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Species abundance and diversity of Burkholderia cepacia complex in the environment.

ABSTRACT: Despite considerable interest in studying Burkholderia cepacia complex in the environment, we still do not have efficient methods to detect, isolate, and screen large numbers of B. cepacia isolates. To better describe the ecology and diversity of B. cepacia complex, a colony hybridization assay was developed to detect specifically all species of the complex based on polymorphism of the variable V3 region of the 16S rRNA sequence. The sensitivity of the assay was dramatically enhanced by using a probe consisting of three repeats of a B. cepacia complex-specific probe, each separated by a phosphoramidite spacer. In addition, a duplex PCR targeting B. cepacia complex-specific recA and 16S rRNA sequences was developed to enable a fast and reliable diagnostic assay for members of the complex. When applied to maize rhizosphere samples, colony hybridization results were in good agreement with those of most-probable-number duplex PCR, both indicating a >100-fold fluctuation of abundance between individual plants. Using restriction analysis of recA for a total of 285 confirmed isolates of the B. cepacia complex, up to seven B. cepacia complex species were identified; however, their diversity and abundance were not evenly distributed among individual plants, and several allelic variants were commonly found from the same rhizosphere sample. These results indicate that not only complex communities of B. cepacia complex species and closely related strains of the same species may coexist at high population levels but also species composition and abundance may dramatically vary between individual plants.

SUBMITTER: Ramette A

PROVIDER: S-EPMC1065178 | biostudies-literature | 2005 Mar

REPOSITORIES: biostudies-literature

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Species abundance and diversity of Burkholderia cepacia complex in the environment.

Ramette Alban A LiPuma John J JJ Tiedje James M JM

Applied and environmental microbiology 20050301 3

Despite considerable interest in studying Burkholderia cepacia complex in the environment, we still do not have efficient methods to detect, isolate, and screen large numbers of B. cepacia isolates. To better describe the ecology and diversity of B. cepacia complex, a colony hybridization assay was developed to detect specifically all species of the complex based on polymorphism of the variable V3 region of the 16S rRNA sequence. The sensitivity of the assay was dramatically enhanced by using a ...[more]

PMID: 15746318

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Project description:Definitive identification of the species in the Burkholderia cepacia complex by routine clinical microbiology methods is difficult. Phenotypic tests to identify B. multivorans and B. vietnamiensis have been established; more recent work indicates B. stabilis may also be identified by growth characteristics and biochemical tests. However, attempts to identify genomovars I and III have, thus far, proved unsuccessful. Previously, we demonstrated the utility of two primer pairs, directed to the rRNA operon, to specifically identify the B. cepacia complex in a PCR. One of these primer pairs, G1-G2, only amplified a DNA fragment from genomovars I and III and B. stabilis in a PCR with genomic DNA isolated from prototypical strains representing the five genomovars. Sequence analysis of the rRNA operon for all the genomovars indicated that this primer pair targeted a region shared by these isolates. Further analysis revealed a region of heterogeneity between genomovar III and B. stabilis internal to the amplified product of G1-G2. Primers designed to target this region were tested with prototypical strains following an initial amplification with the G1-G2 primer pair. New primers specific for the prototypical genomovar III and B. stabilis were designated SPR3 and SPR4, respectively. Analysis of 93 isolates representing 18 genomovar I, 13 B. multivorans, 36 genomovar III, 11 B. stabilis, and 15 B. vietnamiensis isolates was performed. DNA from all isolates of genomovars I and III and B. stabilis was amplified by G1-G2. Genomovar III isolates yielded a product with SPR3/G1 while B. stabilis amplified with SPR4-G1. Genomovar I isolates were amplified by either SPR3-G1 or SPR4-G1, but not both. B. multivorans yielded a product with SPR3-G1 but not G1-G2, and B. vietnamiensis isolates were negative in all PCRs. Thus using an algorithm with G1-G2, SPR3-G1, and SPR4-G1 primers in a PCR analysis, genomovar III isolates can be separated from B. stabilis and the identity of B. multivorans and B. vietnamiensis can be confirmed.

Project description:Ancestral genetic exchange between members of many important bacterial pathogen groups has resulted in phylogenetic relationships better described as networks than as bifurcating trees. In certain cases, these reticulated phylogenies have resulted in phenotypic and molecular overlap that challenges the construction of practical approaches for species identification in the clinical microbiology laboratory. Burkholderia cepacia complex (Bcc), a betaproteobacteria species group responsible for significant morbidity in persons with cystic fibrosis and chronic granulomatous disease, represents one such group where network-structured phylogeny has hampered the development of diagnostic methods for species-level discrimination. Here, we present a phylogeny-informed proteomics approach to facilitate diagnostic classification of pathogen groups with reticulated phylogenies, using Bcc as an example. Starting with a set of more than 800 Bcc and Burkholderia gladioli whole-genome assemblies, we constructed phylogenies with explicit representation of inferred interspecies recombination. Sixteen highly discriminatory peptides were chosen to distinguish B. cepacia, Burkholderia cenocepacia, Burkholderia multivorans, and B. gladioli and multiplexed into a single, rapid liquid chromatography-tandem mass spectrometry multiple reaction monitoring (LC-MS/MS MRM) assay. Testing of a blinded set of isolates containing these four Burkholderia species demonstrated 50/50 correct automatic negative calls (100% accuracy with a 95% confidence interval [CI] of 92.9 to 100%), and 70/70 correct automatic species-level positive identifications (100% accuracy with 95% CI 94.9 to 100%) after accounting for a single initial incorrect identification due to a preanalytic error, correctly identified on retesting. The approach to analysis described here is applicable to other pathogen groups for which development of diagnostic classification methods is complicated by interspecies recombination.

Project description:A novel Gram negative rod-shaped bacterium, designated strain MSh1T, was isolated from Southeast Pahang tropical peat swamp forest soil in Malaysia and characterized using a polyphasic taxonomy approach. The predominant cellular fatty acids (>10.0%) were C16:0 (31.7%), C17:0 cyclo (26.6%), and C19:0 cyclo ?8c (16.1%). The polar lipids detected were phosphatidylglycerol, phosphatidylethanolamine, and diphosphatidylglycerol. The predominant ubiquinone was Q-8. This revealed that strain MSh1T belongs to the genus Burkholderia. The type strain MSh1T can be differentiated from other Burkholderia cepacia complex (Bcc) species by phylogenetic analysis of 16S rRNA gene sequence, multilocus sequence analysis (MLSA), average nucleotide identity (ANI) and biochemical tests. DNA-DNA relatedness values between strain MSh1T and closely related type strains were below the 70% threshold value. Based on this polyphasic study of MSh1T, it can be concluded that this strain represents a novel species within the Bcc, for which the name Burkholderia paludis sp. nov. is proposed. The type strain is MSh1T (= DSM 100703T = MCCC 1K01245T). The dichloromethane extract of MSh1T exhibited antimicrobial activity against four Gram positive bacteria (Enterococcus faecalis ATCC 29212, E. faecalis ATCC 700802, Staphylococcus aureus ATCC 29213, S. aureus ATCC 700699) and a Gram negative bacteria (Escherichia coli ATCC 25922). Further purification work has led to the isolation of Compound 1, pyochelin. Pyochelin demonstrated antimicrobial activity against four S. aureus strains and three E. faecalis strains with MIC-values of 3.13 ?g/ml and 6.26 ?g/ml, respectively. SEM analysis showed that the cellular morphology of E. faecalis ATCC 700802 was not affected by pyochelin; suggesting that it might target the intracellular components. Pyochelin, a siderophore with antimicrobial activity might be useful in treating bacterial infections caused by S. aureus and E. faecalis, however further work has to be done.

Dataset Information

Species abundance and diversity of Burkholderia cepacia complex in the environment.

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Species abundance and diversity of Burkholderia cepacia complex in the environment.

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