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PubMed2002

Single gene target bacterial identification. groEL gene sequencing for discriminating clinical isolates of Burkholderia pseudomallei and Burkholderia thailandensis.

Proper identification of Burkholderia pseudomallei and Burkholderia thailandensis is crucial in guiding clinical management of patients with suspected melioidosis, as more than 99% of cases of melioidosis are caused by B. pseudomallei, whereas B. thailandensis is only responsible for causing less than 1% of the cases. However, the difference between the 16S ribosomal RNA gene sequences of B. pseudomallei and that of B. thailandensis is only 1%, and is therefore not discriminative enough for distinguishing the 2 species confidently. In this study, we amplified and sequenced the groEL genes of 7 strains of B. thailandensis and 6 strains of B. pseudomallei, and compared the sequences with 7 other groEL gene sequences of Burkholderia species. BLAST analysis revealed that the putative protein encoded by the groEL gene of B. thailandensis has 99.6%, 99.5%, 98.4%, 98.5%, and 96.5% amino acid identity with the groEL of B. pseudomallei, B. mallei, B. cepacia, B. vietnamiensis, and B. fungorum respectively. The amino acid sequences of GroEL of the strains of B. thailandensis and B. pseudomallei all showed >99.5% amino acid identity with each other. The nucleotide sequence of the groEL gene of any of the strains of B. thailandensis showed >99.8% nucleotide identity with that of any of the other strains of B. thailandensis, and the nucleotide sequence of the groEL gene of any of the strains of B. pseudomallei showed >99.5% nucleotide identity with that of any of the other strains of B. pseudomallei. However, the nucleotide sequence of any of the strains of B. thailandensis showed <97.6% nucleotide identity with any of the strains of B. pseudomallei. The amino acid sequences of GroEL of the 20 strains of Burkholderia species all showed >96% amino acid identity with each other. Furthermore, the nucleotide sequence of the groEL genes of the 2 strains of B. cepacia showed >99.5% nucleotide identity with each other, and the nucleotide sequence of the groEL gene of B. mallei showed >99.5% nucleotide identity with any of the strains of B. pseudomallei. The groEL gene sequence is therefore good for distinguishing between B. thailandensis and B. pseudomallei, and the GroEL amino acid and groEL nucleotide sequences of this single gene locus may potentially be useful for a 2-tier hierarchical identification of medically important Burkholderia at the genus and species levels respectively.

Base Sequence↗
PubMed1994

Pseudomonas pseudomallei isolates collected over 25 years from a non-tropical endemic focus show clonality on the basis of ribotyping.

Between 1966 and 1991, melioidosis, a disease caused by Pseudomonas pseudomallei that is mostly confined to tropical regions, occurred in farm animals and a farmer in temperate south-west Western Australia. Using an Escherichia coli probe containing a ribosomal RNA operon, P. pseudomallei DNA from isolates from 8 animals, a soil sample and the human case showed an identical ribotype on Southern blotting. The ribotype was different from the 3 commonest ribotypes seen in tropical Australia. This molecular typing supports the theory of clonal introduction of P. pseudomallei into a non-endemic region, with environmental contamination, local dissemination and persistence over 25 years. As melioidosis is often fatal in humans, such persistence in a temperate region is cause for concern.

Animals↗
PubMed2002

An Inv/Mxi-Spa-like type III protein secretion system in Burkholderia pseudomallei modulates intracellular behaviour of the pathogen.

Burkholderia pseudomallei is the causative agent of melioidosis, a serious infectious disease of humans and animals that is endemic in subtropical areas. B. pseudomallei is a facultative intracellular pathogen that may invade and survive within eukaryotic cells for prolonged periods. After internalization, the bacteria escape from endocytic vacuoles into the cytoplasm of infected cells and form membrane protrusions by inducing actin polymerization at one pole. It is believed that survival within phagocytic cells and cell-to-cell spread via actin protrusions is required for full virulence. We have studied the role of a putative type III protein secretion apparatus (Bsa) in the interaction between B. pseudomallei and host cells. The Bsa system is very similar to the Inv/Mxi-Spa type III secretion systems of Salmonella and Shigella. Moreover, B. pseudomallei encodes proteins that are very similar to Salmonella and Shigella Inv/Mxi-Spa secreted proteins required for invasion, escape from endocytic vacuoles, intercellular spread and pathogenesis. Antibodies to putative Bsa-secreted proteins were detected in convalescent serum from a melioidosis patient, suggesting that the system is functionally expressed in vivo. B. pseudomallei mutant strains lacking components of the Bsa secretion and translocation apparatus were constructed. The mutant strains exhibited reduced replication in J774.2 murine macrophage-like cells, an inability to escape from endocytic vacuoles and a complete absence of formation of membrane protrusions and actin tails. These findings indicate that the Bsa type III secretion system plays an essential role in modulating the intracellular behaviour of B. pseudomallei.

Adhesins, Bacterial↗
PubMed2002

Primary melioidotic prostatic abscess: presentation, diagnosis and management.

INTRODUCTION: In South-East Asia and Northern Australia, melioidosis (infection with Burkholderia pseudomallei) is a known cause of severe community-acquired sepsis. However, melioidosis presenting primarily as prostatic abscesses is very rare. METHODS: The presenting features, investigations and management outcome of five patients who developed melioidotic prostatic abscesses from 1997 to 2000 were reviewed in the present study. RESULTS: The mean age at presentation was 53 years (range: 29-69). Old age and diabetes mellitus were predisposing factors. All patients had a fever of at least 38.5 degrees C and presented with obstructive urinary symptoms culminating in urinary retention. Presence of prostatic abscess was demonstrated by transrectal ultrasound in all cases. The abscesses were drained with transurethral resection of the prostate. One patient required re-resection while another patient developed severe septic shock requiring intensive care and -inotropic support. There was no mortality in our series. CONCLUSIONS: Elderly diabetic men presenting with fever and urinary tract obstruction in endemic areas may harbour an unusual but potentially life threatening melioidotic prostatic abscess. Transrectal ultrasound and bacteriological confirmation are mandatory. Prompt surgical drainage coupled with appropriate antibiotics are keys to a favourable outcome.

Abscess↗
PubMed2003

An unusual cause of diabetic ketoacidosis and fulminant septicaemia.

BACKGROUND: Diabetic ketoacidosis (DKA) is a common medical emergency. Resistant and recurrent DKA can be due to underlying infection, and a detailed travel history may be important in determining the cause in such cases. We report here a case of unusual DKA and fulminant septicaemia in a Caucasian male with Type 1 diabetes 2 years after returning from living in Thailand. CASE REPORT: A 39-year-old Caucasian male was diagnosed with Type 1 diabetes whilst working in Thailand where he also subsequently developed a cavitating lung lesion diagnosed locally as pulmonary tuberculosis. Two years after returning to the UK he was admitted with DKA and septicaemia. Despite correction of his DKA his condition deteriorated and he developed a fluid collection anterior to the left hip on computed tomography scanning. Blood and fluid aspirate cultures confirmed a diagnosis of melioidosis, a rare fulminant septicaemia in the UK, but endemic in South-east Asia and tropical Australia. Full recovery followed changing antibiotics to intravenous ceftazidime with no relapse 3 years after acute episode. CONCLUSIONS: Physicians as well as microbiologists should consider melioidosis in anyone presenting with septicaemia and/or resistant DKA, especially if the history includes travel to endemic areas or if the cultures suggest Pseudomonas-like organism. With increasing international travel, it is crucial to remember that good travel history could be life-saving in some cases of septicaemia.

Adult↗
PubMed1999

Combination antimicrobial therapy of acute Burkholderia pseudomallei infection in a mouse model.

Burkholderia pseudomallei is the causative agent of melioidosis, a disease endemic in tropical and subtropical regions of South-East Asia and Northern Australia. Antimicrobial therapy regimens for treatment of acute septicemic melioidosis are of variable efficacy. Ceftazidime is the current antibiotic of choice and is commonly administered with other agents such as cotrimoxazole or doxycycline. The emergence of resistant strains of B. pseudomallei and the persistence of high mortality rates prompted the present study. Using an established mouse model of acute disseminated B. pseudomallei infection, we compared the efficacy of ceftazidime versus cefpirome in combination with cotrimoxazole or chloramphenicol therapy in vivo. Control mice that were infected but did not receive antibiotic therapy died within 96 hours of infection. No deaths occurred in treatment groups receiving either cephalosporin or cotrimoxazole, despite the demonstrated resistance of B. pseudomallei to cotrimoxazole in vitro. The mortality rate in treatment groups receiving either cephalosporin and chloramphenicol was 66%. These results demonstrate a comparable level of efficacy between ceftazidime and cefpirome for treatment of acute B. pseudomallei infection in mice.

Animals↗
PubMed2000

Soluble granzymes are released during human endotoxemia and in patients with severe infection due to gram-negative bacteria.

Extracellular release of granzymes is considered to reflect the involvement of cytotoxic T lymphocytes and NK cells in various disease states. To obtain insight into granzyme release during bacterial infection, granzyme levels were measured during experimental human endotoxemia and in patients with melioidosis, a severe infection due to gram-negative bacteria. Plasma concentrations of granzyme A (GrA) and GrB increased transiently after endotoxin administration, peaking after 2-6 h. In patients with bacteremic melioidosis, GrA and GrB levels were elevated on admission and remained high during the 72-h study period. In whole blood stimulated with heat-killed Burkholderia pseudomallei, neutralization of tumor necrosis factor, interleukin-12, or interleukin-18 inhibited granzyme secretion, which was independent of interferon-gamma. Stimulation with endotoxin and other gram-negative and gram-positive bacteria also strongly induced the secretion of granzymes, suggesting that granzyme release is a general immune response during bacterial infection. The interaction between the cytokine network and granzymes may play an important immunoregulatory role during bacterial infections.

Adolescent↗
PubMed1989

The antimicrobial susceptibility of Pseudomonas pseudomallei. Emergence of resistance in vitro and during treatment.

We have measured the in-vitro activity of 27 antimicrobials against 211 clinical and ten reference strains of Pseudomonas pseudomallei. Imipenem was the most active antibiotic tested, followed by piperacillin, doxycycline, amoxycillin/clavulanic acid, cefixime, cefetamet, azlocillin and ceftazidime, all of which had MICs of less than or equal to 2 mg/l for the majority of strains. The measured MICs were dependent on the media and inocula used, to an extent which varied with the antibiotic class under test; MICs of ureidopenicillins were particularly inoculum-dependent. The beta-lactams and ciprofloxacin were bactericidal, whereas the agents conventionally used to treat melioidosis (doxycycline, chloramphenicol, sulphamethoxazole and trimethoprim) had bacteriostatic activity only. Strains highly resistant to chloramphenicol (MIC greater than or equal to 256 mg/l) emerged during treatment in 7.1% of patients. These strains were fully virulent, and frequently showed cross-resistance to tetracyclines, sulphamethoxazole, trimethoprim and ciprofloxacin, with paradoxical increased susceptibility to beta-lactams and aminoglycosides. Similar resistance patterns were seen in mutants generated in vitro and two reference strains. One strain with isolated ceftazidime resistance, reversible by clavulanic acid, emerged during treatment. Several of the new beta-lactam antibiotics are of potential value in the therapy of P. pseudomallei infections. Patients should be carefully monitored for the emergence of antibiotic-resistant strains during treatment of melioidosis.

Anti-Bacterial Agents↗
PubMed2001

Cloning and characterisation of malE in Burkholderia pseudomallei.

No recombinant protein is available for serodiagnosis or skin test in the diagnosis of melioidosis. This report describes the cloning of the malE gene, which encodes an immunogenic protein of Burkholderia pseudomallei. Bi-directional DNA sequencing of malE revealed that the gene contained a single open reading frame encoding 416 amino acid residues with a predicted molecular mass of 44.4 kDa. BLAST analysis showed that the putative protein encoded by malE is homologous to the maltose-binding protein (MBP) of other bacteria. It has 48% and 63% amino acid identity and similarity with the MBP of Brucella abortus, and malE complementation assay showed that it partially complemented the function of the MBP of Escherichia coli. Several highly conserved regions among the MBP of B. pseudomallei, Br. abortus, Salmonella enterica serotype Typhimurium, E. coli and Enterobacter aerogenes were observed. These regions represent signatures A, B, C, D and F identified in the MBP of E. coli. Further sequence analysis revealed that the first 24 amino acid residues of the MBP of B. pseudomallei probably represent the N-terminal signal peptide of the protein. Similar to the signal peptide of the MBP of E. coli, Ent. aerogenes and S. Typhimurium, the MBP of B. pseudomallei contains two basic residues in the first eight amino acids, followed by a hydrophobic core, with the last three amino acids in the signal peptide being Ala-Gln-Ala, conforming to the consensus sequence Ala-X-Ala at positions -3 to -1 relative to the site of proteolytic cleavage for recognition by signal peptidase I. Further studies on serodiagnosis of melioidosis with recombinant MBP should be performed.

ATP-Binding Cassette Transporters↗
PubMed2002

Passive protection against Burkholderia pseudomallei infection in mice by monoclonal antibodies against capsular polysaccharide, lipopolysaccharide or proteins.

Burkholderia pseudomallei, the aetiological agent of melioidosis, is endemic in south-east Asia and northern Australia, where it is an important cause of human disease. There is no vaccine available and antibiotic therapy is associated with high relapse rates. A panel of seven monoclonal antibodies (MAbs) that recognise capsular polysaccharide, lipopolysaccharide or proteins was produced and their ability to protect mice passively against experimental melioidosis was evaluated. The MAbs were capable of protecting mice against intra-peritoneal challenge with 10(4) cfu/250 MLD of a virulent strain of B. pseudomallei (NCTC 4845), when pooled, and four of the MAbs were individually protective. However, at a higher B. pseudomallei challenge level of 10(6) cfu none of the MAbs afforded protection and only the anti-exopolysaccharide MAbs produced a significantly delayed time to death.

Animals↗
PubMed1996

Biochemical characteristics of clinical and environmental isolates of Burkholderia pseudomallei.

The biochemical characteristics of 213 isolates of Burkholderia pseudomallei from patients with melioidosis and 140 isolates from the soil in central and northeastern Thailand were compared. Whereas the biochemical profiles of all the clinical isolates were similar, all soil isolates from the central area and 25% of isolates from northeastern Thailand comprised a different phenotype. This was characterised by the ability to assimilate L-arabinose (100%), adonitol (100%), 5-keto-gluconate (90%) and D-xylose (84%), but failure to assimilate dulcitol (0%), erythritol (0%) and trehalose (10%). Compared with clinical isolates, these organisms had similar antibiotic susceptibility profiles and were also recognised by a specific polyclonal antibody against B. pseudomallei. As melioidosis is rare in central Thailand, but common in the northeast, this raises the possibility that this biochemical phenotype may be less virulent, or may even represent a different species.

Anti-Bacterial Agents↗
PubMed1997

Ribotype differences between clinical and environmental isolates of Burkholderia pseudomallei.

Burkholderia pseudomallei is isolated frequently from the soil in regions where the disease melioidosis occurs. However, recent surveys in Thailand have shown that the frequency of isolation of the organism from soil samples is not directly related to the incidence of melioidosis in an area. To determine whether strain populations of B. pseudomallei prevalent in soil are gentypically related to strains causing clinical disease, rRNA BamHI restriction fragment length polymorphisms (RFLP) of 139 soil environmental isolates and 228 human isolates were compared. Two groups of ribotype patterns were found. Group I comprised 37 different ribotype patterns which were characterised by five to eight hybridisation bands of 2.8- > 23 kb. All of these ribotypes were identified among the clinical isolates, and 18 of them were also found in 59 environmental isolates. Group II was represented by 12 ribotypes found only in environmental strains. These ribotype patterns comprised one to five bands in the size range 9- > 23 kb. All but one of the 73 isolates in this group grew on a minimal medium supplemented with L-arabinose. In contrast, only 3% of the 66 isolates from the environment with group I ribotype patterns could utilise this sugar as their sole energy source. These findings suggest that B. pseudomallei strains that utilise arabinose constitute a population that is genetically distinct from other environmental and clinical strains.

Arabinose↗
PubMed2004

Burkholderia pseudomallei stimulates low interleukin-8 production in the human lung epithelial cell line A549.

Melioidosis is a life-threatening disease caused by Burkholderia pseudomallei. The lung is the most commonly affected organ, resulting in abscess formation in patients with chronic melioidosis. Previous study has shown that B. pseudomallei was able to invade and multiply in epithelial cells. In the present study, we have demonstrated that B. pseudomallei is able to stimulate interleukin 8 (IL-8) production from the human alveolar lung epithelium cell line A549. However, the level of IL-8 production was significantly lower than when the cells were infected with other Gram-negative bacteria such as Salmonella enterica serovar Typhi (S. typhi) which were used for comparison. The degree of IkappaBalpha degradation in the B. pseudomallei-infected cells was lower than that of the S. typhi-infected cells, suggesting that B. pseudomallei is also a poorer cell activator. Inhibition of B. pseudomallei invasion by cytochalasin D did not interfere with either IL-8 production or IkappaBalpha degradation, indicating that bacterial uptake is not required for the production of this chemokine. Thus, it appears that the signalling initiated by the interaction of B. pseudomallei with the epithelial cell surface is sufficient for epithelial cell activation.

Burkholderia pseudomallei↗
PubMed1997

Melioidotic osteomyelitis in northern Australia.

BACKGROUND: Osteomyelitis is a well-recognized manifestation of melioidosis, a significant disease that is endemic in South-East Asia and northern Australia. Diagnosis requires a high degree of clinical suspicion and is dependent on microbiological confirmation; important clues are travel to or residence in an endemic area. Infection often but not always occurs in well-recognized risk groups, especially diabetics and alcoholics. Subacute presentations often mimic other disease processes and patients may not always be clinically septic. Treatment requires surgical drainage in combination with multiple antibiotic therapy, including at least 2 weeks of intravenous ceftazidime and oral therapy continuing for 3-6 months. Non-compliance usually results in relapse. Due to the potential long latency of the disease and the possibility of reactivation, follow-up should probably be life-long. METHODS: A prospective study of 115 patients presenting with melioidosis between 1989 and 1995 was undertaken. RESULTS: Four patients were found to have osteomyelitis. CONCLUSION: It is important to be aware of this condition so that early treatment can be initiated.

Adolescent↗
PubMed2004

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.

Adult↗
PubMed1990

Experimental infection of normal and immunosuppressed pigs with Pseudomonas pseudomallei.

A single dose of 5 x 10(8) bacilli of Pseudomonas pseudomallei by intratracheal injection resulted in acute (21 cases) or chronic (19 cases) melioidosis in 40 of 48 pigs. Fifteen (10 acute and 5 chronic) had been immunosuppressed by cyclophosphamide before inoculation. The major clinical signs were initial fever, marked neutrophilia and, in the acute cases, respiratory distress. There were no signs of the nasal and ocular discharge, paresis or diarrhoea seen in acute cases in south-east Asia. The cyclophosphamide treatment caused a significant decrease in the neutrophil count by 7 d after inoculation in all 15 immunosuppressed pigs, and all were culture positive at necropsy. Eight of the 33 non-treated pigs were culture negative at necropsy. Pigs overcoming the initial phase of infection had more abscess-like nodules that were bacteriologically sterile at necropsy than the pigs with acute cases of melioidosis. P. pseudomallei was isolated predominantly from the spleen, lungs and the injection site. Although only one strain was used in this study, it is likely that Australian strains of P. pseudomallei are not as virulent as the south-east Asian isolates.

Acute Disease↗
PubMed1991

Pseudomonas pseudomallei, a common pathogen in Thailand that is resistant to the bactericidal effects of many antibiotics.

The purpose of this investigation was to identify newer antimicrobial agents that may be useful in the therapy of melioidosis. The in vitro susceptibilities of 199 clinical isolates of Pseudomonas pseudomallei to 22 antibiotics were determined by standard disk diffusion, and those to 13 antibiotics were determined by agar dilution. Over 90% of the isolates were susceptible to imipenem, piperacillin-tazobactam, piperacillin, ceftazidime, ticarcillin-clavulanate, ampicillin-sulbactam, and carumonam by both methods. Standard disk diffusion yielded unacceptably high false-susceptibility results with aztreonam, ciprofloxacin, and temafloxacin. Piperacillin, ceftazidime, imipenem, and ciprofloxacin were not bactericidal for three selected P. pseudomallei strains as determined by time-kill curve methods. Furthermore, addition of ciprofloxacin to piperacillin, ceftazidime, or imipenem did not enhance bactericidal activity. One hundred ninety-four strains showed weak beta-lactamase production that did not increase upon incubation with cefoxitin. These findings suggest that several newer antimicrobial agents may prove useful in the treatment of melioidosis. However, results of susceptibility studies involving P. pseudomallei and newer agents must be interpreted with caution.

Anti-Bacterial Agents↗