Suppression-subtractive hybridisation reveals variations in gene distribution amongst the Burkholderia cepacia complex, including the presence in some strains of a genomic island containing putative polysaccharide production genes

Parsons, Yasmin N.; Banasko, Rebecca; Detsika, Maria G.; Duangsonk, Kwanjit; Rainbow, Lucille; Winstanley, Craig

doi:10.1007/s00203-003-0518-7

Cited by 25 publications

(25 citation statements)

References 36 publications

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“…Detection involved the use of two primer sets. In accordance with the findings in our previous study (20) comparing PCR-based and hybridization methods for detection, isolates that were found to amplify with either primer set were considered to be PCR positive. Genomovar I isolates did not amplify with either set of primers.…”

Section: Resultssupporting

confidence: 84%

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Molecular Typing of, and Distribution of Genetic Markers among, Burkholderia cepacia Complex Isolates from Brazil

et al. 2003

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PCR tests were used to assign genomovar status to 39 non-cystic fibrosis (non-CF) and 11 CF Burkholderia cepacia complex isolates from patients in hospitals in Recife, Brazil. Non-CF isolates were assigned to genomovar IIIA (71.8%), genomovar I (15.4%), B. vietnamiensis (7.7%), and B. multivorans (5.1%). CF isolates were assigned to genomovar IIIA (18.2%), B. vietnamiensis (18.2%), and genomovar I (9.1%). Six CF isolates sharing recA PCR-restriction fragment length polymorphism (RFLP) and randomly amplified polymorphic DNA (RAPD) patterns could not be assigned to a genomovar. 16S rDNA sequence obtained from these isolates indicated a closest relationship to B. anthina, but the recA sequence was equally divergent from several genomovars. PCR screening indicated the presence of cblA in only two isolates, whereas the B. cepacia epidemic strain marker was found in 22 of 28 genomovar IIIA isolates. A type III secretion gene was detected in all but genomovar I isolates. RAPD and PCR-RFLP assays, targeting both recA and fliC, indicated a large amount of genetic variability among the isolates, with many novel patterns being observed. Nine genomovar IIIA isolates from different non-CF patients and clinical sources had identical genotypes, indicating the presence of a common clone.Burkholderia cepacia is an important opportunistic respiratory pathogen, particularly in patients with cystic fibrosis (CF) (7) and chronic granulomatous disease (23). In addition, B. cepacia causes catheter-associated urinary tract infections, wound infections, intravenous catheter-associated bacteremia, and endocarditis (25,26).What is now known as the B. cepacia complex was subdivided by DNA-DNA hybridization, whole-cell protein pattern similarity, and phenotypic markers into five genomic species or genomovars (28) B. multivorans, genomovars IIIA and IIIB, B. stabilis, B. vietnamiensis, and B. ambifaria (15). Recently, a test for B. anthina has also been published (27).There have been a number of studies aimed at determining the distribution of genomovars among isolates from CF and non-CF patients (1, 2, 13, 24). In this paper, we report the application of PCR tests to the identification of 39 non-CF and 11 CF B. cepacia isolates from patients in hospitals in Brazil. We further report the results of PCR-based fingerprinting to determine genetic variability among the isolates and data concerning the distribution of genetic markers associated with cable pili, type III secretion (TTS), the B. cepacia epidemic strain marker (BCESM), and a putative polysaccharide export gene cluster. MATERIALS AND METHODSBacterial strains. Thirty-nine non-CF isolates came from inpatients at neurological wards or intensive care units of Hospital Português, a large hospital facility in Recife, Brazil. Most patients were old debilitated individuals with respiratory problems and were undergoing ventilation. The CF isolates were recovered from 11 outpatients younger than 14 years, who were attending the CF unit of Instituto Materno Infantil de Pernambuco. All isolates...

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Section: Resultssupporting

confidence: 84%

“…Control strains were taken from the published representative panel of strains (17). PCR assays for detection of putative exopolysaccharide genes with homology to the wcbB and wzm2 genes of the Burkholderia pseudomallei capsule production gene cluster were carried out as described previously (20).…”

Section: Methodsmentioning

confidence: 99%

Molecular Typing of, and Distribution of Genetic Markers among, Burkholderia cepacia Complex Isolates from Brazil

et al. 2003

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“…One approach to identify such virulence factors is to identify genes present in virulent F. tularensis that are absent or have low homology to genes in closely related F. novicida. SSH has been used to successfully identify virulence genes present in pathogens that are absent in closely related species (Bernier & Sokol, 2005;DeShazer et al, 2001;Harakava & Gabriel, 2003;Liu et al, 2003;Newton et al, 2006;Parsons et al, 2003;Winstanley, 2002), and was therefore used to identify genes uniquely expressed by the more virulent type A and B strains. Seventy-six LVS-specific genes with hypothetical or known functions were identified in eight genomic regions in multiple SSH clones (Ahmed & Inzana, 2004).…”

Section: Discussionmentioning

confidence: 99%

Attenuation and protective efficacy of an O-antigen-deficient mutant of Francisella tularensis LVS

Ryder

Mandal

et al. 2007

Microbiology

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Francisella tularensis is a zoonotic, Gram-negative coccobacillus that causes tularemia in humans and animals. F. tularensis subspecies tularensis (type A) and F. tularensis subspecies holarctica (type B) are antigenically similar and more virulent than Francisella novicida in humans. The genetic locus that encodes the LPS O antigen was found to be substantially different between the type B live vaccine strain (LVS) and F. novicida. One LVS-specific gene with homology to a galactosyl transferase was selected for allelic replacement using a sacB-chloramphenicol expression suicide plasmid, and recombinants were screened for colony morphology on Congo red agar that matched that of F. novicida. Two mutants (WbtI S187Y and WbtI G191V ) were isolated that contained substitutions in conserved motifs in the sugar transamine/perosamine synthetase (WbtI) of the O-antigen locus, and the latter mutant was extensively tested and characterized. WbtI G191V grew at the same rate as the parent strain in Chamberlain's defined medium, completely lacked O antigen, was serum-sensitive but could grow in a mouse macrophage cell line, had increased resistance to sodium deoxycholate, and was highly attenuated in mice. Complementation of WbtI G191V with the wild-type wbtI gene in trans restored normal LPS synthesis, phenotypic properties similar to the parent, and virulence in mice. Immunization with WbtI G191V protected mice against a relatively low-dose intraperitoneal challenge with LVS, but was less protective against a high-dose challenge. These results indicate that complete loss of O antigen alters the surface phenotype and abrogates virulence in F. tularensis, but also compromises the induction of full protective immunity against F. tularensis infection in mice.

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“…Genome sequences representing five Bcc species were examined to determine if two previously published EPS gene clusters within B. cenocepacia J2315, the bce gene cluster (Moreira et al, 2003) and the wcb gene cluster (Parsons et al, 2003) (Videira et al, 2005).…”

Section: Investigation Of Conserved Eps Gene Clusters In Bcc Speciesmentioning

confidence: 99%

“…Therefore genome sequences representing five Bcc species (B. ambifaria, B. multivorans, B. vietnamiensis, B. dolosa and Burkholderia sp. 383) were examined to determine if two putative EPS gene clusters within B. cenocepacia J2315, the bce gene cluster (Moreira et al, 2003) and the wcb gene cluster (Parsons et al, 2003), are conserved across the Bcc. The wcb gene cluster was found to be poorly conserved, with between one-third and one-half of J2315 ORFs having no direct homologues within the species examined (data not shown).…”

Section: Investigation Of the Molecular Basis For Eps Biosynthesismentioning

confidence: 99%

Plant host and sugar alcohol induced exopolysaccharide biosynthesis in the Burkholderia cepacia complex

et al. 2008

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The species that presently constitute the Burkholderia cepacia complex (Bcc) have multiple roles; they include soil and water saprophytes, bioremediators, and plant, animal and human pathogens. Since the first description of pathogenicity in the Bcc was based on sour skin rot of onion bulbs, this study returned to this plant host to investigate the onion-associated phenotype of the Bcc. Many Bcc isolates, which were previously considered to be non-mucoid, produced copious amounts of exopolysaccharide (EPS) when onion tissue was provided as the sole nutrient. EPS production was not species-specific, was observed in isolates from both clinical and environmental sources, and did not correlate with the ability to cause maceration of onion tissue. Chemical analysis suggested that the onion components responsible for EPS induction were primarily the carbohydrates sucrose, fructose and fructans. Additional sugars were investigated, and all alcohol sugars tested were able to induce EPS production, in particular mannitol and glucitol. To investigate the molecular basis for EPS biosynthesis, we focused on the highly conserved bce gene cluster thought to be involved in cepacian biosynthesis. We demonstrated induction of the bce gene cluster by mannitol, and found a clear correlation between the inability of representatives of the Burkholderia cenocepacia ET12 lineage to produce EPS and the presence of an 11 bp deletion within the bceB gene, which encodes a glycosyltransferase. Insertional inactivation of bceB in Burkholderia ambifaria AMMD results in loss of EPS production on sugar alcohol media. These novel and surprising insights into EPS biosynthesis highlight the metabolic potential of the Bcc and show that a potential virulence factor may not be detected by routine laboratory culture. Our results also highlight a potential hazard in the use of inhaled mannitol as an osmolyte to improve mucociliary clearance in individuals with cystic fibrosis.

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Suppression-subtractive hybridisation reveals variations in gene distribution amongst the Burkholderia cepacia complex, including the presence in some strains of a genomic island containing putative polysaccharide production genes

Cited by 25 publications

References 36 publications

Molecular Typing of, and Distribution of Genetic Markers among, Burkholderia cepacia Complex Isolates from Brazil

Molecular Typing of, and Distribution of Genetic Markers among, Burkholderia cepacia Complex Isolates from Brazil

Attenuation and protective efficacy of an O-antigen-deficient mutant of Francisella tularensis LVS

Plant host and sugar alcohol induced exopolysaccharide biosynthesis in the Burkholderia cepacia complex

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