence polymorphism and PCR-restriction fragment length polymorphism analysis of the flagellin gene of Burkholderia pseudomallei

Tay ST, Cheah PC, Puthucheary SD

J Clin Microbiol, 2010 Apr;48(4):1465-7.
PMID: 20089759 DOI: 10.1128/JCM.01131-09

Four flagellin allelic types (I to IV) of Burkholderia pseudomallei were identified based on their sequence variation and restriction fragment length polymorphism (RFLP) analysis of the amplified flagellin gene. Flagellin allelic type I was the most predominantly (75.0%) found among the 100 clinical isolates of B. pseudomallei investigated in this study.

Matched MeSH terms: Burkholderia pseudomallei/genetics*
Genome-wide prediction and annotation of Burkholderia pseudomallei AraC/XylS family transcription regulator

Lim BS, Chong CE, Zamrod Z, Nathan S, Mohamed R

In Silico Biol. (Gedrukt), 2007;7(4-5):389-97.
PMID: 18391231

Many members of the AraC/XylS family transcription regulator have been proven to play a critical role in regulating bacterial virulence factors in response to environmental stress. By using the Hidden Markov Model (HMM) profile built from the alignment of a 99 amino acid conserved domain sequence of 273 AraC/XylS family transcription regulators, we detected a total of 45 AraC/XylS family transcription regulators in the genome of the Gram-negative pathogen, Burkholderia pseudomallei. Further in silico analysis of each detected AraC/XylS family transcription regulatory protein and its neighboring genes allowed us to make a first-order guess on the role of some of these transcription regulators in regulating important virulence factors such as those involved in three type III secretion systems and biosynthesis of pyochelin, exopolysaccharide (EPS) and phospholipase C. This paper has demonstrated an efficient and systematic genome-wide scale prediction of the AraC/XylS family that can be applied to other protein families.

Matched MeSH terms: Burkholderia pseudomallei/genetics*
Fulltext Molecular characterization of putative virulence determinants in Burkholderia pseudomallei

Puah SM, Puthucheary SD, Wang JT, Pan YJ, Chua KH

ScientificWorldJournal, 2014;2014:590803.
PMID: 25215325 DOI: 10.1155/2014/590803

The Gram-negative saprophyte Burkholderia pseudomallei is the causative agent of melioidosis, an infectious disease which is endemic in Southeast Asia and northern Australia. This bacterium possesses many virulence factors which are thought to contribute to its survival and pathogenicity. Using a virulent clinical isolate of B. pseudomallei and an attenuated strain of the same B. pseudomallei isolate, 6 genes BPSL2033, BP1026B_I2784, BP1026B_I2780, BURPS1106A_A0094, BURPS1106A_1131, and BURPS1710A_1419 were identified earlier by PCR-based subtractive hybridization. These genes were extensively characterized at the molecular level, together with an additional gene BPSL3147 that had been identified by other investigators. Through a reverse genetic approach, single-gene knockout mutants were successfully constructed by using site-specific insertion mutagenesis and were confirmed by PCR. BPSL2033::Km and BURPS1710A_1419::Km mutants showed reduced rates of survival inside macrophage RAW 264.7 cells and also low levels of virulence in the nematode infection model. BPSL2033::Km demonstrated weak statistical significance (P = 0.049) at 8 hours after infection in macrophage infection study but this was not seen in BURPS1710A_1419::Km. Nevertheless, complemented strains of both genes were able to partially restore the gene defects in both in vitro and in vivo studies, thus suggesting that they individually play a minor role in the virulence of B. pseudomallei.

Matched MeSH terms: Burkholderia pseudomallei/genetics*
Fulltext Multilocus sequence types of clinical Burkholderia pseudomallei isolates from peninsular Malaysia and their associations with disease outcomes

Zueter AR, Rahman ZA, Abumarzouq M, Harun A

BMC Infect Dis, 2018 01 02;18(1):5.
PMID: 29291714 DOI: 10.1186/s12879-017-2912-9

BACKGROUND: Previous studies on the Burkholderia pseudomallei genetic diversity among clinical isolates from melioidosis-endemic areas have identified genetic factors contributing to differential virulence. Although it has been ruled out in Australian and Thai B. pseudomallei populations, it remains unclear whether B. pseudomallei sequence types (STs) correlate with disease in Malaysian patients with melioidosis.
METHODS: In this study, multi-locus sequence typing (MLST) was performed on clinical B. pseudomallei isolates collected from Kelantan state of Malaysia, patients' clinical data were reviewed and then genotype-risk correlations were investigated.
RESULTS: Genotyping of 83 B. pseudomallei isolates revealed 32 different STs, of which 13(40%) were novel. The frequencies of the STs among the 83 isolates ranged from 1 to 12 observations, and ST54, ST371 and ST289 were predominant. All non-novel STs reported in this study have also been identified in other Asian countries. Based on the MLST data analysis, the phylogenetic tree showed clustering of the STs with each other, as well as with the STs from Southeast Asia and China. No evidence for associations between any of B. pseudomallei STs and clinical melioidosis presentation was detected. In addition, the bacterial genotype clusters in relation with each clinical outcome were statistically insignificant, and no risk estimate was reported. This study has expanded the data for B. pseudomallei on MLST database map and provided insights into the molecular epidemiology of melioidosis in Peninsular Malaysia.
CONCLUSION: This study concurs with previous reports concluding that infecting strain type plays no role in determining disease presentation.

Matched MeSH terms: Burkholderia pseudomallei/genetics*
Fulltext Global and regional dissemination and evolution of Burkholderia pseudomallei

Chewapreecha C, Holden MT, Vehkala M, Välimäki N, Yang Z, Harris SR, et al.

Nat Microbiol, 2017 Jan 23;2:16263.
PMID: 28112723 DOI: 10.1038/nmicrobiol.2016.263

The environmental bacterium Burkholderia pseudomallei causes an estimated 165,000 cases of human melioidosis per year worldwide and is also classified as a biothreat agent. We used whole genome sequences of 469 B. pseudomallei isolates from 30 countries collected over 79 years to explore its geographic transmission. Our data point to Australia as an early reservoir, with transmission to Southeast Asia followed by onward transmission to South Asia and East Asia. Repeated reintroductions were observed within the Malay Peninsula and between countries bordered by the Mekong River. Our data support an African origin of the Central and South American isolates with introduction of B. pseudomallei into the Americas between 1650 and 1850, providing a temporal link with the slave trade. We also identified geographically distinct genes/variants in Australasian or Southeast Asian isolates alone, with virulence-associated genes being among those over-represented. This provides a potential explanation for clinical manifestations of melioidosis that are geographically restricted.

Matched MeSH terms: Burkholderia pseudomallei/genetics*
Transcriptome analysis of Burkholderia pseudomallei SCV reveals an association with virulence, stress resistance and intracellular persistence

Al-Maleki AR, Vellasamy KM, Mariappan V, Venkatraman G, Tay ST, Vadivelu J

Genomics, 2020 01;112(1):501-512.
PMID: 30980902 DOI: 10.1016/j.ygeno.2019.04.002

Differences in expression of potential virulence and survival genes were associated with B. pseudomallei colony morphology variants. Microarray was used to investigate B. pseudomallei transcriptome alterations among the wild type and small colony variant (SCV) pre- and post-exposed to A549 cells. SCV pre- and post-exposed have lower metabolic requirements and consume lesser energy than the wild type pre- and post-exposed to A549. However, both the wild type and SCV limit their metabolic activities post- infection of A549 cells and this is indicated by the down-regulation of genes implicated in the metabolism of amino acids, carbohydrate, lipid, and other amino acids. Many well-known virulence and survival factors, including T3SS, fimbriae, capsular polysaccharides and stress response were up-regulated in both the wild type and SCV pre- and post-exposed to A549 cells. Microarray analysis demonstrated essential differences in bacterial response associated with virulence and survival pre- and post-exposed to A549 cells.

Matched MeSH terms: Burkholderia pseudomallei/genetics*
Characterization of Burkholderia pseudomallei isolated in Thailand and Malaysia

Radua S, Ling OW, Srimontree S, Lulitanond A, Hin WF, Yuherman, et al.

Diagn Microbiol Infect Dis, 2000 Nov;38(3):141-5.
PMID: 11109011

A total of 35 Burkholderia pseudomallei isolates from Thailand (16 clinical and eight soil isolates) and Malaysia (seven animal, two isolate each from clinical and soil) were investigated by their antimicrobial resistance, plasmid profiles and were typed by randomly amplified polymorphic DNA analysis. All isolates were found to be resistant to six or more of the 12 antimicrobial agents tested. Only two small plasmids of 1.8 and 2.4 megadalton were detected in two clinical isolates from Thailand. RAPD analysis with primer GEN2-60-09 resulted in the identification of 35 RAPD-types among the 35 isolates. The constructed dendrogram differentiated the 35 isolates into two main clusters and a single isolate. The wide genetic biodiversity among the 35 isolates indicate that RAPD-PCR can be a useful method to differentiate unrelated B. pseudomallei in epidemiological investigation.

Matched MeSH terms: Burkholderia pseudomallei/genetics
Fulltext Genotypic and phenotypic relationship in Burkholderia pseudomallei indicates colonization with closely related isolates

Radu S, Lihan S, Idris A, Ling OW, Al-Haddawi MH, Rusul G

Southeast Asian J Trop Med Public Health, 1999 Dec;30(4):760-3.
PMID: 10928372

Seven isolates of Burkholderia pseudomallei from cases of melioidosis in human (2 isolates) and animal (2 isolates), cat (one isolate) and from soil samples (2 isolates) were examined for in vitro sensitivity to 14 antimicrobial agents and for presence of plasmid DNA. Randomly amplified polymorphic DNA (RAPD) analysis was used to type the isolates, using two arbitrary primers. All isolates were sensitive to chloramphenicol, kanamycin, carbenicillin, rifampicin, enrofloxacin, tetracycline and sulfamethoxazole-trimethoprim. No plasmid was detected in all the isolates tested. RADP fingerprinting demonstrated genomic relationship between isolates, which provides an effective method to study the epidemiology of the isolates examined.

Matched MeSH terms: Burkholderia pseudomallei/genetics*

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