Association of mannose-binding lectin gene heterogeneity with severity of lung disease and survival in cystic fibrosis.
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Mannose-binding lectin (MBL) is a key factor in innate immunity, and lung infections are the leading cause of morbidity and mortality in cystic fibrosis (CF). Accordingly, we investigated whether MBL variant alleles, which are associated with recurrent infections, might be risk factors for CF patients. In 149 CF patients, different MBL genotypes were compared with respect to lung function, microbiology, and survival to end-stage CF (death or lung transplantation). The lung function was significantly reduced in carriers of MBL variant alleles when compared with normal homozygotes. The negative impact of variant alleles on lung function was especially confined to patients with chronic Pseudomonas aeruginosa infection. Burkholderia cepacia infection was significantly more frequent in carriers of variant alleles than in homozygotes. The risk of end-stage CF among carriers of variant alleles increased 3-fold, and the survival time decreased over a 10-year follow-up period. Moreover, by using a modified life table analysis, we estimated that the predicted age of survival was reduced by 8 years in variant allele carriers when compared with normal homozygotes. Presence of MBL variant alleles is therefore associated with poor prognosis and early death in patients with CF. (+info)
Evaluation of three oligonucleotide primer sets in PCR for the identification of Burkholderia cepacia and their differentiation from Burkholderia gladioli.
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AIMS: To evaluate three oligonucleotide primer pairs--two specific for 16S and 23S rRNA sequences of Burkholderia cepacia, and the third specific for internal transcribed spacer region of 16S-23S sequences of B gladioli--for the identification and differentiation of reference and clinical strains of these and other species. METHODS: The three primers sets were applied in polymerase chain reaction (PCR) to a collection of 177 clinical isolates submitted for identification from diagnostic laboratories as presumed B cepacia. RESULTS: At an annealing temperature of 63 degrees C, all eight B cepacia and four B gladioli reference strains reacted with their specific primers. B vandii was the only other species that was positive with both B cepacia primers but five Burkholderia or Ralstonia species reacted with one of these primers. Seventy eight isolates were typical of B cepacia in biochemical tests and 75 of these reacted with specific primers; three, however, were positive with the B gladioli primers. Fifteen asaccharolytic isolates were confirmed as B cepacia by PCR but other non-fermenting Gram negative species were negative with each of the primers. CONCLUSIONS: PCR using 16S rRNA sequences is recommended for identification of B cepacia that give atypical results in biochemical tests. (+info)
Role of ornibactin biosynthesis in the virulence of Burkholderia cepacia: characterization of pvdA, the gene encoding L-ornithine N(5)-oxygenase.
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Burkholderia cepacia is a frequent cause of respiratory infections in cystic fibrosis patients. B. cepacia has been shown to produce at least four siderophores which may play a role in the virulence of this organism. To characterize genes involved in the synthesis of siderophores, Tn5-OT182 mutants were isolated in strain K56-2, which produces two siderophores, salicylic acid (SA) and ornibactins. Two mutants were characterized that did not produce zones on Chrome Azurol S agar in a commonly used assay to detect siderophore activity. These mutants were determined to produce sevenfold more SA than K56-2 yet did not produce detectable amounts of ornibactins. These mutants, designated I117 and T10, had a transposon insertion in genes with significant homology to pyoverdine biosynthesis genes of Pseudomonas aeruginosa. I117 contained an insertion in a pvdA homolog, the gene for the enzyme L-ornithine N(5)-oxygenase, which catalyzes the hydroxylation of L-ornithine. Ornibactin synthesis in this mutant was partially restored when the precursor L-N(5)-OH-Orn was added to the culture medium. T10 contained an insertion in a pvdD homolog, which is a peptide synthetase involved in pyoverdine synthesis. beta-Galactosidase activity was iron regulated in both I117 and T10, suggesting that the transposon was inserted downstream of an iron-regulated promoter. Tn5-OT182 contains a lacZ gene that is expressed when inserted downstream of an active promoter. Both I117 and T10 were deficient in uptake of iron complexed to either ornibactins or SA, suggesting that transposon insertions in ornibactin biosynthesis genes also affected other components of the iron transport mechanism. The B. cepacia pvdA homolog was approximately 47% identical and 59% similar to L-ornithine N(5)-oxygenase from P. aeruginosa. Three clones were identified from a K56-2 cosmid library that partially restored ornibactin production, SA production, and SA uptake to parental levels but did not affect the rate of (59)Fe-ornibactin uptake in I117. A chromosomal pvdA deletion mutant was constructed that had a phenotype similar to that of I117 except that it did not hyperproduce SA. The pvdA mutants were less virulent than the parent strain in chronic and acute models of respiratory infection. A functional pvdA gene appears to be required for effective colonization and persistence in B. cepacia lung infections. (+info)
Lipopolysaccharide chemotypes of Burkholderia cepacia.
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Burkholderia cepacia is an important pathogen in patients with cystic fibrosis (CF) and much is now known of its epidemiology. In contrast, its virulence mechanisms are poorly understood. The lipopolysaccharide (LPS) of B. cepacia, a well-recognised virulence factor of other gram-negative bacteria, is known to be strongly endotoxic in vitro. The aim of this study was to observe if there were any links between the structure of B. cepacia LPS and virulence. This has been investigated by polyacrylamide gel electrophoresis and immunoblotting to define the chemotype and antigenic cross reactivity of B. cepacia LPS. Strains (16) belonging to different genomovars of the B. cepacia complex were selected to represent epidemic and non-epidemic clinical isolates and environmental strains. All strains belonging to genomovars I and II (the latter now renamed B. multivorans) had smooth LPS. However, isolates belonging to genomovar III, the group to which most of the epidemic CF isolates belong - including the highly transmissible strain (ET 12) which has been found in both the UK and North America - were of either rough or smooth LPS chemotype. In this study, B. cepacia J2315 represents the ET 12 lineage, and has a rough chemotype. Rabbit antiserum raised to strain J2315 revealed that the LPS core of this strain was antigenically related to some but not all other genomovar III strains, but it also cross-reacted strongly with all B. multivorans (genomovar II) and most genomovar I strains. Intra-strain phenotypic variation was demonstrated between bacteria grown in broth or on solid agar with a concomitant variation in antigenic cross reactivity. There was no clear evidence to associate any particular LPS phenotype with epidemic or non-epidemic strains, but changes in phenotype in vitro may provide clues to the survival and adaptability of B. cepacia in hostile environments and possibly to its ability to produce an inflammatory response in vivo. (+info)
Cystic fibrosis: inflammatory response to infection with Burkholderia cepacia and Pseudomonas aeruginosa.
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Pulmonary colonization by Burkholderia cepacia in cystic fibrosis (CF) may be associated with enhanced deterioration of pulmonary function. This may be due to a more florid host inflammatory response than in colonization by Pseudomonas aeruginosa, leading to greater lung injury. Circulating markers of inflammation were determined during infective exacerbations and periods of clinical stability in an 18 month prospective study in adults with CF colonized by P. aeruginosa (n=41). B. cepacia (n=13) and in adults who intermittently grew B. cepacia (n=6). There were no differences between the levels of the inflammation markers measured in the three groups (P. aeruginosa, B. cepacia, B. cepacia intermittent) at any of the assessment points. When clinically stable, levels of inflammatory markers in all groups were elevated compared to a matched non-CF population, indicating, continuous inflammation and the potential for lung damage between infective exacerbations. This study does not support the hypothesis that pulmonary colonization with Burkholderia cepacia is associated with a heightened inflammatory response compared with Pseudomonas aeruginosa colonization. (+info)
In vitro susceptibilities of Burkholderia mallei in comparison to those of other pathogenic Burkholderia spp.
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The in vitro antimicrobial susceptibilities of isolates of Burkholderia mallei to 16 antibiotics were assessed and compared with the susceptibilities of Burkholderia pseudomallei and Burkholderia cepacia. The antibiotic susceptibility profile of B. mallei resembled that of B. pseudomallei more closely than that of B. cepacia, which corresponds to their similarities in terms of biochemistry, antigenicity, and pathogenicity. Ceftazidime, imipenem, doxycycline, and ciprofloxacin were active against both B. mallei and B. pseudomallei. Gentamicin was active against B. mallei but not against B. pseudomallei. Antibiotics clinically proven to be effective in the treatment of melioidosis may therefore be effective for treating glanders. (+info)
Identification of Burkholderia species and genomovars from cystic fibrosis patients by AFLP fingerprinting.
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AFLP is a genomic fingerprinting technique based on the selective amplification of restriction fragments from a total double-digest of genomic DNA. The applicability of this method to differentiate between species and genomovars of the genus Burkholderia was tested, with particular emphasis on taxa occurring in cystic fibrosis patients. In this study, 78 well-characterized strains and field isolates were investigated by two methods of AFLP fingerprinting. In the manual procedure, a radioactively labelled primer was used, amplified fragments were separated by conventional PAGE and the patterns were revealed by autoradiography. In the automated procedure, a fluorescently labelled primer was used in combination with electrophoresis and on-line data collection by means of an automated DNA sequencer. Overall, there was good agreement between the two AFLP procedures and the data were mostly consistent with results obtained from SDS-PAGE of whole-cell proteins and DNA-DNA hybridization experiments. The automated AFLP procedure has considerable technical advantages compared with the manual AFLP procedure, but a thorough visual analysis of the DNA profiles was required to avoid misidentification of some Burkholderia cepacia genomovar III strains. (+info)
Molecular epidemiological investigation using a randomly amplified polymorphic DNA assay of Burkholderia cepacia isolates from nosocomial outbreaks.
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We experienced two Burkholderia cepacia outbreaks over a 1-year period. During this period, 28 B. cepacia isolates were obtained from clinical specimens, and 2 were obtained from environmental specimens (i.e., from a nebulizer solution and a nebulizer tube). These 30 isolates were subjected to the PCR-based randomly amplified polymorphic DNA (RAPD) assay as well as to pulsed-field gel electrophoresis (PFGE). In the first outbreak, in which eight patients hospitalized in the Trauma and Critical Care Center were involved, the RAPD assay revealed that all 20 isolates obtained from clinical specimens and the 2 isolates from environmental specimens had identical DNA profiles. These RAPD data enabled us to pinpoint a possible source and to take countermeasures to prevent further spread of the epidemic-causing strain. In the second outbreak, two consecutive B. cepacia infection/colonization cases were seen in the surgery ward. The RAPD profiles of four isolates obtained were again identical, but they were distinct from those seen in the first outbreak, clearly indicating that the second outbreak was not related to the first. Thus, our experience demonstrated that the RAPD assay is a useful and reliable tool for epidemiological studies of B. cepacia isolates from nosocomial outbreaks. Since the RAPD assay could provide discriminatory potential and reproducibility comparable to those of the widely used PFGE assay with less complexity and in a shorter time, the introduction of the RAPD assay into hospital microbiology laboratories as a routine technique may help prevent nosocomial outbreaks. (+info)