Model of Differential Susceptibility to Mucosal Burkholderia pseudomallei Infection

Burkholderia pseudomallei is the causative agent of melioidosis, an infectious disease with protean clinical manifestations. The major route of infection is thought to be through subcutaneous inoculation of contaminated soil and water, although ingestion and inhalation of contaminated aerosols are also possible. This study examines infection through the intranasal route in a murine model to mimic infection through inhalation. Two strains of mice, C57BL/6 and BALB/c, exhibit differential susceptibilities to the infection, with the C57BL/6 mice being considerably more resistant. To examine host factors that could contribute to this difference, bacterial loads and cytokine profiles in the two strains of mice were compared. We found that infected BALB/c mice exhibited higher bacterial loads in the lung and spleen and that they produced significantly higher levels of gamma interferon (IFN-gamma) in the serum than C57BL/6 mice. Although tumor necrosis factor alpha and interleukin-1 could be detected in the nasal washes and sera of both strains of mice, the production in serum was transient and much lower than that of IFN-gamma. C57BL/6 mice also exhibited memory responses to bacteria upon reinfection, with the production of serum immunoglobulin G (IgG) and mucosal IgA antibodies. Thus, it is possible that the production of systemic and mucosal antibodies is important for protection against disease in C57BL/6 mice.     

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.     

Burkholderia pseudomallei Capsular Polysaccharide Conjugates Provide Protection against Acute Melioidosis

Burkholderia pseudomallei, the etiologic agent of melioidosis, is a CDC tier 1 select agent that causes severe disease in both humans and animals. Diagnosis and treatment of melioidosis can be challenging, and in the absence of optimal chemotherapeutic intervention, acute disease is frequently fatal. Melioidosis is an emerging infectious disease for which there are currently no licensed vaccines. Due to the potential malicious use of B. pseudomallei as well as its impact on public health in regions where the disease is endemic, there is significant interest in developing vaccines for immunization against this disease. In the present study, type A O-polysaccharide (OPS) and manno-heptose capsular polysaccharide (CPS) antigens were isolated from nonpathogenic, select-agent-excluded strains of B. pseudomallei and covalently linked to carrier proteins. By using these conjugates (OPS2B1 and CPS2B1, respectively), it was shown that although high-titer IgG responses against the OPS or CPS component of the glycoconjugates could be raised in BALB/c mice, only those animals immunized with CPS2B1 were protected against intraperitoneal challenge with B. pseudomallei. Extending upon these studies, it was also demonstrated that when the mice were immunized with a combination of CPS2B1 and recombinant B. pseudomallei LolC, rather than with CPS2B1 or LolC individually, they exhibited higher survival rates when challenged with a lethal dose of B. pseudomallei. Collectively, these results suggest that CPS-based glycoconjugates are promising candidates for the development of subunit vaccines for immunization against melioidosis.     

Burkholderia pseudomallei: abscess in an unusual site

Melioidosis is an infection caused by Burkholderia pseudomallei. It is an important human pathogen in tropical area. The clinical manifestations are protean and multisystem involvement. We report an unusual case of melioidosis with abscess at root of mesentery in an elderly, non-insulin dependent diabetic Thai women. She presented with prolonged fever and chronic abdominal pain. The early clinical diagnosis was carcinomatous mass with peritonitis. Diagnosis of melioidosis arose from the surgical finding and pus culture. Treatment with surgical drainage and ceftazidime followed by co-trimoxazole plus doxycycline had a good clinical outcome.     

Volatile-Sulfur-Compound Profile Distinguishes Burkholderia pseudomallei from Burkholderia thailandensis

Solid-phase microextraction gas chromatography-mass spectrometry (SPME-GCMS) was used to show that dimethyl sulfide produced by Burkholderia pseudomallei is responsible for its unusual truffle-like smell and distinguishes the species from Burkholderia thailandensis. SPME-GCMS can be safely used to detect dimethyl sulfide produced by agar-grown B. pseudomallei.     

Variations in Ceftazidime and Amoxicillin-Clavulanate Susceptibilities within a Clonal Infection of Burkholderia pseudomallei

A patient with a clonal infection of Burkholderia pseudomallei had subpopulations with ceftazidime and amoxicillin-clavulanate susceptibilities that differed among the clinical specimens. Resistance was associated with a novel Cys69Tyr substitution in the Ambler class A β-lactamase. Susceptibility testing of multiple colony variants from different sites should be performed for patients with culture-confirmed melioidosis.Burkholderia pseudomallei, an environmental bacterium found throughout Southeast Asia and northern Australia, causes melioidosis. Recommended treatment regimens consist of administration of an initial intravenous agent (ceftazidime, imipenem, or amoxicillin-clavulanate) followed by prolonged oral therapy (3). Despite such therapy, recurrent disease occurs in up to 16% of patients; up to 75% of the recurrences are relapses caused by the same strain, while the remainder are new infections by different strains (15, 22). In most cases, even those with an apparently mixed population of colony variants, infection is due to a single strain rather than multiple strains (14, 18).Recurrences are occasionally caused by isolates with acquired antibiotic resistance (4, 6), but detailed analysis of such cases is lacking. In two studies, neither of which determined mechanisms of resistance, randomly amplified polymorphic DNA profiles of resistant isolates from recurrences of disease were the same as those determined for the original strain in some cases (11), while band changes were present in other cases (6). Tribuddharat et al. showed that acquired resistance to ceftazidime and amoxicillin-clavulanate was associated with single mutations in the Ambler class A β-lactamase (21) but did not perform typing of the resistant and susceptible isolates. We noted a patient with multiple B. pseudomallei isolates of various levels of ceftazidime and amoxicillin-clavulanate susceptibilities during a single infection. In this retrospective study, we typed these isolates and determined the molecular basis of β-lactamase-associated resistance.A 57-year-old diabetic male presented with severe pneumonia, which was unresponsive to ampicillin-sulbactam. B. pseudomallei was isolated from three specimens, at days 1, 9, and 17 of admission, during which he was treated with ceftazidime. The three stocked isolates were subcultured, and three to five single colonies from each subculture were picked for pulsed-field gel electrophoresis (PFGE) (19), using SpeI and XbaI and a Chef DR-II system (Bio-Rad, Hercules, CA). Restriction patterns were compared using the Dice coefficient (F value), and cluster analysis was performed by the use of the unweighted-pair group method using average linkages and Bionumerics software, version 5.1 (Applied Maths, Sint-Martens-Latem, Belgium). Etests were used to measure MICs (AB Biodisk, Solna, Sweden), with B. pseudomallei NCTC 13178 and Pseudomonas aeruginosa ATCC 27853 used as controls. PCR amplification and sequencing of the complete class A (penA) and class D (oxa) β-lactamase genes were carried out (9, 17). Sequences were then compared to those of B. pseudomallei strain K96243 (10).A total of five different isolates from the three specimens were studied (Table ​(Table1).1). These showed two patterns of antibiotic susceptibility: intermediate susceptibility to ceftazidime and amoxicillin-clavulanate in the first pattern and ceftazidime resistance and amoxicillin-clavulanate susceptibility in the second pattern. All isolates were susceptible to imipenem, meropenem, chloramphenicol, and trimethoprim-sulfamethoxazole. There was a heterogenous population within the first stocked isolate, with ceftazidime-resistant white and yellow colonies as well as colonies that were intermediately susceptible. The resistant subpopulations had not been noted during primary processing of the specimens. Subsequent isolates, from sputum specimens, showed intermediate ceftazidime susceptibility, followed by ceftazidime resistance. PFGE using XbaI showed indistinguishable profiles for all patient isolates tested (data not shown). With SpeI, all showed the same 18-band profile, except for the 76161y isolate, which had one absent band between 216.9 and 244.4 kbp (F = 0.97) (Fig. ​(Fig.1).1). Thus, all isolates from this patient appear to represent the same strain (or a strain very closely related, in the case of 76161y), despite differences in antibiotic susceptibility and color.Open in a separate windowFIG. 1.PFGE of SpeI-digested DNA of isolates obtained from the patient''s three specimens, showing indistinguishable profiles, except for a 1-band difference in isolate 76161y (lane 16). Lanes 1, 7, 14, and 19: Salmonella enterica serotype Braenderup H9812; lane 2, isolate 76161; lane 3, isolate 0907; lane 4, isolate 3192; lane 5, B. pseudomallei NCTC 13178; lane 6, B. pseudomallei ATCC 23343; lanes 8 to 13 and lanes 17 and 18, other colonies randomly picked from the three specimens; lane 15, isolate 76161w; lane 16, isolate 76161y; lane 20, B. pseudomallei clinical strain 7722 from a different patient.

TABLE 1.

Details of the B. pseudomallei isolates analyzed

Comparison of Ashdown's medium, Burkholderia cepacia medium, and Burkholderia pseudomallei selective agar for clinical isolation of Burkholderia pseudomallei

Ashdown's medium, Burkholderia pseudomallei selective agar (BPSA), and a commercial Burkholderia cepacia medium were compared for their abilities to grow B. pseudomallei from 155 clinical specimens that proved positive for this organism. The sensitivity of each was equivalent; the selectivity of BPSA was lower than that of Ashdown's or B. cepacia medium.     

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.     

Strategies toward vaccines against Burkholderia mallei and Burkholderia pseudomallei

Burkholderia mallei and Burkholderia pseudomallei are Gram-negative, rod-shaped bacteria, and are the causative agents of the diseases glanders and melioidosis, respectively. These bacteria have been recognized as important pathogens for over 100 years, yet a relative dearth of available information exists regarding their virulence determinants and immunopathology. Infection with either of these bacteria presents with nonspecific symptoms and can be either acute or chronic, impeding rapid diagnosis. The lack of a vaccine for either bacterium also makes them potential candidates for bioweaponization. Together with their high rate of infectivity via aerosols and resistance to many common antibiotics, both bacteria have been classified as category B priority pathogens by the US NIH and US CDC, which has spurred a dramatic increase in interest in these microorganisms. Attempts have been made to develop vaccines for these infections, which would not only benefit military personnel, a group most likely to be targeted in an intentional release, but also individuals who may come in contact with glanders-infected animals or live in areas where melioidosis is endemic. This review highlights some recent attempts of vaccine development for these infections and the strategies used to improve the efficacy of vaccine approaches.     

Molecular Procedure for Rapid Detection of Burkholderia mallei and Burkholderia pseudomallei

A PCR procedure for the discrimination of Burkholderia mallei and Burkholderia pseudomallei was developed. It is based on the nucleotide difference T 2143 C (T versus C at position 2143) between B. mallei and B. pseudomallei detected in the 23S rDNA sequences. In comparison with conventional methods the procedure allows more rapid identification at reduced risk for infection of laboratory personnel.     

Functional Characterization of Burkholderia pseudomallei Trimeric Autotransporters

Burkholderia pseudomallei is a tier 1 select agent and the causative agent of melioidosis, a severe and often fatal disease with symptoms ranging from acute pneumonia and septic shock to a chronic infection characterized by abscess formation in the lungs, liver, and spleen. Autotransporters (ATs) are exoproteins belonging to the type V secretion system family, with many playing roles in pathogenesis. The genome of B. pseudomallei strain 1026b encodes nine putative trimeric AT proteins, of which only four have been described. Using a bioinformatic approach, we annotated putative domains within each trimeric AT protein, excluding the well-studied BimA protein, and found short repeated sequences unique to Burkholderia species, as well as an unexpectedly large proportion of ATs with extended signal peptide regions (ESPRs). To characterize the role of trimeric ATs in pathogenesis, we constructed disruption or deletion mutations in each of eight AT-encoding genes and evaluated the resulting strains for adherence to, invasion of, and plaque formation in A549 cells. The majority of the ATs (and/or the proteins encoded downstream) contributed to adherence to and efficient invasion of A549 cells. Using a BALB/c mouse model of infection, we determined the contributions of each AT to bacterial burdens in the lungs, liver, and spleen. At 48 h postinoculation, only one strain, Bp340::pDbpaC, demonstrated a defect in dissemination and/or survival in the liver, indicating that BpaC is required for wild-type virulence in this model.     

Exploitation of host cells by Burkholderia pseudomallei

Intracellular bacterial pathogens have evolved mechanisms to enter and exit eukaryotic cells using the power of actin polymerisation and to subvert the activity of cellular enzymes and signal transduction pathways. The proteins deployed by bacteria to subvert cellular processes often mimic eukaryotic proteins in their structure or function. Studies on the exploitation of host cells by the facultative intracellular pathogen Burkholderia pseudomallei are providing novel insights into the pathogenesis of melioidosis, a serious invasive disease of animals and humans that is endemic in tropical and subtropical areas. B. pseudomallei can invade epithelial cells, survive and proliferate inside phagocytes, escape from endocytic vesicles, form actin-based membrane protrusions and induce host cell fusion. Here we review current understanding of the molecular mechanisms underlying these processes.     

Flagella are virulence determinants of Burkholderia pseudomallei

Burkholderia pseudomallei, a facultatively intracellular pathogen, is a flagellated and motile gram-negative bacterium and is the causative agent of melioidosis in humans. Flagella are commonly recognized as important virulence determinants expressed by bacterial pathogens since the motility phenotype imparted by these organelles often correlates with the ability of an organism to cause disease. We used a virulent isolate of B. pseudomallei, KHW, to construct an isogenic deletion mutant with a mutation in the flagellin gene (fliC) by gene replacement transposon mutagenesis. The KHWDeltafliCKm mutant was aflagellate and nonmotile in semisolid agar. The isogenic KHWDeltafliCKm mutant was not impaired in terms of the ability to invade and replicate in cultured human lung cells compared with the wild type. It was also equally virulent in slow-killing assays involving Caenorhabditis elegans, but it was avirulent during intranasal infection of BALB/c mice. Very few bacteria, if any, were isolated from the lungs and spleens of KHWDeltafliCKm-infected mice. In contrast, the bacterial loads in the lungs and spleens were similar in mice infected with KHW and in mice infected with the complemented mutant, KHWDeltafliCKm/pUCP28TfliC. Unlike the Syrian hamster or diabetic rat models of infection, the B. pseudomallei flagellin was also a virulence factor during intraperitoneal infection of BALB/c mice. In this study, all animals infected with KHWDeltafliCKm remained healthy and did not succumb to disease regardless of the route of infection. The flagellum is therefore an important and necessary virulence determinant of B. pseudomallei during intranasal and intraperitoneal infection of mice.     

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.     

Burkholderia pseudomallei septicaemia - a case report

Burkholderia pseudomallei, a natural saprophyte widely distributed in soil, stagnant waters of endemic areas, is said to infect humans through breaks in the skin or through inhalation causing protean clinical manifestations including fatal septicaemia. A case of septicaemia in a elderly female diabetic due to B. pseudomallei following a history of fall is being reported with complete details.