A 16S rRNA-targeted, Cy3-labeled oligonucleotide probe was designed to detect members of the genus Bdellovibrio by fluorescence in situ hybridization. Specific hybridization conditions were established; however, the detection of bdellovibrios in environmental samples required enrichment, confirming that Bdellovibrio spp. are not present in large numbers in the environment.Bdellovibrio and like organisms (BALOs) are predatory gram-negative bacteria that are ubiquitous in terrestrial and aquatic environments. Prey include free-living bacteria as well as plant, animal, and human pathogens (12,13,17). The life cycle of Bdellovibrio has two major stages: a motile, nonreplicative stage spent searching for prey (the attack phase) and a stage spent inside the periplasm of the gram-negative prey cell after the formation of an osmotically stable body termed the bdelloplast (the growth phase) (15,27). Until recently, all bacteria that exhibited these life cycle traits were included in the genus Bdellovibrio. Phylogenetic studies based on 16S rRNA sequences have demonstrated extensive diversity among the organisms in this genus, and two new genera have been established: Bacteriovorax (4) and Peredibacter (8). To date, the genus Bdellovibrio comprises only one species, Bdellovibrio bacteriovorus. However, a Bdellovibrio-like organism (BLO) with a different life cycle was isolated from sewage on lawns of Caulobacter crescentus cells. This epibiotic strain, JSS, does not enter the periplasmic space of the prey cell (20,26). Phylogenetic studies show that it belongs to the genus Bdellovibrio (8) and is a new species (S. F. Koval, unpublished data). Fluorescence in situ hybridization (FISH) with rRNA-targeted oligonucleotide probes has become a commonly used technique for the direct identification of prokaryotes and is widely used to determine bacterial community composition (2, 10). Previously designed BALO-targeted oligonucleotides for use in FISH were not specific enough for the identification of the genus Bdellovibrio, as the targets detected included non-BALOs (8, 16). However, Bdellovibrio-specific primers were used to detect Bdellovibrio sequences in soil (9). In this study, an oligonucleotide probe designated BDE525, targeting a region of the 16S rRNA gene sequence of Bdellovibrio, was designed. The probe was used in a FISH procedure with B. bacteriovorus strains, other BLO isolates, and environmental samples.Cultures. B. bacteriovorus strain 109J was cultured on Escherichia coli ML35 according to the method of Flannagan et al.(11). B. bacteriovorus 6-5-S was cultured on Aquaspirillum serpens VHL and BLO strains JSS and KL1 were cultured on C. crescentus CB2A as described previously by Koval and Hynes (20). BLO strains DM7C, DM8A, and DM11A were cultured on Burkholderia cenocepacia strains grown in Lennox LuriaBertani medium (Difco) for 24 h at 30°C. For the maintenance of predators, 3 ml of prey cells and 1 ml of predators were mixed in 20 ml of HM buffer (3 mM HEPES [Sigma], pH 7.6, with 1 mM CaCl 2 and 0.1 mM MgSO 4 ...
Bdellovibrio and like organisms are predatory bacteria that have the unusual property of using the cytoplasmic constituents of other Gram-negative bacteria as nutrients. These predators may thus provide an alternative approach to the biocontrol of human and plant pathogens. Predators were isolated on Burkholderia cenocepacia K56-2 and J2315 as prey cells, in enrichment cultures with soil and sewage. Three isolates (DM7C, DM8A, and DM11A) were identified as Bdellovibrio bacteriovorus on the basis of morphology, a periplasmic life cycle, and 16S rRNA gene sequencing. The prey range of these isolates was tested on Burkholderia cepacia complex bacteria and several phytopathogenic bacteria of agricultural importance. Of 31 strains of the Burkholderia cepacia complex tested, only 4 were resistant to predation by strain DM7C. A subset of 9 of the prey tested were also susceptible to strains DM8A and DM11A. Of 12 phytopathogens tested, 4 were resistant to strains DM7C and DM8A, and only 2 were resistant to strain DM11A. Thus, Bdellovibrio bacteriovorus strains retrieved from environmental samples on 2 Burkholderia cenocepacia isolates from cystic fibrosis patients did not distinguish in their prey range between other isolates of that pathogen or phytopathogens. Such strains hold promise as potential wide-spectrum biocontrol agents.
Fresh human sewage was examined from a sewage treatment plant for the presence of members of the Burkholderia cepacia complex (BCC) of bacterial organisms and confirmed the presence of viable B. cenocepacia and B. vietnamiensis, by a combination of cultural, phenotypic and genotypic techniques. Both these organisms are important respiratory pathogens for patients with cystic fibrosis (CF). Presently, the survival dynamics of these organisms in sewage effluent and sludge is, as yet, unknown. Therefore, as this study represents the first report of these CF pathogens in sewage and until such survival data is available, careful risk assessment needs to be undertaken in relation to the end use application of potentially contaminated sewage and where such material comes into association with non-colonised patients with cystic fibrosis, so that any potential transmission of these pathogens from sewage to patient is assessed and minimised/eliminated.
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