Efflux-mediated aminoglycoside and macrolide resistance in Burkholderia pseudomallei.
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Burkholderia pseudomallei, the causative agent of melioidosis, is intrinsically resistant to a wide range of antimicrobial agents including beta-lactams, aminoglycosides, macrolides, and polymyxins. We used Tn5-OT182 to mutagenize B. pseudomallei to identify the genes involved in aminoglycoside resistance. We report here on the identification of AmrAB-OprA, a multidrug efflux system in B. pseudomallei which is specific for both aminoglycoside and macrolide antibiotics. We isolated two transposon mutants, RM101 and RM102, which had 8- to 128-fold increases in their susceptibilities to the aminoglycosides streptomycin, gentamicin, neomycin, tobramycin, kanamycin, and spectinomycin. In addition, both mutants, in contrast to the parent, were susceptible to the macrolides erythromycin and clarithromycin but not to the lincosamide clindamycin. Sequencing of the DNA flanking the transposon insertions revealed a putative operon consisting of a resistance, nodulation, division-type transporter, a membrane fusion protein, an outer membrane protein, and a divergently transcribed regulatorprotein. Consistent with the presence of an efflux system, both mutants accumulated [3H] dihydro streptomycin, whereas the parent strain did not. We constructed an amr deletion strain, B. pseudomallei DD503, which was hypersusceptible to aminoglycosides and macrolides and which was used successfully in allelic exchange experiments. These results suggest that an efflux system is a major contributor to the inherent high-level aminoglycoside and macrolide resistance found in B. pseudomallei. (+info)
Phylogenetic analysis of Ara+ and Ara- Burkholderia pseudomallei isolates and development of a multiplex PCR procedure for rapid discrimination between the two biotypes.
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A Burkholderia pseudomallei-like organism has recently been identified among some soil isolates of B. pseudomallei in an area with endemic melioidosis. This organism is almost identical to B. pseudomallei in terms of morphological and biochemical profiles, except that it differs in ability to assimilate L-arabinose. These Ara+ isolates are also less virulent than the Ara- isolates in animal models. In addition, clinical isolates of B. pseudomallei available to date are almost exclusively Ara-. These features suggested that these two organisms may belong to distinctive species. In this study, the 16S rRNA-encoding genes from five clinical (four Ara- and one Ara+) and nine soil isolates (five Ara- and four Ara+) of B. pseudomallei were sequenced. The nucleotide sequences and phylogenetic analysis indicated that the 16S rRNA-encoding gene of the Ara+ biotype was similar to but distinctively different from that of the Ara- soil isolates, which were identical to the classical clinical isolates of B. pseudomallei. The nucleotide sequence differences in the 16S rRNA-encoding gene appeared to be specific for the Ara+ or Ara- biotypes. The differences were, however, not sufficient for classification into a new species within the genus Burkholderia. A simple and rapid multiplex PCR procedure was developed to discriminate between Ara- and Ara+ B. pseudomallei isolates. This new method could also be incorporated into our previously reported nested PCR system for detecting B. pseudomallei in clinical specimens. (+info)
Characterization of a murine model of melioidosis: comparison of different strains of mice.
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Melioidosis is an infectious disease caused by the saprophytic gram-negative rod Burkholderia pseudomallei. The aim of this study was to establish and characterize a murine model of melioidosis to provide a basis for further investigations on the pathogenesis of the disease. After intravenous infection with B. pseudomallei, C57BL/6 mice were found to be significantly more resistant than BALB/c mice. There was a marked organotropism of B. pseudomallei for the spleen and liver in both strains of mice, with the highest bacterial load in the spleen. Electron microscopic investigations of the spleen clearly demonstrated intracellular replication within membrane-bound phagosomes. Electron micrographs of the liver provided evidence that B. pseudomallei-containing phagosomes in hepatocytes fuse with lysosomes, leading to degradation of bacteria. In both strains of mice, the course of infection was highly dependent on the infective dose and the bacterial strain used, ranging from death within a few days to death after several weeks. In comparison with BALB/c mice, the bacterial counts in C57BL/6 mice were decreased 12 h after infection, which is suggestive of an innate immune mechanism against B. pseudomallei in this early phase of infection contributing to the lower susceptibility of C57BL/6 mice. BALB/c mice developed a more pronounced lymphopenia, granulocytosis, and splenomegaly at a lower infective dose compared to C57BL/6 mice. Analysis of the antibody response against B. pseudomallei 11 days after infection revealed a significantly higher immunoglobulin G2A (IgG2a)/IgG1 ratio in C57BL/6 mice than in BALB/c mice, indicating that a T helper type 1 immune response is associated with resistance to infection with B. pseudomallei. (+info)
Obligatory role of gamma interferon for host survival in a murine model of infection with Burkholderia pseudomallei.
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Burkholderia pseudomallei, the causative agent of melioidosis, is a gram-negative bacterium capable of causing either acute lethal sepsis or chronic but eventually fatal disease in infected individuals. However, despite the clinical importance of this infection in areas where it is endemic, there is essentially no information on the mechanisms of protective immunity to the bacterium. We describe here a murine model of either acute or chronic infection with B. pseudomallei in Taylor Outbred (TO) mice which mimics many features of the human pathology. Intraperitoneal infection of TO mice at doses of >10(6) CFU resulted in acute septic shock and death within 2 days. In contrast, at lower doses mice were able to clear the inoculum from the liver and spleen over a 3- to 4-week period, but persistence of the organism at other sites resulted in a chronic infection of between 2 and 16 months duration which was eventually lethal in all of the animals tested. Resistance to acute infection with B. pseudomallei was absolutely dependent upon the production of gamma interferon (IFN-gamma) in vivo. Administration of neutralizing monoclonal antibody against IFN-gamma lowered the 50% lethal dose from >5 x 10(5) to ca. 2 CFU and was associated with 8,500- and 4,400-fold increases in the bacterial burdens in the liver and spleen, respectively, together with extensive destruction of lymphoid architecture in the latter organ within 48 h. Neutralization of either tumor necrosis factor alpha or interleukin-12 but not granulocyte-macrophage colony-stimulating factor, also increased susceptibility to infection in vivo. Together, these results provide the first evidence of a host protective mechanism against B. pseudomallei. The rapid production of IFN-gamma within the first day of infection determines whether the infection proceeds to an acute lethal outcome or becomes chronic. (+info)
Evidence for the presence in Burkholderia pseudomallei of a type III secretion system-associated gene cluster.
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Burkholderia pseudomallei, the causative agent of melioidosis, contains a cluster of putative genes homologous to those encoding HpaP, HrcQ, HrcR, HrcS and HrpV in the plant pathogen Ralstonia solanacearum. In R. solanacearum, these genes form part of a type III secretion-associated pathogenicity island. The order of the genes in B. pseudomallei is directly equivalent to that found in R. solanacearum. The B. pseudomallei proteins share 49.5% (HpaP), 52.6% (HrcQ), 80.0% (HrcR), 72.1% (HrcS) and 46.7% (HrpV) similarity, respectively, with their equivalent R. solanacearum proteins. The presence of type III secretion-associated genes in B. pseudomallei pathogens suggests a possible role for type III secretion systems in the pathogenicity of this organism. (+info)
Molecular characterization of genetic loci required for secretion of exoproducts in Burkholderia pseudomallei.
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Previous studies have demonstrated that Burkholderia pseudomallei secretes protease, lipase, and phospholipase C (PLC) into the extracellular milieu, but their mechanisms of secretion and roles in pathogenesis have not been elucidated. In this study, we isolated and characterized 29 transposon mutants unable to secrete protease, lipase, and PLC. (+info)
Melioidosis with adrenal gland abscess.
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We report a case of melioidosis with left adrenal gland abscess in a 51-year-old man from Taiwan who traveled to Rangoon, Burma for a four-day tour on July 15, 1997. The patient developed fever and left upper abdominal pain upon returning to Taiwan on July 19, 1997. Ten days after returning to Taiwan, he was admitted to Chang Gung Memorial Hospital in Keelung, Taiwan and blood culture on admission was positive for Burkholderia pseudomallei. Computerized tomography of the abdomen revealed left adrenal gland swelling and suppuration. Treatment with parenteral ceftazidime and cotrimoxazole for three weeks followed by two months of oral cotrimoxazole cured the infection. The patient remained asymptomatic at 12 months follow-up. (+info)
Epidemiology of Burkholderia pseudomallei in Thailand.
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The distribution of Burkholderia pseudomallei in soil collected from four regions of Thailand and the frequency of B. pseudomallei infections in patients attending government hospitals throughout Thailand in 1997 were surveyed. A total of 3,585 soil samples collected from 896 sites in four regions of Thailand were cultured for B. pseudomallei using selective enrichment broth and modified Ashdown's agar. The organism was recovered in 4.4%, 6.1%, 20.4%, and 5.9% of the soil samples collected from the northern, central, northeastern, and southern regions, respectively, of Thailand (P < 0.0001). Burkholderia pseudomallei was cultured from 50.1% of the sites in the northeastern region compared with 13.8%, 24.5%, and 18.4% in the northern, central, and southern regions, respectively (P < 0.0001). The infection rate in patients attending government hospitals in the northeastern region (137.9 per 100,000 inpatients) was significantly higher than those in the northern (18 per 100,000 inpatients), central (13.4 per 100,000 inpatients), and southern (14.4 per 100,000 inpatients) regions, respectively (P < 0.0001). It is suggested that melioidosis, which is endemic in Thailand, is associated with the presence of B. pseudomallei in soil. (+info)