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Peter Vandamme - One of the best experts on this subject based on the ideXlab platform.

  • Isolation of Burkholderia cepacia Complex Genomovars from Waters
    Systematic and Applied Microbiology, 2020
    Co-Authors: Karen Vermis, Peter Vandamme, Mariya Brachkova, Hans J. Nelis
    Abstract:

    Summary The aim of this study was to develop a selective enrichment broth as an aid for the isolation of Burkholderia cepacia complex (Bcc) bacteria from water. To allow growth of all nine Genomovars, mixtures of two carbon sources had to be used, i.e. L-arabinose/D-cellobiose or L-arabinose/L-threonine. Selectivity was provided by polymyxin B and 9-chloro-9-(4-diethylaminophenyl)-10-phenylacridan (C-390). Following enrichment, Bcc bacteria were isolated on a diagnostic O/F agar supplemented with gentamicin. A preliminary bio-diversity study on 28 surface waters yielded five different Genomovars, i.e. B. cepacia(Genomovar I), B. multivorans, B. cenocepacia, B. vietnamiensis and B. anthina. Drinking waters did not contain Bcc bacteria. However, the Genomovar pattern from a given sample varied with the enrichment broth used.

  • Identification of Burkholderia cepacia complex pathogens by rapid-cycle PCR with fluorescent hybridization probes
    Journal of Medical Microbiology, 2006
    Co-Authors: Ralf-peter Vonberg, Peter Vandamme, Susanne Häussler, Ivo Steinmetz
    Abstract:

    Members of the Burkholderia cepacia complex are important bacterial pathogens in cystic fibrosis (CF) patients. The B. cepacia complex currently consists of nine genetic subgroups (Genomovars) of different epidemiological relevance and possibly of different pathogenic potential in humans. In this study, a new approach was developed for the rapid identification of B. cepacia Genomovar I, Burkholderia multivorans (Genomovar II), Burkholderia cenocepacia (lineage III-A and III-B), Burkholderia stabilis (Genomovar IV) and Burkholderia vietnamiensis (Genomovar V), which cause the large majority of infections in CF patients. The method was based on the detection of differences in the recA gene sequence by using rapid-cycle PCR and Genomovar-specific fluorescence resonance energy transfer (FRET) probes. The Genomovar status of all 39 B. cepacia complex strains tested (Genomovars I‐V) was identified by melting-curve analysis. Each FRET probe produced a specific fluorescence signal only with the respective Genomovar, and not with other B. cepacia complex strains and Burkholderia spp. The identification system was easy to handle and revealed B. cepacia complex Genomovar I‐V status from culture isolates within about 1 h.

  • proposal to accommodate burkholderia cepacia Genomovar vi as burkholderia dolosa sp nov
    International Journal of Systematic and Evolutionary Microbiology, 2004
    Co-Authors: Karen Vermis, Eshwar Mahenthiralingam, Hans Nelis, John J. Lipuma, Torn Coenye, Peter Vandamme
    Abstract:

    Phenotypic and genotypic studies revealed new tools for differentiating Burkholderia cepacia Genomovar VI from Burkholderia multivorans and other B. cepacia-complex species. Hence, the name Burkholderia dolosa sp. nov. is proposed, with LMG 18943T (=CCUG 47727T) as the type strain. B. dolosa can be differentiated from other B. cepacia-complex bacteria by its inability to assimilate tryptamine, azelaic acid and salicin and by its failure to grow on the B. cepacia-selective medium PCAT. Both 16S rDNA and recA RFLP analysis revealed unique B. dolosa restriction patterns. In addition, new 16S rDNA- and recA-based PCR assays allowed its specific identification.

  • Burkholderia cepacia complex Genomovars: utilization of carbon sources, susceptibility to antimicrobial agents and growth on selective media.
    Journal of Applied Microbiology, 2003
    Co-Authors: Karen Vermis, Peter Vandamme, Hans J. Nelis
    Abstract:

    AIMS: To investigate the relationship between Genomovar status and carbon source utilization, antibiotic susceptibility and growth ability on selective media of 142 clinical and environmental Burkholderia cepacia complex (Bcc) isolates belonging to all nine Genomovars. METHODS AND RESULTS: Carbon source utilization and growth on selective media were tested by agar plate multipoint inoculation. Antimicrobial minimum inhibitory concentration (MIC) values were determined by agar dilution. Of all carbon sources, l-arabinose was most frequently utilized, supporting growth of 90% of all isolates. Burkholderia cepacia Genomovar VI failed to utilize azelaic acid, penicillin G, phtalate, salicin and tryptamine. Overall, B. vietnamiensis and B. anthina were most susceptible and B. cepacia Genomovar VI most resistant to antimicrobial agents. Burkholderia cepacia selective agar (BCSA) and the Mast B. cepacia medium supported growth of Bcc isolates most efficiently. CONCLUSIONS: This study demonstrates phenotypic heterogeneity within the Bcc. Some trends can be observed at the Genomovar level, but only B. cepacia Genomovar VI could be differentiated unambiguously on the basis of its inability to grow on PCAT. SIGNIFICANCE AND IMPACT OF THE STUDY: This work provides an update on some differential phenotypic characteristics of all nine Bcc Genomovars.

  • Updated version of the Burkholderia cepacia complex experimental strain panel.
    Journal of Clinical Microbiology, 2003
    Co-Authors: Tom Coenye, Peter Vandamme, John J. Lipuma, John R. W. Govan, Eshwar Mahenthiralingam
    Abstract:

    The Burkholderia cepacia complex consists of nine closely related species: Burkholderia cepacia Genomovars I and VI, Burkholderia multivorans (Genomovar II), Burkholderia cenocepacia (Genomovar III), Burkholderia stabilis (Genomovar IV), Burkholderia vietnamiensis (Genomovar V), Burkholderia

Craig A Shoemaker - One of the best experts on this subject based on the ideXlab platform.

  • lack of association between flavobacterium columnare Genomovar and virulence in hybrid tilapia oreochromis niloticus l oreochromis aureus steindachner
    Journal of Fish Diseases, 2015
    Co-Authors: Craig A Shoemaker, Benjamin R. Lafrentz
    Abstract:

    : Columnaris disease can be problematic in tilapia (Oreochromis spp.) production. An understanding of the pathogenesis and virulence of Flavobacterium columnare is needed to develop prevention strategies. The objective of this study was to determine the virulence of genetically defined isolates of F. columnare in sex-reversed hybrid tilapia, Oreochromis niloticus (L.)×O. aureus (Steindachner). A series of immersion challenge trials were performed using isolates of the five established Genomovars of F. columnare: I, II, II-B, III and I/II. The mean per cent mortality of fish challenged with Genomovar I, II and III isolates ranged from 0 to 100, 3.3-78 and 3.3-75%, respectively. The mean per cent mortality of fish challenged with Genomovar II-B ranged from 35 to 96.7%, and the only Genomovar I/II isolate tested caused no mortality. Contrary to previous work in other fish species, there did not appear to be an association between F. columnare Genomovar and virulence in tilapia. The challenge model used resulted in acute mortality. An alternative challenge model was tested by cohabitating healthy fish with dead fish infected with F. columnare. This method resulted in rapid appearance of clinical signs and mortality, suggesting the potential for F. columnare to increase in virulence upon growth on/in a fish host.

  • intragenomic heterogeneity in the 16s rrna genes of flavobacterium columnare and standard protocol for Genomovar assignment
    Journal of Fish Diseases, 2014
    Co-Authors: Benjamin R. Lafrentz, Geoffrey C Waldbieser, Timothy J Welch, Craig A Shoemaker
    Abstract:

    : Genetic variability in 16S rRNA gene sequences has been demonstrated among isolates of Flavobacterium columnare, and a restriction fragment length polymorphism (RFLP) assay is available for genetic typing of this important fish pathogen. Interpretation of restriction patterns can be difficult due to the lack of a formal description of the expected number and sizes of DNA fragments generated for each of the described Genomovars. In this study, partial 16S rRNA gene sequences (ca. 1250-bp fragment) from isolates representing each described Genomovar and isolates generating unique restriction patterns were cloned and sequenced. The results demonstrated that some isolates contained up to three different 16S rRNA genes whose sequences generate different RFLP patterns due to intragenomic heterogeneity within HaeIII restriction sites. The occurrence of HaeIII restriction sites within the portion of the 16S rRNA gene used for typing the F. columnare isolates and intragenomic heterogeneity within these sites explained the restriction patterns observed following RFLP analyses. This research provides a standard protocol for typing isolates of F. columnare by RFLP and a formal description of the expected restriction patterns for the previously described Genomovars I, II, II-B and III. Additionally, we describe a new Genomovar, I/II.

  • Lack of association between Flavobacterium columnare Genomovar and virulence in hybrid tilapia Oreochromis niloticus (L.)×Oreochromis aureus (Steindachner).
    Journal of Fish Diseases, 2014
    Co-Authors: Craig A Shoemaker, Benjamin R. Lafrentz
    Abstract:

    : Columnaris disease can be problematic in tilapia (Oreochromis spp.) production. An understanding of the pathogenesis and virulence of Flavobacterium columnare is needed to develop prevention strategies. The objective of this study was to determine the virulence of genetically defined isolates of F. columnare in sex-reversed hybrid tilapia, Oreochromis niloticus (L.)×O. aureus (Steindachner). A series of immersion challenge trials were performed using isolates of the five established Genomovars of F. columnare: I, II, II-B, III and I/II. The mean per cent mortality of fish challenged with Genomovar I, II and III isolates ranged from 0 to 100, 3.3-78 and 3.3-75%, respectively. The mean per cent mortality of fish challenged with Genomovar II-B ranged from 35 to 96.7%, and the only Genomovar I/II isolate tested caused no mortality. Contrary to previous work in other fish species, there did not appear to be an association between F. columnare Genomovar and virulence in tilapia. The challenge model used resulted in acute mortality. An alternative challenge model was tested by cohabitating healthy fish with dead fish infected with F. columnare. This method resulted in rapid appearance of clinical signs and mortality, suggesting the potential for F. columnare to increase in virulence upon growth on/in a fish host.

  • Reproducible challenge model to investigate the virulence of Flavobacterium columnare Genomovars in rainbow trout Oncorhynchus mykiss.
    Diseases of Aquatic Organisms, 2012
    Co-Authors: Benjamin R. Lafrentz, Craig A Shoemaker, Scott Lapatra, Phillip H Klesius
    Abstract:

    : Flavobacterium columnare is a Gram-negative bacterium that causes columnaris disease and has significant economic impacts on aquaculture production worldwide. Molecular analyses have demonstrated that there is genetic diversity among F. columnare isolates. A review of the published literature that used restriction fragment length polymorphism analysis of the 16S rRNA gene revealed that all isolates typed from salmonids were Genomovar I. Our objective was to develop a laboratory challenge model for F. columnare in rainbow trout Oncorhynchus mykiss (Walbaum) and use the model to determine the virulence of Genomovar I and II isolates. Six F. columnare isolates were obtained from rainbow trout experiencing losses due to columnaris disease and were determined to be Genomovar I. Three of these were chosen for a preliminary assessment of virulence, and isolate 051-10-S5 was chosen for additional experiments to determine the reproducibility of the waterborne challenge model. In 2 independent experiments, cumulative percent mortalities (CPM) were 49 ± 10% and 50 ± 19%. Challenge of rainbow trout with Genomovar I and II isolates demonstrated a difference in the CPM, with the Genomovar II isolates inducing significantly higher CPM. This reproducible waterborne challenge model for columnaris disease in rainbow trout will be useful to investigate host-pathogen interactions, vaccine development, and other potential control strategies. This research also provides a basis for further defining the molecular diversity and virulence associated with F. columnare Genomovars in rainbow trout and other salmonid species.

  • cloning expression and immunogenicity of flavobacterium columnare heat shock protein dnaj
    Journal of Aquatic Animal Health, 2010
    Co-Authors: Oscar Olivaresfuster, Jeffery S Terhune, Craig A Shoemaker, Covadonga R. Arias
    Abstract:

    Abstract The Flavobacterium columnare heat shock protein (HSP) gene dnaJ* was isolated, cloned, expressed, and used as an antigen in a recombinant vaccine strategy for channel catfish Ictalurus punctatus. The F. columnare dnaJ* sequence was obtained from Genomovars I and II and showed intraspecies variability. Recombinant protein was expressed and purified from Escherichia coli cultures and injected intraperitoneally (12 μg of purified DnaJ/fish) into fingerling channel catfish. In addition, induced (expressing the recombinant DnaJ) and uninduced (no recombinant protein being produced) E. coli cultures were also used to immunize fish. At 28 d postimmunization, antibody response was evaluated and the fish were challenged with F. columnare. A specific immune response against DnaJ was observed in fish immunized with DnaJ or E. coli cultures expressing DnaJ. No protection against the disease, however, was observed in F. columnare-challenged fish that had been immunized with DnaJ. Some level of protection was ...

Benjamin R. Lafrentz - One of the best experts on this subject based on the ideXlab platform.

  • Virulence of Flavobacterium columnare Genomovars in rainbow trout Oncorhynchus mykiss.
    Diseases of Aquatic Organisms, 2016
    Co-Authors: Jason P. Evenhuis, Benjamin R. Lafrentz
    Abstract:

    Flavobacterium columnare is the causative agent of columnaris disease and is re - sponsible for significant economic losses in aquaculture. F. columnare is a Gram-negative bac- terium, and 5 genetic types or Genomovars have been described based on restriction fragment length polymorphism of the 16S rRNA gene. Previous research has suggested that Genomovar II isolates are more virulent than Genomovar I isolates to multiple species of fish, including rainbow trout Oncorhynchus mykiss. In addition, improved genotyping methods have shown that some isolates previously classified as Genomovar I, and used in challenge experiments, were in fact Genomovar III. Our objective was to confirm previous results with respect to Genomovar II viru- lence, and to determine the susceptibility of rainbow trout to other Genomovars. The virulence of 8 Genomovar I, 4 Genomovar II, 3 Genomovar II-B, and 5 Genomovar III isolates originating from various sources was determined through 3 independent challenges in rainbow trout using an immersion challenge model. Mean cumulative percent mortality (CPM) of ~49% for Genomovar I isolates, ~1% for Genomovar II, ~5% for the II-B isolates, and ~7% for the III isolates was observed. The inability of Genomovar II isolates to produce mortalities in rainbow trout was unanticipated based on previous studies, but may be due to a number of factors including rainbow trout source and water chemistry. The source of fish and/or the presence of sub-optimal environment may influence the susceptibility of rainbow trout to different F. columnare Genomovars.

  • lack of association between flavobacterium columnare Genomovar and virulence in hybrid tilapia oreochromis niloticus l oreochromis aureus steindachner
    Journal of Fish Diseases, 2015
    Co-Authors: Craig A Shoemaker, Benjamin R. Lafrentz
    Abstract:

    : Columnaris disease can be problematic in tilapia (Oreochromis spp.) production. An understanding of the pathogenesis and virulence of Flavobacterium columnare is needed to develop prevention strategies. The objective of this study was to determine the virulence of genetically defined isolates of F. columnare in sex-reversed hybrid tilapia, Oreochromis niloticus (L.)×O. aureus (Steindachner). A series of immersion challenge trials were performed using isolates of the five established Genomovars of F. columnare: I, II, II-B, III and I/II. The mean per cent mortality of fish challenged with Genomovar I, II and III isolates ranged from 0 to 100, 3.3-78 and 3.3-75%, respectively. The mean per cent mortality of fish challenged with Genomovar II-B ranged from 35 to 96.7%, and the only Genomovar I/II isolate tested caused no mortality. Contrary to previous work in other fish species, there did not appear to be an association between F. columnare Genomovar and virulence in tilapia. The challenge model used resulted in acute mortality. An alternative challenge model was tested by cohabitating healthy fish with dead fish infected with F. columnare. This method resulted in rapid appearance of clinical signs and mortality, suggesting the potential for F. columnare to increase in virulence upon growth on/in a fish host.

  • intragenomic heterogeneity in the 16s rrna genes of flavobacterium columnare and standard protocol for Genomovar assignment
    Journal of Fish Diseases, 2014
    Co-Authors: Benjamin R. Lafrentz, Geoffrey C Waldbieser, Timothy J Welch, Craig A Shoemaker
    Abstract:

    : Genetic variability in 16S rRNA gene sequences has been demonstrated among isolates of Flavobacterium columnare, and a restriction fragment length polymorphism (RFLP) assay is available for genetic typing of this important fish pathogen. Interpretation of restriction patterns can be difficult due to the lack of a formal description of the expected number and sizes of DNA fragments generated for each of the described Genomovars. In this study, partial 16S rRNA gene sequences (ca. 1250-bp fragment) from isolates representing each described Genomovar and isolates generating unique restriction patterns were cloned and sequenced. The results demonstrated that some isolates contained up to three different 16S rRNA genes whose sequences generate different RFLP patterns due to intragenomic heterogeneity within HaeIII restriction sites. The occurrence of HaeIII restriction sites within the portion of the 16S rRNA gene used for typing the F. columnare isolates and intragenomic heterogeneity within these sites explained the restriction patterns observed following RFLP analyses. This research provides a standard protocol for typing isolates of F. columnare by RFLP and a formal description of the expected restriction patterns for the previously described Genomovars I, II, II-B and III. Additionally, we describe a new Genomovar, I/II.

  • Lack of association between Flavobacterium columnare Genomovar and virulence in hybrid tilapia Oreochromis niloticus (L.)×Oreochromis aureus (Steindachner).
    Journal of Fish Diseases, 2014
    Co-Authors: Craig A Shoemaker, Benjamin R. Lafrentz
    Abstract:

    : Columnaris disease can be problematic in tilapia (Oreochromis spp.) production. An understanding of the pathogenesis and virulence of Flavobacterium columnare is needed to develop prevention strategies. The objective of this study was to determine the virulence of genetically defined isolates of F. columnare in sex-reversed hybrid tilapia, Oreochromis niloticus (L.)×O. aureus (Steindachner). A series of immersion challenge trials were performed using isolates of the five established Genomovars of F. columnare: I, II, II-B, III and I/II. The mean per cent mortality of fish challenged with Genomovar I, II and III isolates ranged from 0 to 100, 3.3-78 and 3.3-75%, respectively. The mean per cent mortality of fish challenged with Genomovar II-B ranged from 35 to 96.7%, and the only Genomovar I/II isolate tested caused no mortality. Contrary to previous work in other fish species, there did not appear to be an association between F. columnare Genomovar and virulence in tilapia. The challenge model used resulted in acute mortality. An alternative challenge model was tested by cohabitating healthy fish with dead fish infected with F. columnare. This method resulted in rapid appearance of clinical signs and mortality, suggesting the potential for F. columnare to increase in virulence upon growth on/in a fish host.

  • Reproducible challenge model to investigate the virulence of Flavobacterium columnare Genomovars in rainbow trout Oncorhynchus mykiss.
    Diseases of Aquatic Organisms, 2012
    Co-Authors: Benjamin R. Lafrentz, Craig A Shoemaker, Scott Lapatra, Phillip H Klesius
    Abstract:

    : Flavobacterium columnare is a Gram-negative bacterium that causes columnaris disease and has significant economic impacts on aquaculture production worldwide. Molecular analyses have demonstrated that there is genetic diversity among F. columnare isolates. A review of the published literature that used restriction fragment length polymorphism analysis of the 16S rRNA gene revealed that all isolates typed from salmonids were Genomovar I. Our objective was to develop a laboratory challenge model for F. columnare in rainbow trout Oncorhynchus mykiss (Walbaum) and use the model to determine the virulence of Genomovar I and II isolates. Six F. columnare isolates were obtained from rainbow trout experiencing losses due to columnaris disease and were determined to be Genomovar I. Three of these were chosen for a preliminary assessment of virulence, and isolate 051-10-S5 was chosen for additional experiments to determine the reproducibility of the waterborne challenge model. In 2 independent experiments, cumulative percent mortalities (CPM) were 49 ± 10% and 50 ± 19%. Challenge of rainbow trout with Genomovar I and II isolates demonstrated a difference in the CPM, with the Genomovar II isolates inducing significantly higher CPM. This reproducible waterborne challenge model for columnaris disease in rainbow trout will be useful to investigate host-pathogen interactions, vaccine development, and other potential control strategies. This research also provides a basis for further defining the molecular diversity and virulence associated with F. columnare Genomovars in rainbow trout and other salmonid species.

John J. Lipuma - One of the best experts on this subject based on the ideXlab platform.

  • proposal to accommodate burkholderia cepacia Genomovar vi as burkholderia dolosa sp nov
    International Journal of Systematic and Evolutionary Microbiology, 2004
    Co-Authors: Karen Vermis, Eshwar Mahenthiralingam, Hans Nelis, John J. Lipuma, Torn Coenye, Peter Vandamme
    Abstract:

    Phenotypic and genotypic studies revealed new tools for differentiating Burkholderia cepacia Genomovar VI from Burkholderia multivorans and other B. cepacia-complex species. Hence, the name Burkholderia dolosa sp. nov. is proposed, with LMG 18943T (=CCUG 47727T) as the type strain. B. dolosa can be differentiated from other B. cepacia-complex bacteria by its inability to assimilate tryptamine, azelaic acid and salicin and by its failure to grow on the B. cepacia-selective medium PCAT. Both 16S rDNA and recA RFLP analysis revealed unique B. dolosa restriction patterns. In addition, new 16S rDNA- and recA-based PCR assays allowed its specific identification.

  • Updated version of the Burkholderia cepacia complex experimental strain panel.
    Journal of Clinical Microbiology, 2003
    Co-Authors: Tom Coenye, Peter Vandamme, John J. Lipuma, John R. W. Govan, Eshwar Mahenthiralingam
    Abstract:

    The Burkholderia cepacia complex consists of nine closely related species: Burkholderia cepacia Genomovars I and VI, Burkholderia multivorans (Genomovar II), Burkholderia cenocepacia (Genomovar III), Burkholderia stabilis (Genomovar IV), Burkholderia vietnamiensis (Genomovar V), Burkholderia

  • identification by subtractive hybridization of a novel insertion element specific for two widespread burkholderia cepacia Genomovar iii strains
    Journal of Clinical Microbiology, 2003
    Co-Authors: Theodore Spilker, Tom Coenye, John J. Lipuma
    Abstract:

    Species of the Burkholderia cepacia complex cause chronic and life-threatening infections in persons with cystic fibrosis. Epidemic strains infect multiple patients, reside primarily in Genomovar III, and have an apparent enhanced capacity for human infection and/or interpatient transmission. By using subtractive hybridization, a novel insertion element, designated IS1363, was identified in epidemic strain PHDC, known to infect many cystic fibrosis patients in the mid-Atlantic region of the United States. IS1363 was also found in most isolates of the ET12 lineage, responsible for infecting large numbers of patients in Ontario, Canada, and the United Kingdom. Southern blot analysis demonstrated that whereas multiple copies of IS1363 were present in strain PHDC, only one copy was present in ET12 isolates. IS1363 was used to probe a collection of 943 B. cepacia complex isolates, representing all nine Genomovars, recovered from 761 cystic fibrosis patients or the natural environment. IS1363 was not found in other Genomovar III strains and, with the exception of B. ambifaria, was absent from other B. cepacia complex species. Genotyping analyses of all IS1363-positive isolates demonstrated that strain PHDC was more widely distributed in the United States than previously appreciated; 212 cystic fibrosis patients in 24 states were identified as being infected with PHDC.

  • population structure analysis of burkholderia cepacia Genomovar iii varying degrees of genetic recombination characterize major clonal complexes
    Microbiology, 2003
    Co-Authors: Tom Coenye, John J. Lipuma
    Abstract:

    Infection with bacterial species belonging to the Burkholderia cepacia complex contribute significantly to morbidity and mortality in persons with cystic fibrosis (CF). The majority of isolates recovered from CF patients belong to B. cepacia Genomovar III and several distinct ‘epidemic’ strains have been described. This study examined the population structure of B. cepacia Genomovar III by using multilocus restriction typing, indexing allelic variation at five chromosomal genes by restriction analysis of PCR-amplified genes. A collection of 375 isolates, recovered from CF and non-CF patients and natural environments in North America, Europe and Australia, was examined. Among these isolates 144 different restriction types were found. Overall, the population is at linkage disequilibrium, indicating that it has a clonal structure. The majority (86·7 %) of restriction types grouped into three major clonal complexes, comprising the epidemic ET12, PHDC and Midwest clonal lineages. The analysis indicates that these complexes are geographically widespread and demonstrate varying degrees of genetic recombination. These differences in population structure among major clonal complexes within the same species are likely related to differences in evolutionary history and ecology. The observation that genetic recombination is frequent within some B. cepacia Genomovar III populations has important implications for the biotechnological use of B. cepacia complex species.

  • Lack of cable pili expression by cblA-containing Burkholderia cepacia complex.
    Microbiology, 2002
    Co-Authors: Umadevi S. Sajjan, Theodore Spilker, Janet F. Forstner, Annie Lu, John J. Lipuma
    Abstract:

    The Burkholderia cepacia complex consists of several closely related bacterial species (or Genomovars) which although generally not pathogenic for healthy individuals, contribute significantly to morbidity and mortality among persons with cystic fibrosis (CF). Certain B. cepacia complex strains are more frequently recovered from CF sputum cultures than are others, and these typically reside in Genomovar III. The ET12 clone is a Genomovar III strain that predominates among CF patients in Canada and the United Kingdom and is characterized by distinctive cblA-encoded pili that have a cable-like morphology. In a previous survey of B. cepacia complex isolates recovered from 606 CF patients in the US, a single Genomovar III ET12 isolate (isolate AU0007) was identified; several cblA-containing Genomovar I isolates, however, were also detected. In the study reported here, analysis by PFGE revealed several distinct strain types among these Genomovar I isolates, and sequence analysis of their cblA genes demonstrated 87·8–88·4% identity to the ET12 cblA sequence. Southern analysis indicated that the cblA variant from each Genomovar I isolate resides on a 4 kbp EcoRI fragment, in contrast to ET12 isolates, in which cblA localizes to a 5 kbp EcoRI fragment. Western blot assay indicated expression of the 16 kDa major pilin subunit by ET12 isolates, including AU0007, but neither whole-cell nor surface-protein extracts of the Genomovar I reacted. Electron microscopy revealed the complete absence of pili expression by the Genomovar I isolates. In contrast to typical ET12 isolates, AU0007 appeared to be hyperpiliated with rigid pili that lacked the cable morphology and did not bind cytokeratin 13, which has been previously identified as the epithelial cell receptor for the ET12 cable-pili-associated adhesin.

Eshwar Mahenthiralingam - One of the best experts on this subject based on the ideXlab platform.

  • The multifarious, multireplicon Burkholderia cepacia complex
    Nature Reviews Microbiology, 2005
    Co-Authors: Eshwar Mahenthiralingam, Teresa A. Urban, Joanna B. Goldberg
    Abstract:

    Members of the Burkholderia cepacia complex (Bcc) can cause disease or can be beneficial for plants. The Bcc bacteria are nutritionally diverse and can grow in diverse environments; some can even degrade important pollutants or can use penicillin G as a sole source of carbon. Bcc bacteria can act as opportunistic pathogens and are associated with a variable clinical course in patients with cystic fibrosis (CF) that can include severe lung infections, necrotizing pneumonia and septicaemia. The Bcc currently includes nine species (Genomovars). The most common species that cause infections in CF patients are B. cenocepacia (Genomovar III) and B. multivorans (Genomovar II). Epidemiological studies have identified highly transmissible clones of Bcc bacteria, some of which have been associated with serious outbreaks among CF patients. One of the most prevalent belongs to the ET-12 lineage. However, the factors responsible for the transmission and virulence of these or other epidemic strains have not been identified. It is suggested that, in addition to bacterial factors, the host response is responsible for the variable clinical course among patients. The availability of genome sequence data for the ET-12 lineage strain B. cenocepacia J2315 has led to the identification of novel genomic islands and facilitated the development of genetic tools to investigate the pathogenesis of this bacterium. These tools have been exploited, along with various models systems that include infections in cell cultures, animals, plants, worms, and amoebae, which have led to the identification and characterization of potential virulence factors. The Burkholderia cepacia complex (Bcc) is a collection of genetically distinct but phenotypically similar bacteria that are divided into at least nine species. Bcc bacteria are found throughout the environment, where they can have both beneficial and detrimental effects on plants and some members can also degrade natural and man-made pollutants. Bcc bacteria are now recognized as important opportunistic pathogens that can cause variable lung infections in cystic fibrosis patients, which result in asymptomatic carriage, chronic infection or 'cepacia syndrome', which is characterized by a rapid decline in lung function that can include invasive disease. Here we highlight the unique characteristics of the Bcc, focusing on the factors that determine virulence.

  • proposal to accommodate burkholderia cepacia Genomovar vi as burkholderia dolosa sp nov
    International Journal of Systematic and Evolutionary Microbiology, 2004
    Co-Authors: Karen Vermis, Eshwar Mahenthiralingam, Hans Nelis, John J. Lipuma, Torn Coenye, Peter Vandamme
    Abstract:

    Phenotypic and genotypic studies revealed new tools for differentiating Burkholderia cepacia Genomovar VI from Burkholderia multivorans and other B. cepacia-complex species. Hence, the name Burkholderia dolosa sp. nov. is proposed, with LMG 18943T (=CCUG 47727T) as the type strain. B. dolosa can be differentiated from other B. cepacia-complex bacteria by its inability to assimilate tryptamine, azelaic acid and salicin and by its failure to grow on the B. cepacia-selective medium PCAT. Both 16S rDNA and recA RFLP analysis revealed unique B. dolosa restriction patterns. In addition, new 16S rDNA- and recA-based PCR assays allowed its specific identification.

  • Updated version of the Burkholderia cepacia complex experimental strain panel.
    Journal of Clinical Microbiology, 2003
    Co-Authors: Tom Coenye, Peter Vandamme, John J. Lipuma, John R. W. Govan, Eshwar Mahenthiralingam
    Abstract:

    The Burkholderia cepacia complex consists of nine closely related species: Burkholderia cepacia Genomovars I and VI, Burkholderia multivorans (Genomovar II), Burkholderia cenocepacia (Genomovar III), Burkholderia stabilis (Genomovar IV), Burkholderia vietnamiensis (Genomovar V), Burkholderia

  • evaluation of species specific reca based pcr tests for Genomovar level identification within the burkholderia cepacia complex
    Journal of Medical Microbiology, 2002
    Co-Authors: Karen Vermis, Tom Coenye, Eshwar Mahenthiralingam, Hans Nelis, Peter Vandamme
    Abstract:

    The Burkholderia cepacia complex presently comprises nine Genomovars: B. cepacia (Genomovar I), B. multivorans (Genomovar II), B. cepacia Genomovar III, B. stabilis (Genomovar IV), B. vietnamiensis (Genomovar V), B. cepacia Genomovar VI, B. ambifaria (Genomovar VII), B. anthina (Genomovar VIII) and B. pyrrocinia (Genomovar IX). Strains of each Genomovar can colonise the respiratory tract of cystic fibrosis (CF) patients. However, the majority of infections in CF patients are caused by B. multivorans and B. cepacia Genomovar III isolates. Accurate Genomovar-level identification is best achieved through a polyphasic approach combining phenotypic and genotypic analyses. In the present study, the sensitivity and specificity of recA-based Genomovar specific primer pairs were evaluated with a collection of 508 B. cepacia complex isolates representing all nine Genomovars. The assays for the identification of B. multivorans (sensitivity and specificity, 100%), B. cepacia Genomovar III (sensitivity, 92%; specificity, 100%), and B. ambifaria (sensitivity and specificity, 100%) were the most efficient. However, the B. cepacia Genomovar I assay lacked sensitivity (72%) and cross-reacted with all B. pyrrocinia isolates examined. Several new recA RFLP types were also revealed within the B. cepacia complex. One of these profiles was shared by a clinical and an environmental B. cepacia-like isolate and by the B. ubonensis type strain. The latter organism is a recently described soil bacterium. Its relationship to the various B. cepacia complex Genomovars needs further study.

  • PCR-Based Detection and Identification of Burkholderia cepacia Complex Pathogens in Sputum from Cystic Fibrosis Patients
    Journal of Clinical Microbiology, 2001
    Co-Authors: Andrew Mcdowell, Eshwar Mahenthiralingam, John E Moore, B C Millar, K. Dunbar, A.k. Webb, M. E. Dodd, S.l. Martin, Christopher J. Scott, M Crowe
    Abstract:

    Patients with cystic fibrosis (CF) are extremely susceptible to pulmonary infection with a range of bacterial flora (29). Over the last 20 years, Burkholderia cepacia has emerged as an opportunistic microbial pathogen in patients with CF as well as immunocompromised patients without CF (13, 25). B. cepacia can be transmitted between patients, is frequently resistant to a wide range of antimicrobial treatments, and produces an increase in pulmonary symptoms and a decrease in long-term survival (9, 10, 27, 28). In addition, approximately 20% of all CF patients infected with B. cepacia succumb to cepacia syndrome, a necrotizing pneumonia with bacteremia which leads to an acute and frequently fatal clinical decline (17). In response to these serious problems, CF centers now segregate patients so that cross-infection with the organism is reduced (12, 20). The taxonomy of B. cepacia has proved to be very complex. Initial phylogenetic investigations demonstrated that isolates previously classified as B. cepacia comprised at least five genotypically distinct Genomovars, collectively referred to as the B. cepacia complex (30). B. cepacia Genomovar V has been identified as Burkholderia vietnamiensis, a nitrogen-fixing organism associated with rice roots (11), while B. cepacia Genomovar II and Genomovar IV have been proposed as the new species Burkholderia multivorans and Burkholderia stabilis, respectively (30, 31). At present, B. cepacia Genomovar III awaits assignment of a binomial species name pending the availability of suitable phenotypic identification criteria. Strains of B. cepacia Genomovar I (which contains the type strain) will be known as B. cepacia when taxonomic reappraisal is complete. Very recently, the description of B. cepacia Genomovar VI and Genomovar VII (also known as Burkholderia ambifaria sp. nov.) as members of the B. cepacia complex has further demonstrated the extraordinary diversity of this group of organisms (6, 7, 16). Ribotyping of serial B. cepacia complex strains has revealed that CF patients are infected and colonized with a single Genomovar strain (3, 22). Although all species of the B. cepacia complex have been cultured from CF patients, the majority of infections result from strains of B. cepacia Genomovar III and B. multivorans (21, 30). B. cepacia complex organisms also show differences with respect to transmissibility and virulence, with strains of B. cepacia Genomovar III being responsible for most epidemic outbreaks as well as cases of cepacia syndrome (8, 30). Knowledge of the B. cepacia complex Genomovar species responsible for pulmonary infections is extremely important for appropriate segregation and grouping of CF patients into cohorts. We routinely use the recA-based diagnostic scheme recently described by Mahenthiralingam et al. (23) to identify B. cepacia complex isolates. Particular advantages of this multifaceted approach are its capacity to identify all Genomovars of the B. cepacia complex and to differentiate B. cepacia Genomovar III isolates into the two distinct recA cluster groups, known as III-A and III-B. This diagnostic approach provides the clinical microbiologist with a variety of experimental methods to identify Genomovar-specific polymorphisms within B. cepacia complex isolates. These include restriction fragment length polymorphism (RFLP) analysis of the PCR-amplified recA gene, the use of Genomovar-specific recA primers, and direct nucleotide sequencing of recA. Due to the flexibility of the approach, these tests can be used individually or, if desired, can be applied for multiple complementary analyses. We now describe the novel application of recA-based PCR-RFLP analysis for the rapid detection and identification of B. cepacia complex Genomovars directly from CF sputum samples. The ability, using a single PCR, to both detect and differentiate all members of the B. cepacia complex in sputum may prove particularly valuable for diagnostic laboratories.