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Revised approach for identification of isolates within the Burkholderia cepacia complex and description of clinical isolates not assigned to any of the known genomovars.

ABSTRACT: One hundred thirty-eight clinical isolates of the Burkholderia cepacia complex (Bcc) were identified using a modified strategy that involved PCR detection of the cblA gene for the ET12 lineage simultaneously with detection of the Bcc recA PCR product; recA sequence cluster analysis also was part of the strategy. Four strains could not be assigned to any of the known genomovars.

SUBMITTER: Turton JF

PROVIDER: S-EPMC2045300 | biostudies-literature | 2007 Sep

REPOSITORIES: biostudies-literature

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Revised approach for identification of isolates within the Burkholderia cepacia complex and description of clinical isolates not assigned to any of the known genomovars.

Turton Jane F JF Arif Nazia N Hennessy Daneeta D Kaufmann Mary E ME Pitt Tyrone L TL

Journal of clinical microbiology 20070711 9

One hundred thirty-eight clinical isolates of the Burkholderia cepacia complex (Bcc) were identified using a modified strategy that involved PCR detection of the cblA gene for the ET12 lineage simultaneously with detection of the Bcc recA PCR product; recA sequence cluster analysis also was part of the strategy. Four strains could not be assigned to any of the known genomovars. ...[more]

PMID: 17626169

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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:Members of the Burkholderia cepacia complex (Bcc) are an important cause of opportunistic or nosocomial infections that may be hard to treat due to a high incidence of multidrug resistance. We characterised a collection of 51 clinical isolates from this complex, assigning them to 18 sequence types using multi-locus sequence type analysis. Resistance to eight commonly used antibiotics was assessed using by using agar-dilution assays to calculate MICs and widespread and heterogeneous multidrug resistance was confirmed, with eight strains proving resistant to all antibiotics tested. Disc diffusion screening of antimicrobial activity of a range of plant essential oils against these Bcc isolates identified six oils with significant activity (lavender, lemongrass, marjoram, peppermint, tea tree and rosewood) and broth microdilution assays indicated that of these lemongrass and rosewood oils had the highest activity, with MIC50 values of 0.5% and MIC90 values of 1%. Comparison of MIC and MBC values showed that four of these six oils, including lemongrass and rosewood, were bacteriocidal rather than bacteriostatic in their effects. Qualitative analysis of the four bacteriocidal essential oils via GC/MS indicated the presence of 55 different component compounds, mostly monoterpenes. We assessed selected essential oil components as anti-Bcc agents and demonstrated that terpinen-4-ol and geraniol were effective with MICs of 0.125-0.5% (v/v) and 0.125-1% (v/v), respectively. Time-kill studies indicate that these two alcohols are effective against non-growing cells in an efflux-dependent manner. Analysis of bacterial leakage of potassium ions and 260 nm UV-absorbing material on treatment with terpinen-4-ol and geraniol suggested that the observed anti-Bcc activity was a consequence of membrane disruption. This finding was supported by a gas chromatography analysis of bacterial fatty acid methyl esters, which indicated changes in membrane fatty acid composition caused by terpinen-4-ol and geraniol. These essential oils or oil components may ultimately prove useful as therapeutic drugs, for example to treat Bcc infections in CF patients.

Project description:Burkholderia cepacia complex (Bcc) infection, associated with cystic fibrosis (CF) is intrinsically multidrug resistant to antibiotic treatment making eradication from the CF lung virtually impossible. Infection with Bcc leads to a rapid decline in lung function and is often a contraindication for lung transplant, significantly influencing morbidity and mortality associated with CF disease. Standard treatment frequently involves antibiotic combination therapy. However, no formal strategy has been adopted in clinical practice to guide successful eradication. A new class of direct-acting, large molecule polycationic glycopolymers, derivatives of a natural polysaccharide poly-N-acetyl-glucosamine (PAAG), are in development as an alternative to traditional antibiotic strategies. During treatment, PAAG rapidly targets the anionic structural composition of bacterial outer membranes. PAAG was observed to permeabilize bacterial membranes upon contact to facilitate potentiation of antibiotic activity. Three-dimensional checkerboard synergy analyses were used to test the susceptibility of eight Bcc strains (seven CF clinical isolates) to antibiotic combinations with PAAG or ceftazidime. Potentiation of tobramycin and meropenem activity was observed in combination with 8-128 ?g/mL PAAG. Treatment with PAAG reduced the minimum inhibitory concentration (MIC) of tobramycin and meropenem below their clinical sensitivity breakpoints (?4 ?g/mL), demonstrating the ability of PAAG to sensitize antibiotic resistant Bcc clinical isolates. Fractional inhibitory concentration (FIC) calculations showed PAAG was able to significantly potentiate antibacterial synergy with these antibiotics toward all Bcc species tested. These preliminary studies suggest PAAG facilitates a broad synergistic activity that may result in more positive therapeutic outcomes and supports further development of safe, polycationic glycopolymers for inhaled combination antibiotic therapy, particularly for CF-associated Bcc infections.

Dataset Information

Revised approach for identification of isolates within the Burkholderia cepacia complex and description of clinical isolates not assigned to any of the known genomovars.

Publications

Revised approach for identification of isolates within the Burkholderia cepacia complex and description of clinical isolates not assigned to any of the known genomovars.

Similar Datasets

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