Burkholderia anthina sp. nov. and Burkholderia pyrrocinia,two additional Burkholderia cepacia complex bacteria,may confound results of new molecular diagnostic tools

Nineteen Burkholderia cepacia-like isolates of human and environmental origin could not be assigned to one of the seven currently established genomovars using recently developed molecular diagnostic tools for B. cepacia complex bacteria. Various genotypic and phenotypic characteristics were examined. The results of this polyphasic study allowed classification of the 19 isolates as an eighth B. cepacia complex genomovar (Burkholderia anthina sp. nov.) and to design tools for its identification in the diagnostic laboratory. In addition, new and published data for Burkholderia pyrrocinia indicated that this soil bacterium is also a member of the B. cepacia complex. This highlights another potential source for diagnostic problems with B. cepacia-like bacteria.     

Phenotypic and genotypic identification of bacteria from the Burkholderia cepacia complex

AIM: Evaluation of the diagnostic value of pheno- and genotypic characteristics of B. cepacia strains collection. MATERIALS AND METHODS: Phenotypic and genetic methods of identification and differentiation of 25 strains of the B. cepacia complex. RESULTS: Polyphasic taxonomic approach utilizing multiple diagnostic tests was used for accurate identification of Burkholderia species. Algorithm for identification of microorganisms from the B. cepacia complex was developed. CONCLUSION: Combined use of phenotypic and molecular genetic tests, such as recA gene PCR, is recommended for differentiation of the B. cepacia complex genomovars.     

Burkholderia cepacia complex: distribution of genomovars among isolates from the maize rhizosphere in Italy

Burkholderia cepacia is a 'complex' in which seven genomic species or genomovars have so far been identified. It appears that all seven B. cepacia genomovars are capable of causing infections in vulnerable persons; in particular, the importance of Burkholderia multivorans (genomovar II) and B. cepacia genomovar III among cystic fibrosis isolates, especially epidemic ones, has been emphasized. In order to acquire a better comprehension of the genomovar composition of environmental populations of B. cepacia, 120 strains were isolated from the rhizosphere of maize plants cultivated in fields located in northern, central and southern Italy. The identification of the different genomovars was accomplished by a combination of molecular polymerase chain reaction (PCR)-based techniques, such as restriction fragment length polymorphism (RFLP) analysis of 16S rDNA (ARDRA), genomovar-specific PCR tests and RFLP analyses based on polymorphisms in the recA gene whole-cell protein electrophoresis. ARDRA analysis allowed us to distinguish between all B. cepacia genomovars except B. cepacia genomovar I, B. cepacia genomovar III and Burkholderia ambifaria (genomovar VII). The latter genomovars were differentiated by means of recA PCR tests and RFLP analyses. Among the rhizospheric isolates of B. cepacia, we found only B. cepacia genomovar I, B. cepacia genomovar III, Burkholderia vietnamiensis (genomovar V) and B. ambifaria. B. cepacia genomovars I and III and B. ambifaria were recovered from all three fields, whereas B. vietnamiensis was detected only in the population isolated from the field located in central Italy. Among strains isolated from northern and southern Italy, the most abundant genomovars were B. ambifaria and B. cepacia genomovar III respectively; in contrast, the population isolated in central Italy showed an even distribution of strains among genomovars. These results indicate that it is not possible to differentiate clinical and environmental strains, or pathogenic and non-pathogenic strains, of the B. cepacia complex simply on the basis of genomovar status, and that the environment may serve as a reservoir for B. cepacia genomovar III infections in vulnerable humans.     

Burkholderia cepacia genomovar III Is a common plant-associated bacterium

A polyphasic taxonomic study involving DNA-DNA hybridization, whole-cell protein electrophoresis, and 16S ribosomal DNA sequence analysis revealed that a group of Burkholderia cepacia-like organisms isolated from the rhizosphere or tissues of maize, wheat, and lupine belong to B. cepacia genomovar III, a genomic species associated with "cepacia syndrome" in cystic fibrosis patients. The present study also revealed considerable protein electrophoretic heterogeneity within this species and demonstrated that the B. cepacia complex consists of two independent phylogenetic lineages.     

Biofilm formation and acyl homoserine lactone production in the Burkholderia cepacia complex

Acyl homoserine lactone (acyl-HSL)-mediated gene regulation has been shown to influence biofilm formation in one Burkholderia cepacia cystic fibrosis isolate, but it is not known whether this relationship is a consistent feature of the several genomic species that make up the B. cepacia complex (BCC). We screened strains belonging to genomovars I to V of the BCC for biofilm formation on an abiotic surface and for acyl-HSL synthesis. We determined that organisms from each of these genomovars were capable of biofilm formation. Similarly, acyl-HSL was synthesized by organisms from each of genomovars I to V, with most isolates producing octanoyl-HSL in greatest abundance. When biofilms were grown in Luria broth, acyl-HSL synthesis and biofilm formation appeared to be associated, but these phenotypes were independent when the biofilms were grown in basal salts containing citrate. Genomovar V strains synthesized the greatest quantities of acyl-HSL, and genomovar II and III-A strains elaborated the most abundant biofilms. Quorum sensing may play a role in BCC pathogenesis, but it may not regulate biofilm formation under all growth conditions.     

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.     

Interspecies biofilms of Pseudomonas aeruginosa and Burkholderia cepacia.

The leading cause of morbidity and mortality in cystic fibrosis (CF) continues to be lung infections with Pseudomonas aeruginosa biofilms. Co-colonization of the lungs with P aeruginosa and Burkholderia cepacia can result in more severe pulmonary disease than P. aeruginosa alone. The interactions between P. aeruginosa biofilms and B. cepacia are not yet understood; one possible association being that mixed species biofilm formation may be part of the interspecies relationship. Using the Calgary Biofilm Device (CBD), members of all genomovars of the B. cepacia complex were shown to form biofilms, including those isolated from CF lungs. Mixed species biofilm formation between CF isolates of P. aeruginosa and B. cepacia was readily achieved using the CBD. Oxidation-fermentation lactose agar was adapted as a differential agar to monitor mixed biofilm composition. Scanning electron micrographs of the biofilms demonstrated that both species readily integrated in close association in the biofilm structure. Pseudomonas aeruginosa laboratory strain PAO1, however, inhibited mixed biofilm formation of both CF isolates and environmental strains of the B. cepacia complex. Characterization of the soluble inhibitor suggested pyocyanin as the active compound.     

Burkholderia cepacia complex genomovars: utilization of carbon sources, susceptibility to antimicrobial agents and growth on selective media

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.     

The Burkholderia cepacia rpoE gene is not involved in exopolysaccharide production and onion pathogenicity

Burkholderia cepacia was originally described as the causative agent of bacterial rot of onions, and it has now emerged as an important opportunistic pathogen causing severe chronic lung infections in patients having cystic fibrosis. Burkholderia cepacia is now classified into nine very closely related species (previously designated as genomovars), all of which have been isolated from both environmental and clinical sources and are collectively known as the B. cepacia complex. The alternative extracytoplasmic function sigma factor, sigmaE, has been determined in several bacterial species as making substantial contributions to bacterial survival under stress conditions. Here, we report the identification and characterization of the rpoE gene, encoding sigmaE, of B. cepacia. It is highly similar to sigmaE of other bacteria, including Escherichia coli and Pseudomonas aeruginosa. Studies using an rpoE knockout mutant of B. cepacia revealed that many stress adaptations, including osmotic, oxidative, desiccation, carbon, and nitrogen stress, were independent of sigmaE. Similarly, biofilm formation; production of exopolysaccharides, N-acyl homoserine lactones, and several exoenzymes; and onion pathogenicity were not affected by the absence of sigmaE. In contrast, sigmaE contributed to the adaptation to heat stress and phosphate starvation.     

Sensitivity of the Burkholderia cepacia complex and Pseudomonas aeruginosa to transducing bacteriophages

Burkholderia cepacia is now recognised as a life-threatening pathogen among several groups of immunocompromised patients. In this context, the proposed large-scale use of these bacteria in agriculture has increased the need for a better understanding of the genetics of the species forming the B. cepacia complex. Until now, little information has been available on the bacteriophages of the B. cepacia complex. Transducing phages, named NS1 and NS2, were derived from the lysogenic B. cepacia strains ATCC 29424 and ATCC 17616. The frequency of transduction per phage particle ranged from 1.0x10(-8) to 7.0x10(-6) depending on the phage and recipient strain used. The host range of NS1 and NS2 differed but in each case included environmental and clinical isolates, and strains belonging to several species and genomovars of the B. cepacia complex. The host range of both phages also included Pseudomonas aeruginosa. Some B. cepacia complex isolates were sensitive to the well-characterised P. aeruginosa transducing phages, B3, F116L and G101. The lytic activity of NS1 and NS2 was inhibited by B. cepacia lipopolysaccharide suggesting that this moiety is a binding site for both phages. The molecular size of the NS1 and NS2 genomes was approximately 48 kb.     

Development of a recA gene-based identification approach for the entire Burkholderia genus

Burkholderia is an important bacterial genus containing species of ecological, biotechnological, and pathogenic interest. With their taxonomy undergoing constant revision and the phenotypic similarity of several species, correct identification of Burkholderia is difficult. A genetic scheme based on the recA gene has greatly enhanced the identification of Burkholderia cepacia complex species. However, the PCR developed for the latter approach was limited by its specificity for the complex. By alignment of existing and novel Burkholderia recA sequences, we designed new PCR primers and evaluated their specificity by testing a representative panel of Burkholderia strains. PCR followed by restriction fragment length polymorphism analysis of an 869-bp portion of the Burkholderia recA gene was not sufficiently discriminatory. Nucleotide sequencing followed by phylogenetic analysis of this recA fragment differentiated both putative and known Burkholderia species and all members of the B. cepacia complex. In addition, it enabled the design of a Burkholderia genus-specific recA PCR that produced a 385-bp amplicon, the sequence of which was also able to discriminate all species examined. Phylogenetic analysis of 188 novel recA genes enabled clarification of the taxonomic position of several important Burkholderia strains and revealed the presence of four novel B. cepacia complex recA lineages. Although the recA phylogeny could not be used as a means to differentiate B. cepacia complex strains recovered from clinical infection versus the natural environment, it did facilitate the identification of clonal strain types of B. cepacia, B. stabilis, and B. ambifaria capable of residing in both niches.     

Isolation of Burkholderia cepacia complex genomovars from waters

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.     

A putative type III secretion gene cluster is widely distributed in the Burkholderia cepacia complex but absent from genomovar I

Using probes constructed from Ralstonia solanacearum and Burkholderia pseudomallei, putative type III secretion (TTS) genes were identified in Burkholderia cepacia J2315 (genomovar III). A cosmid clone containing DNA with homology to five TTS genes was sub-cloned and regions were sequenced in order to design oligonucleotides for polymerase chain reaction assays. These indicated that two putative TTS genes (bcscQ and bcscV) were present in all members of the B. cepacia complex with the exception of strains from genomovar I. Southern blot assays confirmed this observation, suggesting that the lack of a TTS gene cluster may define a major difference between B. cepacia genomovar I and other members of the B. cepacia complex, including genomovar III. In contrast to TTS gene clusters in other bacteria, a putative gene homologous to the virB1 gene of Brucella suis was located directly downstream of bcscQR.     

Sequence divergence in type III secretion gene clusters of the Burkholderia cepacia complex

The Burkholderia cepacia complex (BCC) comprises a group of bacteria associated with opportunistic infections, especially in cystic fibrosis patients. B. cenocepacia J2315, of the transmissible ET12 lineage, contains a type III secretion (TTS) gene cluster implicated in pathogenicity. PCR and hybridisation assays indicate that the TTS gene cluster is present in all members of the BCC except B. cepacia (formerly genomovar I). The TTS gene clusters of B. cenocepacia J2315 and B. multivorans are similar in organisation but have variable levels of gene identity. Nucleotide sequence data obtained for the equivalent region of the B. cepacia genome indicate the absence of TTS structural genes due to a rearrangement likely to involve more than one step.     

Virulent properties of hospital strains of bacteria of the Burkholderia cepacia complex, isolated in hospitals of Moscow

The results of the study of hospital strains of the B. cepacia complex, isolated in hospitals of Moscow, with the use of phenotypical and molecular-genetic methods are presented. The phenotypical methods made it possible to differentiate Russian strains and classify them with a group of genomovars (I, III, IV). As the result the epidemic importance of the strains with epidemic markers, having specific characteristics for every clinic, was determined. The detection of the collection of genes cepI and cepR in the strains made confirmed the epidemic importance of the stains which had, due to the regulatory "quorum sensing" (QS) system, the potential capacity for inducing infection and persisting in the patient's body. The presence of gene cepR in all strains and the absence of gene cepl in 33% of strains gave evidence to suggest that in some strains the activation of the production of pathogenicity factors required the presence of other bacteria having the fully developed QS system. Thus, the new complex approach with the use of phenotypical and molecular-genetic methods permits more precise identification of the source of hospital infection induced by the bacteria of the B. cepacia complex.     

Selective medium for Pseudomonas cepacia containing 9-chloro-9-(4-diethylaminophenyl)-10-phenylacridan and polymyxin B sulfate.

Contamination of solutions and lotions with Pseudomonas cepacia is a growing concern among health professionals. The identification of P. cepacia usually requires a long series of biochemical tests. In an effort to develop a more direct method, we evaluated plate count agar containing 9-chloro-9-(4-diethylaminophenyl)-10-phenylacridan and polymyxin B sulfate at respective concentrations of 1 and 75 micrograms/ml as a medium for selectively isolating P. cepacia. The medium inhibited the growth of all gram-negative bacilli and gram-positive cocci tested except P. cepacia and Serratia marcescens. These two microorganisms could easily be differentiated by their colony morphology and their reactions in the oxidase test. When nonsterilized water samples were inoculated with P. cepacia and spread or streaked on the selective medium, all P. cepacia organisms were recovered. These results demonstrate the usefulness of 9-chloro-9-(4-diethylaminophenyl)-10-phenylacridan and polymyxin B sulfate in the detection of P. cepacia. We believe that this selective medium could be useful in isolating P. cepacia from mixed bacterial flora that might be present in environmental water and water-related samples, such as solutions and lotions.     

Detection of cultured and uncultured Burkholderia cepacia complex bacteria naturally occurring in the maize rhizosphere

The species composition of a Burkholderia cepacia complex population naturally occurring in the maize rhizosphere was investigated by using both culture-dependent and culture-independent methods. B. cepacia complex isolates were recovered from maize root slurry on the two selective media Pseudomonas cepacia azelaic acid tryptamine (PCAT) and trypan blue tetracycline (TB-T) and subjected to identification by a combination of restriction fragment length polymorphism (RFLP) analysis and species-specific polymerase chain reaction (PCR) tests of the recA gene. DNA extracted directly from root slurry was examined by means of nested PCR to amplify recA gene with species-specific B. cepacia complex primers and to obtain a library of PCR amplified recA genes. Using the culture-dependent method the species Burkholderia cepacia, Burkholderia cenocepacia, Burkholderia ambifaria and Burkholderia pyrrocinia were identified, whereas using the culture-independent method also the species Burkholderia vietnamiensis was detected. The latter method also allowed us to highlight a higher diversity within the B. cenocepacia species. In fact, by using the culture-independent method the species B. cenocepacia recA lineages IIIA and IIID besides B. cenocepacia recA lineage IIIB were detected. Moreover, higher heterogeneity of recA RFLP patterns was observed among clones assigned to the species B. cenocepacia than among B. cenocepacia isolates from selective media.     

A novel strategy for the isolation and identification of environmental Burkholderia cepacia complex bacteria

The purpose of this study was to develop a novel strategy for the isolation and identification of Burkholderia cepacia complex bacteria from the home environment of cystic fibrosis (CF) patients. Water and soil samples were enriched in a broth containing 0.1% l-arabinose, 0.1% l-threonine, and a mixture of selective agents including 1 microgml(-1) C-390, 600U ml(-1) polymyxin B sulfate, 10 microgml(-1) gentamycin, 2 microgml(-1) vancomycin and 10 microgml(-1) cycloheximide. On selective media (consisting of the same components as above plus 1.8% agar), several dilutions of the enrichment broth were inoculated and incubated for 5 days at 28 degrees C. Isolates with different randomly amplified polymorphic DNA patterns were inoculated in Stewart's medium. Putative B. cepacia complex bacteria were confirmed by means of recA PCR and further identified by HaeIII-recA restriction fragment length polymorphism analysis. Our results suggest that these organisms may be more widespread in the home environment than previously assumed and that plant associated soil and pond water may be reservoirs of B. cepacia complex infection in CF patients.     

Conservation of a novel protein associated with an antibiotic efflux operon in Burkholderia cenocepacia

Burkholderia cenocepacia is a significant problem in individuals with cystic fibrosis and is a member of the B. cepacia complex of closely related antibiotic resistant bacteria. A salicylate-regulated antibiotic efflux operon has been identified in B. cenocepacia and one of its four genes, llpE, is without parallel in previously reported efflux operons. PCR amplification and sequencing of llpE from B. cepacia complex isolates demonstrated the highest prevalence in B. cenocepacia with a high degree of sequence conservation. While at least one non-synonymous mutation was identified between isolates from different genomovars, only synonymous differences were identified within the IIIA and IIIB sub-groups of B. cenocepacia. Structural modeling suggests that LlpE is a member of the alpha/beta hydrolase enzyme family. Identification of strong structural homology to hydrolases and a high degree of conservation in B. cenocepacia suggests an enzymatic function for LlpE, benefiting survival in the cystic fibrosis lung.