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Comparison of Pseudomonas pseudomallei from humans, animals, soil and water by restriction endonuclease analysis.

Pseudomonas pseudomallei isolates from 62 human, 17 animal, 3 soil and 3 water samples were examined by genomic DNA digestion with PstI. Five major (RE I, II, III, IV, V) reproducible restriction patterns were observed, with most (56/62) of the human isolates displaying RE I (30/62), II (5/62), III (15/62), IV (4/62), V (2/62), and the animal (16/17), soil (2/3), water (3/3) isolates showing predominantly RE II profiles. Six human and one soil isolates showed patterns different from those of RE I to V. Restriction endonuclease analysis may be applied in epidemiological studies of melioidosis.

Animals↗

Characterization of Pseudomonas pseudomallei antigens by SDS-polyacrylamide gel electrophoresis and western blot.

Immunological characterization of various Pseudomonas pseudomallei preparations was carried out by SDS-PAGE and Western blot using sera from infected humans and from patients with other bacterial infections. Somatic (SOM) and partially purified cell extracts (PCE) gave more complex SDS-PAGE patterns: M(r) ranged from 86 to 12.7 and 48 to 10 kDa, respectively. The culture-filtrated antigens (CF) from 3 different kinds of synthetic media consisted of fairly simple profiles with common bands M(r) of 40, 26 and 16 kDa. PCE and CF reacted specifically with infected human sera; SOM did not. The components with M(r) of 40 kDa in CF reacted consistently with all infected sera but failed to react with sera infected with Escherichia coli, Enterobacter spp., Klebsiella pneumoniae, Proteus mirabilis, Salmonella spp., Staphylococcus aureus, Streptococcus spp., Pseudomonas aeruginosa and P. stutzeri. This peptide was demonstrated to be a major component in CF thus suggesting its potential for development of immunodiagnostic methods for melioidosis.

Antigens, Bacterial↗

Seroepidemiological survey of hospital-associated populations in Colombo, Sri Lanka.

Almost 1,500 sera from hospital-associated groups in Colombo, Sri Lanka were tested for antibodies against melioidosis, scrub typhus, influenza, and group B arboviruses. A low prevalence of antibodies was found against meliodosis and scrub typhus. Crude prevalence rates of more than 50% were encountered for antibodies against A influenza, and there was no apparent difference in rates when these were analysed according to age, sex, or ethnic background. Influenza B antibodies were more prevalent in older individuals. Antibodies against group B arboviruses were found in all groups tested, and were significantly more prevalent in older Tamils, who has estimated attack rates of more than 5% per year.

Adolescent↗

[Pseudomonas pseudomallei and Pseudomonas mallei--capsule-forming bacteria].

The specific features of the submicroscopic structure of the causative agents of melioidosis and glanders, grown in culture media and at the initial stages of experimental infection in guinea pigs and golden hamsters, were studied with the use of electronic cytochemistry and immunocytochemistry. P. pseudomallei and P. mallei were found to be capsule-forming bacteria. According to the data obtained with the use of electronic cytochemistry, the basis of the capsular substance P. pseudomallei and P. mallei is formed by polysaccharide biopolymers protecting these bacteria from phagocytosis.

Animals↗

Emerging & re-emerging bacterial pathogens in India.

In spite of major successes against infectious diseases in the 20th century, new infectious diseases have emerged and old ones re-emerged in recent decades in different parts of the world. A brief survey of emerging and re-emerging bacterial diseases of public health importance in India is presented in this paper. Plague re-appeared in two outbreaks in Maharashtra and Gujarat in 1994, indicating a breakdown of the public health measures that had prevented its occurrence for several decades. Leptospirosis appears to be on the increase in Kerala, Tamilnadu and the Andamans during the last 2 decades, probably due to increased farming and inadequate rodent control. It is suggested that melioidosis due to the soil organism Burkholderia pseudomallei may be prevalent in many parts of India, but is under-diagnosed and under-reported. Since 1991, a completely new choleragenic Vibrio cholerae, designated 0139 has emerged in southern India and spread to other parts of India and to neighbouring countries, setting in motion the 8th cholera pandemic. Animal anthrax is very common in many parts of India, but human anthrax is recognised in only certain limited locations. In the Chittoor and North Arcot districts, its prevalence had increased in recent years. Since 1990, a multi-drug resistant variety of typhoid fever had been prevalent in many parts of India, caused by Salmonella typhi resistant to chloramphenicol, ampicillin and trimethoprim-sulphamethoxazole. Nosocomial methicillin-resistant Staphylococcus aureus infection seems to be widely prevalent in hospitals in many regions in India, and its prevalence seems to be on the rise. These pathogens pose new threats to public health, and call for appropriate responses. Microbiological expertise and epidemiological surveillance are deficient in the health care and public health systems in India; therefore even infections and diseases that have been under control elsewhere remain prevalent in the country, but are also under-diagnosed and under-reported. Without improving microbiological expertise and application as well as epidemiological skills and practices, emerging and re-emerging diseases may not be recognised, identified or intercepted in their early stages.

Animals↗

Multifactorial pathogenic mechanisms of Burkholderia pseudomallei as suggested from comparison with Burkholderia cepacia.

With the purpose to elucidate the pathogenesis of disease due to Burkholderia pseudomallei some biological and biochemical properties of this species were studied in comparison with B. cepacia, since the difference in the level of virulence between the two species is remarkable despite of their toxonomic closeness. B. pseudomallei was distinct from B. cepacia in the capability to grow under anaerobic conditions, with positive nitrate respiration, excretion of high-molecular polysaccharides into liquid culture, and cytotoxicity against cultured tissue cells. From these observations together with our previous finding that B. pseudomallei can grow and survive in an acidic environment, we suggest multifactorial mechanisms for the pathogenesis of melioidosis due to B. pseudomallei.

Animals↗

Burkholderia pseudomallei infection in the Singapore Armed Forces from 1987 to 1994--an epidemiological review.

Between 1987 and 1994, twenty-three cases of Burkholderia pseudomallei infection (melioidosis) were diagnosed in persons serving in the Singapore Armed Forces. There were four deaths resulting from complications of the infection. Unlike the situation in the general population, where the affected are mainly the elderly with underlying illness, the majority of cases in the Singapore Armed Forces were otherwise fit and healthy young servicemen. Serological surveys have shown the prevalence of the infection in Singapore to be 0.2% in the military as well as civilian population. As physical contact with soil is an unavoidable part of military training, military personnel continue to be at risk of exposure to this soil-related disease.

Adolescent↗

Tropical infection and the lung.

The term "tropical lung" has been used to describe the lungs that are vulnerable to those indigenous diseases that occur with particular prevalence in tropical countries. International travel, student and cultural exchanges and changing immigration patterns are insidiously transforming the face of medicine in Europe and other developed countries. The climates of tropical countries provide an ideal environment for pathogenic organisms, and their vectors and intermediate hosts, to flourish. In an average out-patient department, 20-40% of patients have been seen with respiratory complaints, and 20-30% of hospital medical admissions are for disorders predominantly affecting the lungs. Pulmonary tuberculosis is common in tropical countries as well as the rest of the world. The other principal environmentally related tropical pulmonary diseases are melioidosis, paragonimiasis, amoebiasis, leptospirosis, gnathostomiasis and tropical eosinophilia. It is essential that the practising clinician be aware of the increasing prevalence of various new and exotic tropical lung diseases. Clinicians in developing countries can now use their clinical skills together with recent developments in immunology, molecular biology and biochemistry to improve the diagnostic accuracy and therapeutic effectiveness related to tropical lung infections. Treatment, if inappropriate, may not only be worthless but, in many cases, extremely harmful and even fatal.

Amebiasis↗

Specific binding of Burkholderia pseudomallei cells and their cell-surface acid phosphatase to gangliotetraosylceramide (asialo GM1) and gangliotriaosylceramide (asialo GM2).

Specific binding between bacterial cells and host tissue is an early step of the pathogenesis of infection. Burkholderia pseudomallei cells, the causative micro-organisms of melioidosis, were demonstrated to bind specifically to tissue glycolipids (asialo GM1 and asialo GM2) by solid phase binding assay on thin layer chromatograms. The detection limit was around 400 pmol of the glycolipids. Acid phosphatase purified from the culture filtrate of B. pseudomallei was tested for such binding properties, and the same results were obtained. According to our previous studies, the enzyme is a glycoprotein located on the cell surface, and hydrolysed tyrosine phosphate most actively among the substrates so far tested. The mode of binding between the enzyme and the glycolipids was analyzed by comparison of binding levels among three samples different in protein content, sugar content and specific phosphatase activities per protein and sugar residue. The results suggested the possibility of a receptor-ligand relationship between the bacterial enzyme and the host-cell glycolipids (asialo GM).

Acid Phosphatase↗

An ultrastructural study of the phagocytosis of Burkholderia pseudomallei.

Burkholderia pseudomallei causes melioidosis and is believed to be an intracellular pathogen in human and animal disease. The uptake of B. pseudomallei by mouse peritoneal macrophages and cells in tissue culture was examined by electron microscopy. In all the systems studied B. pseudomallei were phagocytosed and apparently inhibited the normal processes of intracellular killing. Destruction of the phagosome membrane occurred and the bacteria escaped into the cytoplasm.

Animals↗

[Isolation and primary characteristics of a Pseudomonas (Burkholderia) pseudomallei plasmid].

Plasmid screening of reference Pseudomonas pseudomallei strains isolated from patients and animals revealed cryptic plasmids with different molecular weights in 30 strains. Plasmids were investigated by restriction analysis and DNA-DNA hybridization. Cryptic plasmids were denoted as pPM1, pCM2, pCM3, and pCM4. The presence of plasmids in melioidosis agent permits their use for intraspecies typing of strains and for genetic studies.

Animals↗

A simple method to detect and differentiate Burkholderia pseudomallei and Burkholderia thailandensis using specific flagellin gene primers.

We have previously shown that Burkholderia pseudomallei, the causative pathogen of melioidosis, may be discriminated from the closely related non-pathogenic species Burkholderia thailandensis by the presence of a 15 base pair deletion in the flagellin gene of B. thailandensis. Using specific flagellin gene primers flanking the distinctive region, PCR products of 191 and 176 bp in size were detected for B. pseudomallei and B. thailandensis, respectively. The sensitivity of detection is 20-80 colony forming units/reaction of B. pseudomallei and B. thailandensis cell suspension. To mimic the expected environmental situation, mixed populations of the two species were analyzed. The results showed that the PCR-based method could be use to distinguish the two species in a duplex reaction. In addition, we have developed a simplified method for direct PCR-based detection from soil samples. The result indicated that about 200 colonies of bacteria per reaction could be detected. This method can be applied to epidemiological studies, especially for investigating the ecological relationship between these two species in the environment.

Asia, Southeastern↗

Fever of unknown origin in Taiwan.

BACKGROUND: Fever of unknown origin (FUO) is a challenging problem worldwide. There was no prospective study of FUO in the past two decades in Taiwan. A prospective study was conducted. MATERIALS AND METHODS: The prospective study was undertaken from March 2001 to May 2002. All patients fulfilling the modified criteria for FUO, either admitted, referred or consulted in a medical center in southern Taiwan, were enrolled for analysis. RESULTS: A total of 94 cases met the criteria of FUO. The final diagnoses of FUO consisted of 54 infectious diseases (57.4%), 8 hematologic/neoplastic (8.5%), 7 noninfectious inflammatory (7.4%), 8 miscellaneous (8.5%) and 17 undiagnosed (18.1%) cases. The single most common cause of FUO was tuberculosis. Some infectious diseases, such as rickettsiosis and melioidosis, were rarely reported in western countries. Three patients with hemophagocytotic syndrome without ascertainable etiologies were present with FUO in this study. Between the patients with and those without a final diagnosis, the short-term survival (3 months) was compared by the Kaplan-Meier analysis, which revealed no difference. CONCLUSIONS: Mycobacteriosis is still the leading cause of FUO in Taiwan and it is important to identify this treatable disease from all causes of FUO. This study has showed geographical variation among the studies of FUO.

Adolescent↗

Isolation of Pseudomonas pseudomallei from soil in north-eastern Thailand.

In order to optimize the recovery from soil of Pseudomonas pseudomallei, the cause of melioidosis, 3 selective broths were compared. A basal salt solution containing L-threonine (TBSS) performed significantly better than trypticase soy broth containing crystal violet and colistin 50 mg/L (CVC50), both in isolation rate and suppression of overgrowth of other organisms, but the addition of colistin to TBSS gave the best results overall. In a survey in north-eastern Thailand, P. pseudomallei was recovered from 114 (68%) of the 167 sites tested. A detailed study of a single rice farm showed that the isolation rate increased with depth of soil sample, and P. pseudomallei could still be isolated during the dry season, although only from moist soil in areas where other crops were cultivated and around the water source.

Animals↗

Biophysical characterization of the catalytic domain of guanine nucleotide exchange factor BopE from Burkholderia pseudomallei.

BopE is a type III secreted protein from Burkholderia pseudomallei, the aetiological agent of melioidosis. Like its Salmonella homologues SopE and SopE2, BopE is a guanine nucleotide exchange factor for Rho GTPases. It is thought that, in order to be secreted by the type III system, proteins must be unfolded or only partially folded. As part of a study of B. pseudomallei virulence proteins, we have expressed, purified and characterized the catalytic domain of BopE (amino acids 78-261). Analytical ultracentrifugation experiments in conjunction with analytical size exclusion chromatography show that BopE(78-261) is monomeric in aqueous solution. CD spectroscopy indicates that the protein is predominantly alpha-helical, with predicted secondary structure composition of 59% alpha-helix and 7% beta-strand. NMR spectroscopy confirms that BopE(78-261) adopts a single, stable conformation. In differential scanning calorimetry experiments, thermal denaturation of BopE(78-261) (T(m) 52 degrees C) is reversible. Also, the secondary structure composition of BopE(78-261) changes little over a range of pH values from 3.5 to 10.5. BopE may therefore fold spontaneously to a functional form upon secretion into the host cell cytoplasm, and retains a native or native-like fold in varied environments. These properties are likely to be advantageous for a secreted bacterial effector protein.

Amino Acid Sequence↗

Detection and differentiation of Burkholderia pseudomallei, Burkholderia mallei and Burkholderia thailandensis by multiplex PCR.

Burkholderia pseudomallei, a Gram-negative bacterium that causes melioidosis may be differentiated from closely related species of Burkholderia mallei that causes glanders and non-pathogenic species of Burkholderia thailandensis by multiplex PCR. The multiplex PCR consists of primers that flank a 10-bp repetitive element in B. pseudomallei and B. mallei amplifying PCR fragment of varying sizes between 400-700 bp, a unique sequence in B. thailandensis amplifying a PCR fragment of 308 bp and the metalloprotease gene amplifying a PCR fragment of 245 bp in B. pseudomallei and B. thailandensis. The multiplex PCR not only can differentiate the three Burkholderia species but can also be used for epidemiological typing of B. pseudomallei and B. mallei strains.

Animals↗

The Burkholderia pseudomallei RpoE (AlgU) operon is involved in environmental stress tolerance and biofilm formation.

Burkholderia pseudomallei, the causative agent of melioidosis, can be isolated from soil and water. To persist, adapt and survive within and outside their human host, bacteria rely on regulatory mechanisms that allow them to respond rapidly to stressful situations. We have examined the possible role of B. pseudomallei alternative sigma factor sigma(E) (RpoE) in the stress response and found that rpoE and its putative regulators (bprE-rseB-mucD) are transcribed in a single transcriptional unit. Inactivation of the rpoE operon changed the B. pseudomallei phenotype. Changes included increased susceptibility to killing by menadione and H(2)O(2), susceptibility to high osmolarity, reduced ability to form biofilms, and reduced survival in macrophage J774A.1. Therefore, we conclude that rpoE controls gene expression that contributes, at least in part, to B. pseudomallei adaptation to adverse environmental conditions.

Adaptation, Physiological↗

Genome-wide expression analysis of iron regulation in Burkholderia pseudomallei and Burkholderia mallei using DNA microarrays.

Burkholderia pseudomallei and B. mallei are the causative agents of melioidosis and glanders, respectively. As iron regulation of gene expression is common in bacteria, in the present studies, we have used microarray analysis to examine the effects of growth in different iron concentrations on the regulation of gene expression in B. pseudomallei and B. mallei. Gene expression profiles for these two bacterial species were similar under high and low iron growth conditions irrespective of growth phase. Growth in low iron led to reduced expression of genes encoding most respiratory metabolic systems and proteins of putative function, such as NADH-dehydrogenases, cytochrome oxidases, and ATP-synthases. In contrast, genes encoding siderophore-mediated iron transport, heme-hemin receptors, and a variety of metabolic enzymes for alternative metabolism were induced under low iron conditions. The overall gene expression profiles suggest that B. pseudomallei and B. mallei are able to adapt to the iron-restricted conditions in the host environment by up-regulating an iron-acquisition system and by using alternative metabolic pathways for energy production. The observations relative to the induction of specific metabolic enzymes during bacterial growth under low iron conditions warrants further experimentation.

Adaptation, Physiological↗