Novel selective medium for isolation of Burkholderia pseudomallei

Isolation of Burkholderia pseudomallei currently relies on the use of Ashdown's selective agar (ASA). We designed a new selective agar (Burkholderia pseudomallei selective agar [BPSA]) to improve recovery of the more easily inhibited strains of B. pseudomallei. B. pseudomallei, Burkholderia cepacia, and Pseudomonas aeruginosa were used to determine the selectivity and sensitivity of BPSA. BPSA was more inhibitory to P. aeruginosa and B. cepacia and should make recognition of Burkholderia species easier due to distinctive colony morphology. BPSA also inhibited Enterococcus, Escherichia, Staphylococcus, and Streptococcus: These results indicate that BPSA is a potential replacement for ASA.     

Novel pan-genomic analysis approach in target selection for multiplex PCR identification and detection of Burkholderia pseudomallei, Burkholderia thailandensis, and Burkholderia cepacia complex species: a proof-of-concept study

Burkholderia pseudomallei, Burkholderia thailandensis, and the Burkholderia cepacia complex differ greatly in pathogenicity and epidemiology. Yet, they are occasionally misidentified by biochemical profiling, and even 16S rRNA gene sequencing may not offer adequate discrimination between certain species groups. Using the 23 B. pseudomallei, four B. thailandensis, and 16 B. cepacia complex genome sequences available, we identified gene targets specific to each of them (a Tat domain protein, a 70-kDa protein, and a 12-kDa protein for B. pseudomallei, B. thailandensis, and the B. cepacia complex, respectively), with an in-house developed algorithm. Using these targets, we designed a robust multiplex PCR assay useful for their identification and detection from soil and simulated sputum samples. For all 43 B. pseudomallei, seven B. thailandensis, and 20 B. cepacia complex (B. multivorans, n = 6; B. cenocepacia, n = 3; B. cepacia, n = 4; B. arboris, n = 2; B. contaminans, B. anthina, and B. pyrrocinia, n = 1 each; other unnamed members, n = 2) isolates, the assay produced specific products of predicted size without false positives or negatives. Of the 60 soil samples screened, 19 (31.6%) and 29 (48.3%) were positive for B. pseudomallei and the B. cepacia complex, respectively, and in four (6.7%) soil samples, the organisms were codetected. DNA sequencing confirmed that all PCR products originated from their targeted loci. This novel pan-genomic analysis approach in target selection is simple, computationally efficient, and potentially applicable to any species that harbors species-specific genes. A multiplex PCR assay for rapid and accurate identification and detection of B. pseudomallei, B. thailandensis, and the B. cepacia complex was developed and verified.     

Comparison of diagnostic laboratory methods for identification of Burkholderia pseudomallei

Limited experience and a lack of validated diagnostic reagents make Burkholderia pseudomallei, the cause of melioidosis, difficult to recognize in the diagnostic microbiology laboratory. We compared three methods of confirming the identity of presumptive B. pseudomallei strains using a collection of Burkholderia species drawn from diverse geographic, clinical, and environmental sources. The 95 isolates studied included 71 B. pseudomallei and 3 B. thailandensis isolates. The API 20NE method identified only 37% of the B. pseudomallei isolates. The agglutinating antibody test identified 82% at first the attempt and 90% including results of a repeat test with previously negative isolates. Gas-liquid chromatography analysis of bacterial fatty acid methyl esters (GLC-FAME) identified 98% of the B. pseudomallei isolates. The agglutination test produced four false positive results, one B. cepacia, one B. multivorans, and two B. thailandensis. API produced three false positive results, one positive B. cepacia and two positive B. thailandensis. GLC-FAME analysis was positive for one B. cepacia isolate. On the basis of these results, the most robust B. pseudomallei discovery pathway combines the previously recommended isolate screening tests (Gram stain, oxidase test, gentamicin and polymyxin susceptibility) with monoclonal antibody agglutination on primary culture, followed by a repeat after 24 h incubation on agglutination-negative isolates and GLC-FAME analysis. Incorporation of PCR-based identification within this schema may improve percentages of recognition further but requires more detailed evaluation.     

Accuracy of Burkholderia pseudomallei identification using the API 20NE system and a latex agglutination test

In an evaluation of the API 20NE for the identification of Burkholderia spp., 792/800 (99%) Burkholderia pseudomallei and 17/19 (89%) B. cepacia isolates were correctly identified but 10 B. mallei and 98 B. thailandensis isolates were not correctly identified. A latex agglutination test was positive for 796/800 (99.5%) B. pseudomallei isolates and negative for 120 other oxidase-positive gram-negative bacilli.     

Use of a real-time PCR TaqMan assay for rapid identification and differentiation of Burkholderia pseudomallei and Burkholderia mallei

A TaqMan allelic-discrimination assay designed around a synonymous single-nucleotide polymorphism was used to genotype Burkholderia pseudomallei and Burkholderia mallei isolates. The assay rapidly identifies and discriminates between these two highly pathogenic bacteria and does not cross-react with genetic near neighbors, such as Burkholderia thailandensis and Burkholderia cepacia.     

Burkholderia pseudomallei endophthalmitis

Melioidosis is an infectious disease caused by Burkholderia pseudomallei, a gram-negative bacillus. We report a case of endogenous endophthalmitis caused by B. pseudomallei that was treated with systemic and intravitreal ceftazidime. Finally, the patient achieved a final visual acuity of 20/60 without complication.     

Cellular fatty acid profile distinguishes Burkholderia pseudomallei from avirulent Burkholderia thailandensis

Burkholderia pseudomallei, the cause of melioidosis, can be distinguished from the closely related but nonpathogenic Burkholderia thailandensis by gas chromatography (GC) analysis of fatty acid derivatives. A 2-hydroxymyristic acid derivative (14:0 2OH) was present in 95% of B. pseudomallei isolates and no B. thailandensis isolates. GC mass spectrophotometry confirmed that 2-hydroxymyristic acid was present in B. pseudomallei. GC-fatty acid methyl ester analysis may be useful in distinguishing these two closely related species.     

Use of antigens derived from Burkholderia pseudomallei, B. thailandensis, and B. cepacia in the indirect hemagglutination assay for melioidosis

The serological diagnosis of melioidosis is carried out using the indirect hemagglutination assay. We looked at the reactivity of sera from culture-proven cases of melioidosis from north Queensland against antigens derived from Burkholderia pseudomallei, B. thailandensis, and B. cepacia. Cross-reactivity between sera from culture-positive cases of melioidosis and B. thailandensis was demonstrated.     

Use of 16S rRNA gene sequencing for rapid identification and differentiation of Burkholderia pseudomallei and B. mallei

Burkholderia pseudomallei and B. mallei, the causative agents of melioidosis and glanders, respectively, are designated category B biothreat agents. Current methods for identifying these organisms rely on their phenotypic characteristics and an extensive set of biochemical reactions. We evaluated the use of 16S rRNA gene sequencing to rapidly identify these two species and differentiate them from each other as well as from closely related species and genera such as Pandoraea spp., Ralstonia spp., Burkholderia gladioli, Burkholderia cepacia, Burkholderia thailandensis, and Pseudomonas aeruginosa. We sequenced the 1.5-kb 16S rRNA gene of 56 B. pseudomallei and 23 B. mallei isolates selected to represent a wide range of temporal, geographic, and origin diversity. Among all 79 isolates, a total of 11 16S types were found based on eight positions of difference. Nine 16S types were identified in B. pseudomallei isolates based on six positions of difference, with differences ranging from 0.5 to 1.5 bp. Twenty-two of 23 B. mallei isolates showed 16S rRNA gene sequence identity and were designated 16S type 10, whereas the remaining isolate was designated type 11. This report provides a basis for rapidly identifying and differentiating B. pseudomallei and B. mallei by molecular methods.     

Contribution of gene loss to the pathogenic evolution of Burkholderia pseudomallei and Burkholderia mallei

Burkholderia pseudomallei is the causative agent of melioidosis. Burkholderia thailandensis is a closely related species that can readily utilize l-arabinose as a sole carbon source, whereas B. pseudomallei cannot. We used Tn5-OT182 mutagenesis to isolate an arabinose-negative mutant of B. thailandensis. Sequence analysis of regions flanking the transposon insertion revealed the presence of an arabinose assimilation operon consisting of nine genes. Analysis of the B. pseudomallei chromosome showed a deletion of the operon from this organism. This deletion was detected in all B. pseudomallei and Burkholderia mallei strains investigated. We cloned the B. thailandensis E264 arabinose assimilation operon and introduced the entire operon into the chromosome of B. pseudomallei 406e via homologous recombination. The resultant strain, B. pseudomallei SZ5028, was able to utilize l-arabinose as a sole carbon source. Strain SZ5028 had a significantly higher 50% lethal dose for Syrian hamsters compared to the parent strain 406e. Microarray analysis revealed that a number of genes in a type III secretion system were down-regulated in strain SZ5028 when cells were grown in l-arabinose, suggesting a regulatory role for l-arabinose or a metabolite of l-arabinose. These results suggest that the ability to metabolize l-arabinose reduces the virulence of B. pseudomallei and that the genes encoding arabinose assimilation may be considered antivirulence genes. The increase in virulence associated with the loss of these genes may have provided a selective advantage for B. pseudomallei as these organisms adapted to survival in animal hosts.     

Intracellular survival of Burkholderia pseudomallei.

Burkholderia pseudomallei is the causative agent of melioidosis, a disease being increasingly recognized as an important cause of morbidity and mortality in many regions of the world. Several features of melioidosis suggest that B. pseudomallei is a facultative intracellular pathogen. This study was designed to assess the ability of B. pseudomallei to invade and survive in eukaryotic cells. We have shown that B. pseudomallei has the capacity to invade cultured cell lines, including HeLa, CHO, A549, and Vero cells. We have demonstrated intracellular survival of B. pseudomallei in professional phagocytic cells, including rat alveolar macrophages. B pseudomallei was localized inside vacuoles in human monocyte-like U937 cells, a histiocytic lymphoma cell line with phagocytic properties. Additionally, electron microscopic visualization of B. pseudomallei-infected HeLa cells and polymorphonuclear leukocytes confirmed the presence of intracellular bacteria within membrane-bound vacuoles. B. pseudomallei was found to be resistant to the cationic peptide protamine and to purified human defensin HNP-1.     

Virulence of Burkholderia pseudomallei does not correlate with biofilm formation

Burkholderia pseudomallei is the causative agent of melioidosis but currently the pathogenesis of the disease is still poorly understood. One of the virulent factors of gram-negative bacteria is the ability to produce biofilm to evade host defense. As B. pseudomallei has also been reported to develop the biofilm [1], in the present study, we therefore, quantified the biofilm formation in 50 strains of B. pseudomallei and compared with 50 strains of its avirulent counterpart Burkholderia thailandensis using a modified microtiter-plate test. The results showed that the quantity of biofilm produced by B. pseudomallei was statistically higher (P< 0.01) than that of B. thailandensis (means and SEs of the corrected OD630 were 2.17+/-0.29 and 0.59+/-0.05, respectively). Transmission electron micrographs of the B. pseudomallei strain with high biofilm formation exhibited microcolonies of bacterial cells surrounded by dense extracellular slime matrix comparing with only trace quantity in the low biofilm-producing strain or the biofilm mutants generated by Tn5-OT182 mutagenesis. However, no correlation could be observed between the biofilm formation and virulence, judging from the LD50 values in BALB/c mice. The data obtained with these naturally occurring Burkholderia species and the biofilm mutants are incompatible with the possibility that the biofilm plays a role in the pathogenesis of B. pseudomallei infection.     

CD4+ T-cell immunity to the Burkholderia pseudomallei ABC transporter LolC in melioidosis

Burkholderia pseudomallei causes melioidosis, a disease with a wide range of possible outcomes, from seroconversion and dormancy to sepsis and death. This spectrum of host-pathogen interactions poses challenging questions about the heterogeneity in immunity to B. pseudomallei. Models show protection to be dependent on CD4(+) cells and IFN-γ, but little is known about specific target antigens. Having previously implicated the ABC transporter, LolC, in protective immunity, we here use epitope prediction, HLA-binding studies, HLA-transgenic models and studies of T cells from seropositive individuals to characterize HLA-restricted LolC responses. Immunized mice showed long-lasting memory to the protein, whereas predictive algorithms identified epitopes within LolC that subsequently demonstrated strong HLA class II binding. Immunization of HLA-DR transgenics with LolC stimulated T-cell responses to four of these epitopes. Furthermore, the responsiveness of HLA transgenics to LolC revealed a hierarchy supportive of HLA polymorphism-determined differential susceptibility. Seropositive human donors of diverse HLA class II types showed T-cell responses to LolC epitopes, which are conserved among Burkholderia species including Burkholderia cenocepacia, associated with life-threatening cepacia complex in cystic fibrosis patients and Burkholderia mallei, which causes glanders. These findings suggest a role for LolC epitopes in multiepitope vaccine design for melioidosis and related diseases.     

The use of selective media for the isolation of Pseudomonas pseudomallei in clinical practice

Ashdown's selective-differential agar medium, with or without preenrichment in selective broth, was evaluated for the isolation of Pseudomonas pseudomallei from 1972 clinical specimens obtained from 643 subjects in Northeast Thailand; 226 patients proved to have meliodosis. The use of Ashdown's medium significantly increased the frequency of recovery of P. pseudomallei from sites or specimens with an extensive normal flora (throat, rectum, wounds and sputum) as compared to the recovery on blood and MacConkey agars (p less than 0.01). The isolation frequency from throat, rectal and wound swabs was further increased by the use of the broth pre-enrichment. The colonial morphology of P. pseudomallei on Ashdown's medium was sufficiently characteristic to allow presumptive identification. With the use of these selective media it was possible to culture P. pseudomallei from throat swabs taken from 87% of the patients from whom the organism could also be isolated from corresponding tracheal aspirates or sputum specimens. P. pseudomallei was isolated from rectal swabs taken from 51 patients, the first time that faecal excretion of the organism has been demonstrated in man. The diagnosis of melioidosis would not have been confirmed bacteriologically in eight patients (3.5%) without the use of the selective media. It is suggested that, in areas endemic for melioidosis, all sputum specimens should be cultured on selective media, such as Ashdown's. For the investigation of clinically suspected cases of melioidosis, and for follow-up during treatment of the disease, the use of broth pre-enrichment is recommended for specimens obtained from sites with an extensive normal flora.     

Flagellum-mediated adhesion by Burkholderia pseudomallei precedes invasion of Acanthamoeba astronyxis

In this study we investigated the role of the bacterial flagellum in Burkholderia pseudomallei entry to Acanthamoeba astronyxis trophozoites. B. pseudomallei cells were tethered to the external amoebic surface via their flagella. MM35, the flagellum-lacking fliC knockout derivative of B. pseudomallei NCTC 1026b did not demonstrate flagellum-mediated endocytosis in timed coculture, confirming that an intact flagellar apparatus assists B. pseudomallei entry into A. astronyxis.     

Virulent Burkholderia pseudomallei is more efficient than avirulent Burkholderia thailandensis in invasion of and adherence to cultured human epithelial cells

Burkholderia pseudomallei, a causative agent of melioidosis, is a facultative intracellular gram-negative bacillus that is closely related to its avirulent counterpart, Burkholderia thailandensis. However, pathogenic mechanisms and virulence factors of B. pseudomallei remain elusive. In the present study, we compared the invasiveness, adherence, and replication of B. pseudomallei and B. thailandensis in human respiratory epithelial cells A549. Invasion was determined after 4 h of coculturing using antibiotic protection assay. Adherence was demonstrated by coculturing the cells with fluorescein-labeled bacteria for 1 h and the number of positive cells was analyzed by flow cytometry. The results obtained with this in vitro study demonstrated that compared with its avirulent counterpart, B. pseudomallei is significantly more efficient (P<0.01) in invasion, adherence and inducing cellular damage, as represented by plaque formation.     

Development and evaluation of a real-time PCR assay targeting the type III secretion system of Burkholderia pseudomallei

Here we report on the development of a discriminatory real-time assay for the rapid identification of Burkholderia pseudomallei isolates and the evaluation of this assay for sensitivity against related species and detection in spiked human blood samples. The assay targets a 115-base-pair region within orf2 of the B. pseudomallei type III secretion system gene cluster and distinguishes B. pseudomallei from other microbial species. Assay performance was evaluated with 224 geographically, temporally, and clinically diverse B. pseudomallei isolates from the Centers for Disease Control and Prevention strain collection. This represents the first real-time PCR for rapid and sensitive identification of B. pseudomallei that has been tested for cross-reactivity with 23 Burkholderia mallei, 5 Burkholderia thailandensis, and 35 Burkholderia and 76 non-Burkholderia organisms which have historically presented diagnostic challenges. The assay performed with 100% specificity. The limit of detection was found to be 76 femtograms of DNA (equivalent to 5.2 x 10(3) genome equivalents per ml) in a single PCR. In spiked human blood, the assay could detect as few as 8.4 x 10(3) CFU per ml. This rapid assay is a valuable tool for identification of B. pseudomallei and may improve diagnosis in regions endemic for melioidosis.     

Genome-wide identification and mapping of variable sequences in the genomes of Burkholderia mallei and Burkholderia pseudomallei

Burkholderia mallei and Burkholderia pseudomallei, closely related Gram-negative bacteria, are the causative agents of such serious infectious diseases of humans and animals as glanders and melioidosis, respectively. Despite numerous studies of these pathogens, the detailed mechanisms of their pathogenesis is still poorly understood. One of the serious obstacles to revealing factors responsible for pathogenicity lies in the considerable natural variability of B. pseudomallei and B. mallei, which is also a challenge to development of rapid and efficient diagnostic tools facilitating unambiguous identification of the infectious agents. To gain a deeper insight into B. mallei and B. pseudomallei interspecies divergence and intraspecies polymorphism, we compared the genomes of B. mallei C-5 and B. pseudomallei C-141 strains using a subtractive hybridization technique. A library of DNA fragments specific for B. mallei C-5 and absent from B. pseudomallei C-141 was obtained and analyzed. Some of the differential sequences detected were also not found in the recently sequenced genome of B. pseudomallei K96243. However, a multitude of B. mallei C-5 sequences absent from the B. pseudomallei C-141 genome were detected in the genome of B. pseudomallei K96243. On the other hand, some sequences identified as constituents of the B. mallei C-5 genome were not found in the genome of B. mallei ATCC 23344. Some of the differential DNA fragments displayed similarity to different mobile elements that have not yet been described for B. mallei, whereas the others matched fragments of various prophages, or, when translated into protein sequences, components of active transport systems and different enzymes. A substantial proportion of the differential clones had no database matches either at the nucleotide or amino acid sequence level. The results suggest great genome-wide intra- and interspecies variability of B. mallei and B. pseudomallei. The differences identified may be useful as molecular signatures for identification of B. mallei strains.     

groEL encodes a highly antigenic protein in Burkholderia pseudomallei.

No recombinant protein is available for serodiagnosis of melioidosis. In this study, we report the cloning of the groEL gene, which encodes an immunogenic protein of Burkholderia pseudomallei. Bidirectional DNA sequencing of groEL revealed that the gene contained a single open reading frame encoding 546 amino acid residues with a predicted molecular mass of 57.1 kDa. Basic Local Alignment Search Tool analysis showed that the putative protein encoded by groEL is homologous to the chaperonins encoded by the groEL genes of other bacteria. It has 98% amino acid identity with the GroEL of Burkholderia cepacia, 98% amino acid identity with the GroEL of Burkholderia vietnamiensis, and 82% amino acid identity with the GroEL of Bordetella pertussis. Furthermore, it was observed that patients with melioidosis develop a strong antibody response against GroEL, suggesting that the recombinant protein and its monoclonal antibody may be useful for serodiagnosis in patients with melioidosis and that the protein may represent a good cell surface target for host humoral immunity. Further studies in these directions would be warranted.     

Exposure to Burkholderia pseudomallei induces cell-mediated immunity in healthy individuals

Melioidosis is an emerging tropical infection caused by the intracellular bacterium Burkholderia pseudomallei, and is associated with high mortality rates. Previous studies investigating the prevalence of melioidosis have based conclusions on serological evidence. However, cell-mediated immunity is more relevant for protection against an intracellular pathogen such as B. pseudomallei. This is the first demonstration that exposure to B. pseudomallei may lead to the formation of specific antibodies and the development of cell-mediated immunity in a healthy individual.