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PubMed1985

[The polysaccharide component of Pseudomonas pseudomallei slime].

The polysaccharide component contained in the slime of the causative agent of melioidosis was obtained. This component was found to be a biopolymer, mainly of the carbohydrate nature, consisting of galactose, glucose, mannose, rhamnose and two unidentified carbohydrates. The slime polysaccharide component contained two thermostable and acid-resistant antigens. The action of alkali led to the loss of one of these antigens. Rabbit antisera to the preparations of the slime polysaccharide component with titers of 1:64 to 1:256, determined in the immunodiffusion test, were obtained. In the precipitation test the slime polysaccharide component reacted with antisera from sick experimental animals, thus confirming the suggestion of its secretion in the process of the development of melioidosis infection.

Antigens, Bacterial↗
PubMed1978

[Phagocytability of Pseudomonas pseudomallei].

Experiments were conducted on the cell culture of macrophages of animals significantly differing by the extent of resistance to melioidosis; the presence of correlation between the extent of the animal natural immunity and the intensity of dying of the microbes in the test system was demonstrated. The causative agent of melioidosis proved to be more resistant to phagocytosis with guinea pig macrophages than E. coli. Ps. aeruginosa and A. mallei. It was impossible to establish any relationship between the efficacy of phagocytosis by animal macrophages and the virulence or morphology of the colonies in the Ps. pseudomallei species.

Animals↗
PubMed1995

Localised melioidotic osteomyelitis.

Melioidosis is an uncommon infection caused by a Gram-negative bacillus, Pseudomonas pseudomallei. Only a few case reports of orthopaedic infection have been published in English, and most were of isolated septic arthritis or secondary to melioidosis of another organ. We have reviewed ten patients with localised melioidotic osteomyelitis; six had underlying conditions. We discuss the importance of obtaining a bacteriological diagnosis, and of surgical debridement as well as appropriate antibiotic therapy.

Adult↗
PubMed2003

Liver abscess caused by Stenotrophomonas maltophilia: report of a case.

We report the case of a melioidosis-like abscess of the liver caused by Stenotrophomonas (Xanthomonas) maltophilia infection in a Chinese man living in Hungary. Although this appears to be the first documentation of a liver abscess of this origin in a nonimmunocompromised patient, our case report demonstrates that this common facultative pathogen can also cause liver abscess and sepsis. After repeated negative blood cultures, histological examinations of liver biopsies suggested the possibility of chronic melioidosis, but the microbiological examination performed directly on the same specimen identified a Stenotrophomonas maltophilia infection. Surgical drainage was performed and sulphamethoxazole/trimethoprim therapy was commenced, after which the patient recovered fully. The facultative pathogen S. maltophilia, which most often causes nosocomial infections, may cause severe sepsis and liver abscess. We wish to draw attention to the fact that the antibiotic sensitivity of S. maltophilia is not necessarily the same in vivo and in vitro. This can create difficulties in both diagnosis and treatment.

Adult↗
PubMed2002

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.

Anti-Bacterial Agents↗
PubMed2006

A live experimental vaccine against Burkholderia pseudomallei elicits CD4+ T cell-mediated immunity, priming T cells specific for 2 type III secretion system proteins.

Burkholderia pseudomallei is the etiological agent of melioidosis, a serious human disease for which no vaccine is available. Immunization of susceptible BALB/c mice with the live attenuated mutant B. pseudomallei ilvI (referred to as "2D2") generated significant, although incomplete, immunity. Splenic B. pseudomallei-specific T cells, detected in immunized mice, proliferated and produced interferon-gamma in vitro in response to dead bacteria. Assessment of T cell antigen specificity indicated that subpopulations of B. pseudomallei-reactive T cells were responsive to BopE, a type III secretion system (TTSS) effector protein, and to a lesser extent to BipD, a TTSS translocator protein. Increased survival of severe combined immunodeficient mice adoptively transferred with T cells from immunized mice, compared with that of naive T cell recipients, demonstrated that immunization with 2D2 generated T cell-mediated immunity. CD4+ and CD8+ cell depletion studies demonstrated that CD4+ cells, but not CD8+ cells, mediated this protection in vivo. Thus, CD4+ T cells can mediate vaccine-induced immunity to experimental melioidosis.

Animals↗
PubMed2004

Development of 5' nuclease real-time PCR assays for the rapid identification of the burkholderia mallei//burkholderia pseudomallei complex.

Burkholderia pseudomallei is the causative agent of melioidosis and was classified as a biologic agent by the Centers for Disease Control and Prevention (Atlanta, GA). Acute melioidosis has a case fatality rate of >40%, and septicemia is fatal in up to 90%. The aim of the study was to design 5'-nuclease real-time PCR assays for the rapid and reliable identification of the B. mallei/B. pseudomallei complex. Real-time PCR assays using TaqMan probes targeting the 16S rDNA and fliC were developed on an ABI Prism 7000 sequence detection system (Applied Biosystems, Foster City, CA). Specificity was assessed with 64 B. pseudomallei, nine B. mallei, 126 other Burkholderia strains of 29 species, and 45 clinically relevant non-Burkholderia organisms. Sensitivity, specificity, and positive and negative predictive value of the assays were 100%. Discrimination between B. pseudomallei and B. mallei, an organism which can be regarded as a clone of B. pseudomallei, could not be achieved. A probit analysis revealed that 7.5 and 52 genome equivalents (GE) of B. pseudomallei could be detected using the fliC and the 16S rDNA assays (P = .05), respectively. In spiked blood samples, the detection limit was approximately 300 and 3.000 GE for fliC and the 16S rDNA, respectively. In conclusion, we recommend the simultaneous use of the 16S rDNA and fliC real-time PCR assays for the rapid and specific identification of the B. mallei/B. pseudomallei complex in positive blood cultures or from suspicious bacterial colonies allowing the early onset of appropriate antibiotic therapy.

Animals↗
PubMed1998

Antigenic differences between clinical and environmental isolates of Burkholderia pseudomallei.

Burkholderia pseudomallei is a free-living organism that causes the potentially lethal tropical infection melioidosis. The disease is endemic in many parts of eastern Asia and northern Australia. The presence of two distinct biotypes in soil can be reliably distinguished by their ability to assimilate L-arabinose. Whereas some soil isolates could utilize this substrate (Ara+), the remaining soil isolates and all clinical isolates tested so far could not (Ara-). Only the Ara- isolates were virulent in animal models. We have raised a murine monoclonal antibody (MAb) that can readily distinguish Ara- from Ara+ biotypes. The MAb reacted with a high molecular weight component present only on the Ara- biotype. With this MAb, clinical and soil Ara- isolates gave identical positive reactions in agglutination, immunofluorescence, ELISA and immunoblot assays. Using these same assay systems, the soil Ara+ biotype did not react with the MAb. Similar but distinct immunoblot patterns were also noted when these two Ara biotypes were probed with sera from patients with melioidosis or with polyclonal immune rabbit sera. These data showed that the Ara- biotype from both clinical and environmental isolates is antigenically different from its Ara+ environmental counterpart. The SDS-PAGE protein and lectin-binding profiles of both groups of Ara- isolates were also found to be different from those of the Ara+ biotype.

Animals↗
PubMed1995

Purification and characterization of an exopolysaccharide of Burkholderia (Pseudomonas) pseudomallei.

Burkholderia pseudomallei (basonym Pseudomonas pseudomallei) is the causative organism of melioidosis, a disease which is recognized as a major public health problem primarily in Southeast Asia and Northern Australia. In this paper, we report on the identification, purification, and characterization of a species-specific exopolysaccharide of B. pseudomallei. After immunization of mice with a B. pseudomallei strain exhibiting mucoid growth characteristics, we isolated an immunoglobulin G1 monoclonal antibody (MAb) (3015) with specificity for a carbohydrate structure as determined by immunoblotting following sodium dodecyl sulfatepolyacrylamide gel electrophoresis. Electron microscopy studies with MAb 3015 revealed reactivity with an exopolysaccharide with a capsule-like appearance in the immunizing strain. All of the mucoid and nonmucoid B. pseudomallei strains tested from geographically different tropical regions were recognized by MAb 3015 in an enzyme-linked immunosorbent assay or immunoblot, indicating that the exopolysaccharide is constitutively expressed among this species. Intensive testing for cross-reactivity including members of all the Pseudomonas rRNA groups showed no cross-reactivity except in the case of the closely related species Burkholderia mallei. A protocol for purification of the exopolysaccharide which is based principally on mechanical separation from the cell surface followed by repetitive ethanol precipitation steps and finally affinity chromatography using MAb 3015 was established. The exopolysaccharide yielded was of high purity. Gel permeation chromatography was performed, and the molecular mass was estimated to be > 150 kDa. Sera from patients with melioidosis were strongly reactive with the purified exopolysaccharide, indicating its in vivo expression and immunogenicity in natural infection. The diagnostic value of the exopolysaccharide and its role in the pathogenesis of disease must still be determined.

Animals↗
PubMed1996

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.

Animals↗
PubMed1998

Purification and characterization of a cytotoxic exolipid of Burkholderia pseudomallei.

Burkholderia pseudomallei is the causative agent of melioidosis, an infectious disease, which is increasingly recognized as an important public health problem in various tropical regions. This study describes the identification and characterization of a heat-stable extracellular toxin of B. pseudomallei. After cultivation of B. pseudomallei in liquid media, the heated cell-free supernatant was concentrated by ultrafiltration. The concentrate exhibited a cytotoxic and hemolytic activity which showed remarkable resistance against alkaline and acidic treatments. For further purification, reversed-phase chromatography using a fast-performance liquid chromatography system was performed. After elution with an acetonitrile gradient, a single cytotoxic and hemolytic peak was detected. Structural characterization of the toxin was performed by a combination of mass spectrometric and nuclear magnetic resonance spectroscopic techniques. A highly purified glycolipid, 2-O-alpha-L-rhamnopyranosyl-alpha-L-rhamnopyranosyl-beta-hydroxytetradec anoyl-beta-hydroxytetradecanoate (Rha-Rha-C14-C14), with a molecular mass of 762 Da was identified. The purified exolipid showed a time- and dose-dependent cytotoxic effect on phagocytic (HL60) and nonphagocytic (HeLa) cell lines. In addition, a time- and dose-dependent hemolysis of erythrocytes from various species was observed. The toxin structure makes a detergentlike action most probable. Interestingly, the cytotoxic and hemolytic activities of the glycolipid could be neutralized by albumin. Future studies will concentrate on the role of this exolipid as a virulence factor in the pathogenesis of melioidosis.

Animals↗
PubMed2006

Use of a variable amplicon typing scheme reveals considerable variation in the accessory genomes of isolates of Burkholderia pseudomallei.

Melioidosis, a disease caused by the bacterium Burkholderia pseudomallei, is endemic in southeast Asia and northern Australia. We used suppression subtractive hybridization (SSH) to identify sequences that varied between two B. pseudomallei isolates from Australia and determined the distribution of 45 SSH-derived sequences among a panel of B. pseudomallei and B. thailandensis isolates. Sequences exhibiting variable prevalence were included in a variable amplicon typing (VAT) scheme designed to score the presence or absence of 14 PCR amplicons. VAT analysis was carried out with 48 isolates from Thailand, which were typed by multilocus sequence typing (MLST), and 44 isolates from Australia of known MLST type. The VAT scheme could be used to divide the 48 isolates from Thailand into 23 VAT types and the 44 isolates from Australia into 36 VAT types. Some of the sequences included in the VAT scheme were more commonly PCR positive among isolates from Australia than among isolates from Thailand, and vice versa. No isolate from Australia was PCR positive for genomic island 11 or a putative transposase sequence, whereas four SSH-derived sequences were far more prevalent among the Australian isolates. Analysis based on the VAT scheme indicated that the isolates clustered into groups, some of which were mainly or exclusively from one geographical origin. One cluster included Australian isolates that were mostly associated with severe disease, including rare neurological melioidosis, suggesting that the content of the accessory genome may play an important role in determining the clinical manifestation of the disease.

Bacterial Typing Techniques↗
PubMed2000

Antigenic heterogeneity of lipopolysaccharide among Burkholderia pseudomallei clinical isolates.

Burkholderia pseudomallei (BP) causes melioidosis, a potentially fatal human infection in the tropics. Clinical isolates from different geographical locations have similar morphological and biochemical characteristics. Although BP has been reported to possess 2 types of lipopolysaccharide (LPS) differing in the chemical structure of their O-polysaccharide (O-PS) component, earlier report demonstrated that the clinical strains exhibited identical LPS moieties. Recently, we reported antigenic similarity between the pathogenic (Ara-) and nonpathogenic (Ara+) biotypes. However, a few clinical isolates showed atypical SDS-PAGE profiles. In this study, LPS from 739 BP isolated from patients and animals in different geographical areas were extracted by proteinase K digestion method. Their SDS-PAGE profiles and their immunoreactivities with patients' sera and monoclonal antibody (MAb) to LPS were analyzed. The isolates showed 3 LPS patterns differing in the number and electrical mobility of bands in silver-stained gel. A majority of BP (711) isolates exhibited identical typical ladder pattern, 21 isolates showed atypical ladder pattern and 7 isolates did not exhibit ladder appearance. However, all LPS preparations exhibited similar endotoxic activity as determined by Limulus amebocyte lysate assay. On the other hand, there were no immunological cross reactivity between typical and atypical LPS, as judged from Western blot against homologous and heterologous sera from melioidosis patients from whom the typical and atypical LPS were isolated. Nevertheless, a Western blot profile of the typical LPS showed some variations when probed with MAb against BP LPS (9D5). Heat-killed bacteria from all LPS groups could similarly activate mouse macrophage cell line to produce nitric oxide (NO) and inducible NO synthase (iNOS).

Animals↗
PubMed2001

Diagnostic role of pleural fluid adenosine deaminase in tuberculous pleural effusion.

Between June 1998 and June 2000, 132 consecutive patients with symptomatic exudative lymphocytic pleural effusion were studied to evaluate the diagnostic role of pleural fluid adenosine deaminase (ADAPF) levels. The mean age was 52.2 (SD 16.3) years. The male to female ratio was 1.4:1. The analysis of ADAPF levels was measured base on Giusti's method. Tuberculous pleural effusion was diagnosed in 50 patients (37.9%). Another 59 patients (44.7%) had malignancies, 23 patients (17.4%) had miscellaneous other etiologies (including; 19 with chronic inflammations, 3 with melioidosis, and 1 with systemic lupus erythrematosus). The percentages of pleural fluid lymphocytes and pleural fluid protein in the tuberculous pleural effusion were similar to those with malignancies, but higher than those in the miscellaneous group. The mean value of ADAPF in the tuberculosis group was 93.2 (SD 56.5) U/l, which was significantly higher than for the malignancy and miscellaneous groups (p<0.05, one-way ANOVA). The mean values of ADAPF in the malignancy group were 36.7 (SD 39.2) U/l, and 31.3 (SD 23.4) U/l in miscellaneous group. Three patients were diagnosed with melioidosis and had ADAPF levels of 15, 46.9, and 49.8 U/l, respectively. One patient with systemic lupus erythrematosus had ADAPF levels of 24.1 U/l. A receiver operating characteristic (ROC) curve identified ADAPF level of 48 U/l as the best cut-off value, which in turn yielded a sensitivity of 80% (95% CI, 73 to 87%) and specificity of 80.5% (95% CI, 73.6 to 87.4%). The positive and negative predictive values at this cut-off value were 71.4% and 86.8%, respectively. The likelihood ratios for the diagnosis of tuberculous pleural effusion in patients with ADAPF levels less than 45 U/ l were 1:4, between 45 and 100 U/l were 5:2, and greater than 100 U/l were 7:1. We concluded that ADAPF levels are a useful diagnostic test for tuberculous pleural effusion. In addition, The analyis of ADA levels can be done simply, quickly, and cheaply.

Adenosine Deaminase↗
PubMed1991

Pulmonary infections in immigrants and refugees.

Immigrants and refugees are susceptible to a wide array of pulmonary diseases, including familiar infections that are acquired after entry and unusual infections that are imported from abroad. The challenges are great; tuberculosis (TB) is now 13 times more prevalent among immigrants than among the general US population. Melioidosis, paracoccidioidomycosis, and paragonimiasis are often mistaken for TB. Pulmonary disease caused by parasites is uncommon and particularly perplexing unless a peripheral blood eosinophilia suggests helminthic infection. TB, melioidosis, fungal infections, and strongyloidiasis may remain inapparent for years and then produce devastating illness in the setting of immunosuppressive therapy or the acquired immunodeficiency syndrome. Proper health care of immigrants requires an understanding of unusual exposures and infections and should include preventive measures as well.

Acquired Immunodeficiency Syndrome↗
PubMedDate not supplied

[Transport medium for pathogenic pseudomonads].

A transporting medium was designed for the transportation and preservation for 5 to 7 days of material infected with agents of malleus and melioidosis. Foreign contaminating microorganisms gradually die in this medium, whereas solitary cells of malleus and melioidosis agents (100 bacterial cells per 0.1 ml) survive.

Burkholderia pseudomallei↗
PubMedDate not supplied

[A methodology for determining reservoirs of the causative agents of sapronoses].

Comparative ecological strategy for choosing the causative agents of melioidosis and glanders, pseudotuberculosis and plague after McArthur [correction of Macartura] and Wilson were studied. The kinetic analysis of the populations of these bacilli in the steppe neocultured chestnut soil formed a basis of the study with the consideration of data on their growth and death in artificial culture media. To identify the types of selection, the authors defined the rate of growth, the level of population stabilization, the dependence of the object growth rate on their population density, the survival capacity under competitive conditions. The kinetic analysis indicated that the causative agents of melioidosis and pseudotuberculosis have all necessary potentials for active soil existence and this substrate may be considered to be their reservoir; the plague bacillus has no such potentials and the telluric theory of plague does not withstand experimental tests. It is concluded that to define a selection strategy may be regarded as a new guideline for studying and identifying the reservoirs of causative agents of sapronoses and infections with the supposed sources of non-animal origin.

Bacteriological Techniques↗
PubMed2003

Effects of soil pH, temperature and water content on the growth of Burkholderia pseudomallei.

Optimum conditions were determined for the growth of Burkholderia pseudomallei in natural soils or waters. It grows better in paddy soil, crop-covered and fallow field than in fresh and salty water. Although the optimal temperature and pH for the growth were 37 or 42 degrees C, and 6.5 or 7.5 in an environmental-mimicking soil medium, this bacterium can still grow at 4 degrees C, which was suggested to be related with the occurrence of melioidosis in some cold areas. In soil media with water content < 15. B. pseudomallei did not grow until 60 d of incubation, suggesting that water contents of soils in which it dwelled would be one important factor in determining the growth rate.

Bacteriological Techniques↗