Effect of reduced pH on inorganic polyphosphate accumulation by Burkholderia cepacia complex isolates.

Aims: Burkholderia cepacia complex (Bcc) isolates causing pulmonary infection in cystic fibrosis (CF) patients grow within an acidic environment in the lung. As exposure to acid pH has been shown to increase intracellular inorganic polyphosphate (polyP) formation in some bacteria, we investigated the inter-relationship between acidic pH and polyP accumulation in Bcc isolates.

Use of breakpoint combination sensitivity testing as a simple and convenient method to evaluate the combined effects of ceftazidime and tobramycin on Pseudomonas aeruginosa and Burkholderia cepacia complex isolates in vitro

An in vitro method of determining the activity of antibiotics in combination which is simple and convenient to perform and which could be used routinely in clinical microbiology laboratories is desirable. We investigated the activity, against Pseudomonas aeruginosa and Burkholderia cepacia complex clinical isolates, of ceftazidime and tobramycin in combination using a broth macrodilution sensitivity method based on breakpoint minimum inhibitory concentrations and compared the results obtained using this method with those obtained using the microtitre checkerboard method. There was good agreement in interpretation of results between the two methods for both P. aeruginosa (90%) and B. cepacia complex isolates (70%) with tobramycin and for P. aeruginosa isolates (70%) with ceftazidime. As the breakpoint combination sensitivity testing method employs only four tubes and does not require initial determination of individual antibiotic minimum inhibitory concentrations, it is simpler and more convenient for determining the activity of antibiotics in combination than the microtitre checkerboard method. The use of this method in routine microbiology laboratories to determine the activity of antibiotic combinations against clinical isolates should optimise treatment of infection by ensuring that appropriate antibiotic combinations are prescribed. (C) 2004 Elsevier B.V. All rights reserved.

Interactions of Burkholderia cenocepacia and other Burkholderia cepacia complex bacteria with epithelial and phagocytic cells

Burkholderia cenocepacia is a member of the B. cepacia complex (Bcc), a group of opportunistic bacteria that infect the airways of patients with cystic fibrosis (CF) and are extraordinarily resistant to almost all clinically useful antibiotics. Infections in CF patients with Bcc bacteria generally lead to a more rapid decline in lung function, and in some cases to the 'cepacia syndrome', a virtually deadly exacerbation of the lung infection with systemic manifestations. These characteristics of Bcc bacteria contribute to higher morbidity and mortality in infected CF patients. In the last 10 years considerable progress has been made in understanding the interactions between Bcc bacteria and mammalian host cells. Bcc isolates can survive either intracellularly within eukaryotic cells or extracellularly in host tissues. They survive within phagocytes and respiratory epithelial cells, and they have the ability to breach the respiratory epithelium layer. Survival and persistence of Bcc bacteria within host cells and tissues are believed to play a key role in pulmonary infection and to contribute to the persistent inflammation observed in patients with CF. This review summarizes recent findings concerning the interaction between Bcc bacteria and epithelial and phagocytic cells.

A quorum-quenching approach to investigate the conservation of quorum-sensing-regulated functions within the Burkholderia cepacia complex

Taxonomic studies of the past few years have shown that the Burkholderia cepacia complex, a heterogeneous group of B. cepacia-like organisms, consists of at least nine species. B. cepacia complex strains are ubiquitously distributed in nature and have been used for biocontrol, bioremediation, and plant growth promotion purposes. At the same time, B. cepacia complex strains have emerged as important opportunistic pathogens of humans, particularly those with cystic fibrosis. All B. cepacia complex species investigated thus far use quorum-sensing (QS) systems that rely on N-acylhomoserine lactone (AHL) signal molecules to express certain functions, including the production of extracellular proteases, swarming motility, biofilm formation, and pathogenicity, in a population-density-dependent manner. In this study we constructed a broad-host-range plasmid that allowed the heterologous expression of the Bacillus sp. strain 240B1 AiiA lactonase, which hydrolyzes the lactone ring of various AHL signal molecules, in all described B. cepacia complex species. We show that expression of AiiA abolished or greatly reduced the accumulation of AHL molecules in the culture supernatants of all tested B. cepacia complex strains. Phenotypic characterization of wild-type and transgenic strains revealed that protease production, swarming motility, biofilm formation, and Caenorhabditis elegans killing efficiency was regulated by AHL in the large majority of strains investigated.

Diverse pathogenicity of Burkholderia cepacia complex strains in the Caenorhabditis elegans host model

A fast screening method was developed to assess the pathogenicity of a diverse collection of environmental and clinical Burkholderia cepacia complex isolates in the nematode Caenorhabditis elegans. The method was validated by comparison with the standard slow-killing assay. We observed that the pathogenicity of B. cepacia complex isolates in C. elegans was strain-dependent but species-independent. The wide range of observed pathogenic phenotypes agrees with the high degree of phenotypic variation among species of the B. cepacia complex and suggests that the taxonomic classification of a given strain within the complex cannot predict pathogenicity.

Burkholderia cepacia complex isolates survive intracellularly without replication within acidic vacuoles of Acanthamoeba polyphaga

We have previously demonstrated that isolates of the Burkholderia cepacia complex can survive intracellularly in murine macrophages and in free-living Acanthamoeba. In this work, we show that the clinical isolates B. vietnamiensis strain CEP040 and B. cenocepacia H111 survived but did not replicate within vacuoles of A. polyphaga. B. cepacia-containing vacuoles accumulated the fluid phase marker Lysosensor Blue and displayed strong blue fluorescence, indicating that they had low pH. In contrast, the majority of intracellular bacteria within amoebae treated with the V-ATPse inhibitor bafilomycin A1 localized in vacuoles that did not fluoresce with Lysosensor Blue. Experiments using bacteria fluorescently labelled with chloromethylfluorescein diacetate demonstrated that intracellular bacteria remained viable for at least 24 h. In contrast, Escherichia coli did not survive within amoebae after 2 h post infection. Furthermore, intracellular B. vietnamiensis CEP040 retained green fluorescent protein within the bacterial cytoplasm, while this protein rapidly escaped from the cytosol of phagocytized heat-killed bacteria into the vacuolar lumen. Transmission electron microscopy analysis confirmed that intracellular Burkholderia cells were structurally intact. In addition, both Legionella pneumophila- and B. vietnamiensis-containing vacuoles did not accumulate cationized ferritin, a compound that localizes within the lysosome. Thus, our observations support the notion that B. cepacia complex isolates can use amoebae as a reservoir in the environment by surviving without intracellular replication within an acidic vacuole that is distinct from the lysosomal compartment.

Construction and evaluation of plasmid vectors optimized for constitutive and regulated gene expression in Burkholderia cepacia complex isolates

Genetic studies with Burkholderia cepacia complex isolates are hampered by the limited availability of cloning vectors and by the inherent resistance of these isolates to the most common antibiotics used for genetic selection. Also, some of the promoters widely employed for gene expression in Escherichia coli are inefficient in B. cepacia. In this study, we have utilized the backbone of the vector pME6000, a derivative of the pBBR1 plasmid that was originally isolated from Bordetella bronchiseptica, to construct a set of vectors useful for gene expression in B. cepacia. These vectors contain either the constitutive promoter of the S7 ribosomal protein gene from Burkholderia sp. strain LB400 or the arabinose-inducible P(BAD) promoter from E. coli. Promoter sequences were placed immediately upstream of multiple cloning sites in combination with the minimal sequence of pME6000 required for plasmid maintenance and mobilization. The functionality of both vectors was assessed by cloning the enhanced green fluorescent protein gene (e-gfp) and determining the levels of enhanced green fluorescent protein expression and fluorescence emission for a variety of clinical and environmental isolates of the B. cepacia complex. We also demonstrate that B. cepacia carrying these constructs can readily be detected intracellularly by fluorescence microscopy following the infection of Acanthamoeba polyphaga.