Burkholderia

Appl Environ Microbiol, 2003 Dec, 69(12), 7257 - 65
Molecular and culture-based analyses of aerobic carbon monoxide oxidizer diversity; King GM; Isolates belonging to six genera not previously known to oxidize CO were obtained from enrichments with aquatic and terrestrial plants . DNA from these and other isolates was used in PCR assays of the gene for the large subunit of carbon monoxide dehydrogenase (coxL) . CoxL and putative coxL fragments were amplified from known CO oxidizers (e.g., Oligotropha carboxidovorans and Bradyrhizobium japonicum), from novel CO-oxidizing isolates (e.g., Aminobacter sp . strain COX, Burkholderia sp . strain LUP, Mesorhizobium sp . strain NMB1, Stappia strains M4 and M8, Stenotrophomonas sp . strain LUP, and Xanthobacter sp . strain COX), and from several well-known isolates for which the capacity to oxidize CO is reported here for the first time (e.g., Burkholderia fungorum LB400, Mesorhizobium loti, Stappia stellulata, and Stappia aggregata) . PCR products from several taxa, e.g., O . carboxidovorans, B . japonicum, and B . fungorum, yielded sequences with a high degree (>99.6%) of identity to those in GenBank or genome databases . Aligned sequences formed two phylogenetically distinct groups . Group OMP contained sequences from previously known CO oxidizers, including O . carboxidovorans and Pseudomonas thermocarboxydovorans, plus a number of closely related sequences . Group BMS was dominated by putative coxL sequences from genera in the Rhizobiaceae and other alpha-PROTEOBACTERIA: PCR analyses revealed that many CO oxidizers contained two coxL sequences, one from each group . CO oxidation by M . loti, for which whole-genome sequencing has revealed a single BMS-group putative coxL gene, strongly supports the notion that BMS sequences represent functional CO dehydrogenase proteins that are related to but distinct from previously characterized aerobic CO dehydrogenases.

FEMS Microbiol Lett, 2003 Dec 5, 229(1), 9 - 14
Improved flagellin genotyping in the Burkholderia cepacia complex; Winstanley C; Oligonucleotide primers designed to N- and C-terminal sequences of Burkholderia cepacia complex fliC genes were used to amplify and sequence fliC genes from a strain of Burkholderia vietnamiensis and three isolates of Burkholderia multivorans with large fliC genes . Alignments incorporating the new sequences enabled the design of polymerase chain reaction (PCR) primers for extension of a published PCR/restriction fragment length polymorphism typing method, to include isolates that previously failed to yield fliC amplicons . Most B . vietnamiensis isolates and hitherto non-typable Burkholderia cenocepacia isolates contained much smaller fliC genes than previously reported . Although B . multivorans strains with larger fliC genes clustered together, relationships between strains based on fliC sequences did not generally correlate with genomovar status.

Cas Lek Cesk, 2001 Dec 6, 140(24), 752 - 4
{Glanders--an eradicable disease--or a threat?}; Pospisil L; Glanders (malleus), attacking equids and transmissible to humans, does not occur in our geographical area any more, but world-wide eradication has not yet been achieved . Cases of glanders have been reported from India, Iraq, Mongolia and China and in 2001 also from South America . The disease is caused by Burkholderia mallei (earlied known as Bacillus, Pfeiferella, Loefflerella, Malleomyces, Actinobacillus, or Pseudomonas mallei) . The continual interest of microbiologists in the causative agents indicates that glanders cannot be regarded as a closed historic episode . Occupational infections of laboratory personnel occurred during World War II and the years thereafter and the last accident was reported in May 2000 . Topical problems of glanders include the development of a vaccine and antibiotic therapy tested in experimentally infected subjects.

J Environ Sci Health B, 2003 Nov, 38(6), 771 - 82
Phylogenetic and degradation characterization of Burkholderia cepacia WZ1 degrading herbicide quinclorac; Lu Z et al.; Strain WZI capable of degrading quinclorac was isolated from a pesticide manufactory soil and considered to be Burkholderia cepacia, belonged to bacteria, Proteobacteria, beta-Proteobacteria, based on morphology, physio-biochemical properties, whole cell fatty acid analysis and a partial sequencing of 16S rDNA . Strain WZ1 decomposed 90% of quinclorac at original concentration of 1000 mg L(-1) within 11 days . GC/MS analysis showed that the strain degraded quinclorac to 3,7-dichloro-8-quinoline and the cracked residue 2-chloro, 1,4-benzenedicarboxylic acid, indicating that the metabolic pathway was initiated by process of decarboxylation followed by cleavage of the aromatic ring . Stain WZ1 was also able to degrade some other herbicides and aromatic compounds, including 2,4,5-T, phenol, naphthalene and hydrochinone etc . This paper describes for the first time Phylogenetic and degradation characterization of a pure bacterium which, is able to mineralize quinclorac.

Thorax, 2003 Dec, 58(12), 1087 - 91
Burkholderia pseudomallei: another emerging pathogen in cystic fibrosis; O'Carroll MR et al.; BACKGROUND: Burkholderia pseudomallei is an important cause of acute fulminant pneumonia and septicaemia in tropical regions of northern Australia and south east Asia . Subacute and chronic forms of the disease also occur . There have been three recent reports of adults with cystic fibrosis (CF) who presumably acquired B pseudomallei infection during extended vacations or residence in either Thailand or northern Australia . METHODS: The clinical course, molecular characteristics, serology and response to treatment are described in four adult CF patients infected with B pseudomallei . Polymerase chain reaction (PCR) based methods were used to confirm B pseudomallei and exclude B cepacia complex . Genotyping was performed using randomly amplified polymorphic DNA (RAPD) PCR and pulsed field gel electrophoresis (PFGE) . RESULTS: Four patients are described with a mean duration of infection of 32 months . All but one patient lived in tropical Queensland . Two patients (with the longest duration of infection) deteriorated clinically and one subsequently died of respiratory failure . Both responded to intravenous treatment specifically targeting B pseudomallei . Another patient suffered two severe episodes of acute bronchopneumonia following acquisition of B pseudomallei . Eradication of the organism was not possible in any of the cases . PFGE of a sample isolate from each patient revealed the strains to be unique and RAPD analysis showed retention of the same strain within an individual over time . CONCLUSIONS: These findings support a potential pathogenic role for B pseudomallei in CF lung disease, producing both chronic infection and possibly acute bronchopneumonia . Identical isolates are retained over time and are unique, consistent with likely environmental acquisition and not person to person spread . B pseudomallei is emerging as a significant pathogen for patients with CF residing and holidaying in the tropics.

J Bacteriol, 2003 Dec, 185(24), 7266 - 72
Legume symbiotic nitrogen fixation by beta-proteobacteria is widespread in nature; Chen WM et al.; Following the initial discovery of two legume-nodulating Burkholderia strains (L . Moulin, A . Munive, B . Dreyfus, and C . Boivin-Masson, Nature 411:948-950, 2001), we identified as nitrogen-fixing legume symbionts at least 50 different strains of Burkholderia caribensis and Ralstonia taiwanensis, all belonging to the beta-subclass of proteobacteria, thus extending the phylogenetic diversity of the rhizobia . R . taiwanensis was found to represent 93% of the Mimosa isolates in Taiwan, indicating that beta-proteobacteria can be the specific symbionts of a legume . The nod genes of rhizobial beta-proteobacteria (beta-rhizobia) are very similar to those of rhizobia from the alpha-subclass (alpha-rhizobia), strongly supporting the hypothesis of the unique origin of common nod genes . The beta-rhizobial nod genes are located on a 0.5-Mb plasmid, together with the nifH gene, in R . taiwanensis and Burkholderia phymatum . Phylogenetic analysis of available nodA gene sequences clustered beta-rhizobial sequences in two nodA lineages intertwined with alpha-rhizobial sequences . On the other hand, the beta-rhizobia were grouped with free-living nitrogen-fixing beta-proteobacteria on the basis of the nifH phylogenetic tree . These findings suggest that beta-rhizobia evolved from diazotrophs through multiple lateral nod gene transfers.

Environ Microbiol, 2003 Dec, 5(12), 1328 - 40
Conservation of the response regulator gene gacA in Pseudomonas species; de Souza JT et al.; The response regulator gene gacA influences the production of several secondary metabolites in both pathogenic and beneficial Pseudomonas spp . In this study, we developed primers and a probe for the gacA gene of Pseudomonas species and sequenced a 425 bp fragment of gacA from ten Pseudomonas strains isolated from different plant-associated environments . Polymerase chain reaction analysis and Southern hybridization showed that gacA is highly conserved within the genus Pseudomonas: multiple strains of different Pseudomonas species all responded positively to the probe, whereas no response was obtained from 18 other strains representing 14 species that belong to eight different genera of Gram-negative bacteria other than Pseudomonas . Furthermore, from a total of approximately 550 indigenous bacterial isolates obtained from the rhizosphere of wheat, all isolates that hybridized with the gacA probe were classified as Pseudomonas spp . by group-specific primers . Isolates that did not respond with the gacA probe and primers were identified as bacterial genera other than Pseudomonas, including Stenotrophomonas, Cryseomonas and Comamonas spp . These results indicate that gacA can be used as a complementary genetic marker for detection of Pseudomonas spp . in environmental samples . Phylogenetic relationships inferred from the newly sequenced gacA fragments and the sequences of gacA homologues present in the databases, showed six distinct clusters that correspond to the following bacterial families: Pseudomonaceae, Enterobacteriaceae, Alteromonadaceae, Vibrionaceae, Burkholderia and Xanthomonas . Within the Pseudomonadaceae and Enterobacteriaceae, polymorphisms within gacA and its homologues allowed identification of six and five subclusters respectively . Comparison of the gacA gene and GacA protein-based trees with the tree inferred from 16S rDNA sequences yielded a similar overall clustering . These results suggest that gacA and its homologues may provide complementary markers for phylogenetic studies of Pseudomonas spp . and Gram-negative bacteria other than Pseudomonas.

FEMS Microbiol Lett, 2003 Nov 21, 228(2), 287 - 97
Adherence and autoaggregation phenotypes of a Burkholderia cenocepacia cable pilus mutant; Tomich M et al.; Cable pili are unique peritrichous adherence organelles expressed by certain strains of the opportunistic human pathogen Burkholderia cenocepacia . Cable pili have been proposed to facilitate binding to human epithelial cells and mucin, and may play a role in the ability of B . cenocepacia to colonise the respiratory tract of compromised hosts . In this study, a genetic approach was undertaken to assess the role of cable pili in mediating adherence as well as bacterial cell-cell interactions . The cblA gene, encoding the major pilin subunit, was insertionally inactivated, and the resulting mutant was shown to be blocked in CblA expression and in cable pilus morphogenesis . Although non-piliated, the cblA mutant was not defective in adherence to either porcine mucin or to cultured A549 human respiratory epithelial cells . Microscopic and flow cytometric analyses of B . cenocepacia cultures revealed that cable pilus expression facilitated the formation of diffuse cell networks, whereas disruption of cable pilus biogenesis enhanced autoaggregation and the formation of compact cell aggregates . Autoaggregation was observed both in culture and during B . cenocepacia infection of A549 epithelial cell monolayers . These findings indicate that cable pilus expression plays an important role in mediating B . cenocepacia cell-cell interactions, and that both cable pilus-dependent and cable pilus-independent mechanisms may contribute to B . cenocepacia adherence to cellular and acellular surfaces.

Biochem Biophys Res Commun, 2003 Dec 12, 312(2), 323 - 33
Identification and physical organization of the gene cluster involved in the biosynthesis of Burkholderia cepacia complex exopolysaccharide; Moreira LM et al.; Bacteria belonging to the Burkholderia cepacia complex (BCC) are important opportunistic pathogens in patients with cystic fibrosis (CF) . Since approximately 80% of the CF isolates examined produce exopolysaccharide (EPS), it was hypothesized that this EPS may play a role in the colonization and persistence of these bacteria in the CF lung . The present study describes the identification and physical organization of the EPS biosynthetic gene cluster . This bce gene cluster was identified following the isolation of three EPS-defective mutants from the highly mucoid CF isolate IST408, belonging to BCC genomovar I, based on random plasposon insertion mutagenesis and comparison of the nucleotide sequence of the interrupted genes with the available genome of Burkholderia cenocepacia J2315 . This 16.2 kb cluster includes 12 genes and is located on chromosome 2 . Database searches for homologous proteins and secondary structure analysis for the deduced Bce amino acid sequences revealed genes predicted to encode enzymes required for the formation of nucleotide sugar precursors, glycosyltransferases involved in the repeat-unit assembly, and other proteins involved in polymerization and export of bacterial surface polysaccharides.

J Appl Microbiol, 2003, 95(6), 1191 - 9
Burkholderia cepacia complex genomovars: utilization of carbon sources, susceptibility to antimicrobial agents and growth on selective media; Vermis K et al.; 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.

J Bacteriol, 2003 Dec, 185(23), 7008 - 14
Role of the stationary growth phase sigma factor RpoS of Burkholderia pseudomallei in response to physiological stress conditions; Subsin B et al.; The Burkholderia pseudomallei rpoS gene was identified, and an rpoS null mutant was constructed . The mutant was shown to have an increased sensitivity to carbon starvation and oxidative stress . By using rpoS-lacZ fusions, transcription of rpoS was shown to be growth phase regulated, reaching a peak upon entry into stationary phase.

J Bacteriol, 2003 Dec, 185(23), 6976 - 80
Pinpointing biphenyl dioxygenase residues that are crucial for substrate interaction; Zielinski M et al.; Three regions of the biphenyl dioxygenase (BDO) of Burkholderia sp . strain LB400 have previously been shown to significantly influence the interaction between enzyme and substrates at the active site . For a further discrimination within these regions, we investigated the effects of 23 individual amino acid exchanges . The regiospecificity of substrate dioxygenation was used as a sensitive means to monitor changes in the steric-electronic structure of the active site . Replacements of residues that, according to a model of the BDO three-dimensional structure, directly interact with substrates in most, but not all, cases (Met231, Phe378, and Phe384) very strongly altered this parameter (by factors of >7) . On the other hand, a number of amino acids (Ile243, Ile326, Phe332, Pro334, and Trp392) which have no contacts with substrates also strongly changed the site preference of dioxygenation (by factors of between 2.6 and 3.5) . This demonstrates that residues which had not been predicted to be influential can play a pivotal role in BDO specificity.

Arch Microbiol, 2003 Dec, 180(6), 498 - 502 Epub 2003 Nov 12.
Compensatory increase in ahpC gene expression and its role in protecting Burkholderia pseudomallei against reactive nitrogen intermediates; Loprasert S et al.; In the human pathogen Burkholderia pseudomallei, katG encodes the antioxidant defense enzyme catalase-peroxidase . Interestingly, a B . pseudomallei mutant, disrupted in katG, is hyperresistant to organic hydroperoxide . This hyperresistance is due to the compensatory expression of the alkyl hydroperoxide reductase gene ( ahpC) and depends on a global regulator OxyR . The KatG-deficient mutant is also highly resistant to reactive nitrogen intermediates (RNI) . When overproduced, the B . pseudomallei AhpC protein, protected cells against killing by RNI . The levels of resistance to both organic peroxide and RNI returned to those of the wild-type when the katG mutant was complemented with katG . These studies establish the partially overlapping defensive activities of KatG and AhpC.

J Med Microbiol, 2003 Dec, 52(Pt 12), 1109 - 15
Experimental aerogenic Burkholderia mallei (glanders) infection in the BALB/c mouse; Lever MS et al.; The object of this study was to develop and characterize experimental Burkholderia mallei aerosol infection in BALB/c mice . Sixty-five mice were infected with 5000 {approx . 2.5 median lethal doses (MLD)} B . mallei strain ATCC 23344(T) bacteria by the aerosol route . Bacterial counts within lung, liver, spleen, brain, kidney and blood over 14 days were determined and histopathological and immunocytochemical profiles were assessed . Mortality due to B . mallei infection occurred between days 4 and 10 post-infection . Bacterial numbers were consistently higher in the lungs than in other tissues, reaching a maximum of approximately 1.0 x 10(6) c.f.u . ml(-1) at 5 days post-infection . Bacterial counts in liver and spleen tissue remained approximately equal, reaching a maximum of approximately 1.0 x 10(4) c.f.u . ml(-1) at day 4 post-infection . By day 14 post-infection, bacterial counts were in the range 1.0 x 10(3)-1.0 x 10(4) c.f.u . ml(-1) for all tissues . Infection of the lungs by B . mallei resulted in foci of acute inflammation and necrosis . As infection progressed, the inflammatory process became subacute or chronic; this was associated with the development of extensive consolidation . Lesions in liver and spleen tissue were typical of those that might be expected in bacteraemia or bacterial toxaemia . These results suggest that the BALB/c mouse is susceptible to B . mallei when delivered by the aerosol route and that this represents a model system of acute human glanders that is suitable for research into the pathogenesis of and vaccines against this disease.

FEMS Microbiol Lett, 2003 Nov 7, 228(1), 57 - 62
Evidence of transmission of Burkholderia cepacia, Burkholderia multivorans and Burkholderia dolosa among persons with cystic fibrosis; Biddick R et al.; Previous studies have identified specific Burkholderia cepacia complex strains that are common to multiple persons with cystic fibrosis (CF) . Such so-called epidemic strains have an apparent enhanced capacity for inter-patient spread and reside primarily in Burkholderia cenocepacia (formerly B . cepacia complex genomovar III) . We sought to identify strains from B . cepacia complex species other than B . cenocepacia that are similarly shared by multiple CF patients . We performed genotype analysis of 360 recent sputum culture isolates from 360 persons residing in 29 cities by using repetitive extragenic palendromic polymerase chain reaction (rep-PCR) and pulsed field gel electrophoresis . The results indicate that sharing of a common Burkholderia multivorans strain occurs relatively infrequently; however, several small clusters of patients infected with the same strain were identified . A cluster of seven patients infected with the same B . cepacia (genomovar I) strain was found . We also identified a large group of 28 patients receiving care in the same treatment center and infected with the same Burkholderia dolosa strain . These observations suggest that B . cepacia complex strains in species other than B . cenocepacia may be spread among CF patients.

Int J Biol Macromol, 2003 Dec, 33(4-5), 175 - 82
Burkholderia genome analysis reveals new enzymes belonging to the nitrilase superfamily . The amidase of Burkholderia cepacia (hospital isolate); Novo C et al.; Burkholderia cepacia (formerly Pseudomonas cepacia) grows in media containing acetamide or propionamide as C and N sources . Chromosomal DNA from a hospital isolate of B . cepacia served as a template in PCRs using primers designed for the amplification of the P . aeruginosa amiE gene that encodes an aliphatic amidase . Partial sequencing of the PCR products gave a translated sequence 100% identical with the amino acid sequence of P . aeruginosa amidase . A search of Burkholderia genomes detected a putative amidase in B . cepacia J2315 with high identity to the P . aeruginosa amidase and predicted that other Burkholderia species also possessed CN_hydrolases that use the same catalytic triad (Glu-Lys-Cys) as amidase . Superimposition of theoretical three-dimensional models suggested that differences in the amino acid sequences between amidases from B . cepacia (hospital isolate) and B . cepacia J2315 do not affect their three-dimensional structure.

Planta, 2004 Feb, 218(4), 647 - 57 Epub 2003 Nov 06.
Early perception responses of Nicotiana tabacum cells in response to lipopolysaccharides from Burkholderia cepacia; Gerber IB et al.; Lipopolysaccharides (LPS) are cell surface components of Gram-negative bacteria and, as microbe-/pathogen-associated molecular patterns, have diverse roles in plant-microbe interactions, e.g . LPS are able to promote plant disease tolerance through activation of induced or acquired resistance . However, little is known about the mechanisms of signal perception and transduction in response to elicitation by these bio-active lipoglycans . The present study focused on the involvement of LPS isolated from the outer cell wall of the Gram-negative bacterium Burkholderia cepacia (strain ASP B 2D) in the molecular mechanisms and components involved in signal perception and transduction and defense-associated responses in suspension-cultured tobacco (Nicotiana tabacum L.) cells . The purified LPS(B.cep.) was found to trigger a rapid influx of Ca2+ into the cytoplasm of aequorin-transformed tobacco cells . An oxidative burst, concomitant with the production of reactive oxygen and nitrogen species was measured by chemiluminescence and fluorescence . These early perception responses were accompanied by K+/H+ exchange and alkalinization of the extracellular medium . Through the use of various inhibitors of the oxidative burst reaction, as well as scavengers of produced radicals, the biochemical basis of the cellular response to LPS(B.cep.) elicitation was dissected, elucidated and compared to that induced by a yeast elicitor . These results suggest that LPS(B.cep.) interacts with tobacco cells in a manner different from the response elicited by yeast elicitor.

Cutis, 2003 Oct, 72(4), 310 - 2
A case of nonfatal cutaneous melioidosis; Thng TG et al.; Melioidosis is a tropical infectious disease caused by the gram-negative bacterium Burkholderia pseudomallei . It is endemic in many parts of the world, including Southeast Asia, and has a mortality rate of about 45% . We report a case of localized nonfatal cutaneous melioidosis presenting as a persistent ulcer in an otherwise healthy young woman.

J Antimicrob Chemother, 2003 Dec, 52(6), 915 - 9 Epub 2003 Oct 29.
In vitro activity and synergy of bismuth thiols and tobramycin against Burkholderia cepacia complex; Veloira WG et al.; OBJECTIVES: To determine the susceptibility of Burkholderia multivorans and Burkholderia cenocepacia to bismuth-thiols (BTs), and to examine the synergistic effects of tobramycin and subinhibitory concentrations of BTs against these organisms . METHODS: The susceptibilities of 25 clinical isolates each of B . multivorans and B . cenocepacia to six BTs were measured by broth dilution in accordance with NCCLS protocols . Ten strains were selected to evaluate the antimicrobial interaction between BTs and tobramycin . Fractional inhibitory concentration (FIC) and fractional bactericidal concentration (FBC) indices were calculated to assess synergy . RESULTS: B . multivorans and B . cenocepacia showed a wide range of susceptibilities to BTs . Bismuth ethanedithiol (BisEDT) was one of the more potent BTs against these organisms (MIC50 7.8 microM), and was selected for synergy studies . Selected strains were highly resistant to tobramycin . The addition of subinhibitory concentrations of BisEDT (2 microM) reduced the MIC and MBC of tobramycin against all strains, achieving synergy in many instances . The FIC index was in the range 0.28-0.66 and the FBC in the range 0.12-0.85 . Most strains became susceptible to tobramycin at clinically achievable concentrations in the presence of non-toxic BisEDT levels . CONCLUSIONS: Treatment with subinhibitory BisEDT and tobramycin reduces the MICs and MBCs for B . multivorans and B . cenocepacia . BTs may represent an important adjunctive therapy for resistant Burkholderia cepacia complex.

Acta Haematol, 2003, 110(2-3), 86 - 92
Gene therapy for chronic granulomatous disease; Goebel WS et al.; Identification of gene mutations responsible for leukocyte dysfunction along with the application of gene transfer technology has made genetic correction of such disorders possible . Much of the research into molecular therapy for inherited disorders of phagocytes has been focused on chronic granulomatous disease (CGD) . CGD results from mutations in any one of the four genes encoding essential subunits of respiratory burst NADPH oxidase, the enzyme complex required for the production of reactive oxygen intermediates in phagocytes . The absence of phagocyte oxidants results in a predisposition to recurrent bacterial and fungal infections and inflammatory granulomas in CGD patients, associated with significant morbidity and mortality . Allogeneic bone marrow transplantation can cure CGD, but transplant-related toxicity and the limited availability of matched donors have restricted its wider application . Because the gene defects causing CGD are known, and CGD is a stem cell disorder treatable by marrow transplantation, CGD has emerged as a promising disease for somatic gene therapy targeted at the hematopoietic system . Multiple reports have demonstrated the reconstitution of NADPH oxidase activity by gene transfer to human CGD marrow and cell lines cultured in vitro . CGD mouse models have been developed by gene disruption, and preclinical studies on these animals using recombinant retroviral vectors have demonstrated reconstitution of functionally normal neutrophils and increased resistance to pathogens such as Aspergillus fumigatus, Burkholderia cepacia and Staphylococcus aureus . Although the results of these murine studies are encouraging, human phase-I clinical studies in CGD patients have yet to produce clinically beneficial numbers of corrected neutrophils for extended periods . Efforts to improve gene transfer efficiency into human hematopoietic stem cells and to increase engraftment of transduced stem cells are ongoing .

Indian J Med Res, 2003 Mar, 117, 119 - 21
Septicaemic melioidosis in a tertiary care hospital in south India; Jesudason MV et al.; BACKGROUND & OBJECTIVES: Melioidosis and the causative organism Burkholderia pseudomallei are being recognized gradually in various centres in India . In the septicaemic form, melioidosis is a serious and life threatening condition which requires early detection and specific treatment to avoid case fatality . A review of patients with septicaemic melioidosis at a tertiary care hospital in south India was carried out with a view to define the clinical features, predisposing conditions, if any, and the outcome . METHODS: A total of 28 patients with culture proven septicaemic melioidosis during December 1993 to December 2002 were included . Information on clinical details and outcome was obtained and antibiotic susceptibility of the isolates studied . RESULTS: Of the 28 patients of blood culture proven septicaemic melioidosis, the organism was also isolated from pus in two patients . The presenting clinical features were varied, most presenting as pyrexia of unknown origin or visceral abscesses, or septic arthiritis . Associated/predisposing conditions were present in 50 per cent of the patients, and diabetes mellitus was the commonest one . mortality was 58 per cent in our series . INTERPRETATION & CONCLUSION: Melioidosis is an emerging infection in India . The magnitude of the problem can only be assessed by increasing awareness, both of its existence in the clinical setting and its identification in the laboratory.

Biodegradation, 2003 Oct, 14(5), 357 - 65
Biodegradation of 2-chlorobenzoate by recombinant Burkholderia cepacia expressing Vitreoscilla hemoglobin under variable levels of oxygen availability; Urgun-Demirtas M et al.; The influence of bacterial hemoglobin, VHb, on dechlorination and degradation of 2-chlorobenzoate (2-CBA) by recombinant Burkholderia sp . under variable oxygen availability with an initial dissolved oxygen concentration of 0.27 mM-0.72 mM was investigated in batch and continuous culture . Ability to express VHb was provided to recombinant Burkholderia by transformation with the VHb gene, vgb, on plasmid pSC160 . 100% of 0.5 mM CBA was degraded in cultures with 85% and 70% of total volume as headspace air in closed reactors by both wild type and recombinant Burkholderia . The recombinant cultures were able to dechlorinate and degrade 100% of the 2-CBA in less than 48 hours at 30 degrees C compared to more than 120 hours for wild type cultures . The rate and extent of CBA degradation by recombinant cultures with 40% of total volume as headspace air was higher than those achieved by wild type cells at the end of the 168 hours of incubation period, 98 and 73%, respectively . The chloride released: CBA degraded molar ratio for cultures with 40% of total volume headspace air was nearly stoichiometric (molar ratio = 1.0) for recombinant strains, whereas it was non-stoichiometric (molar ratio = 0.24) for wild type cells . The results suggest a suicidal meta-pathway for wild type cells and a complete dechlorination and degradation pathway for recombinant cells under hypoxic conditions . The degradation and dechlorination ability of both types of cells was also investigated in continuous reactor studies by varying the dilution rate under hypoxic conditions . Regarding potential of the recombinant strain for 2-CBA degradation in either open ecosystems or closed bioreactor bioremediation systems, the stability of the plasmid containing vgb in the recombinant cells was also studied; the plasmid was 100% stable at 0.025 h(-1) dilution rate (approximately 1.7 d hydraulic retention time), even after one month.

Curr Microbiol, 2003 Sep, 47(3), 174 - 9
AHL-deficient mutants of Burkholderia ambifaria BC-F have decreased antifungal activity; Zhou H et al.; Burkholderia ambifaria BC-F, a biocontrol strain reported previously to exhibit broad-spectrum antifungal activity, was highly active in formation of N-acyl homoserine lactones (AHLs) . We constructed AHL-deficient derivatives of strain BC-F in which the genes specifying AHL synthase (bafI) and AHL-binding transcriptional activator (bafR) were inactivated by allelic exchange . The resulting AHL-deficient mutants had decreased antifungal activity.

Mol Immunol, 2003 Nov, 40(7), 431 - 43
Antimicrobial peptides in defence of the oral and respiratory tracts; Devine DA; Antimicrobial peptides (AMPs) are components of complex host secretions, acting synergistically with other innate defence molecules to combat infection and control resident microbial populations throughout the oral cavity and respiratory tract . AMPs are directly antimicrobial, bind lipopolysaccharide (LPS) and lipoteichoic acid, and are immunomodulatory signals . Pathogenic and commensal organisms display a variety of resistance mechanisms, which are related to structure of cell wall components (e.g . LPS) and cytoplasmic membranes, and peptide breakdown mechanisms . For example, LPS of the AMP-resistant cystic fibrosis pathogen Burkholderia cepacia is under-phosphorylated and highly substituted with charge-neutralising 4-deoxy-4-aminoarabinose . Additionally, host mimicry by addition of phosphorylcholine contributes to resistance in oral and respiratory organisms . Porphyromonas gingivalis, Pseudomonas aeruginosa and other pathogens produce extracellular and membrane-bound proteases that degrade AMPs . Many of these bacterial properties are environmentally regulated . Their modulation in response to host defences and inflammation can result in altered sensitivity to AMPs, and may additionally change other host-microbe interactions, e.g . binding to Toll-like receptors . The diversity and breadth of antimicrobial cover and immunomodulatory function provided by AMPs is central to the ability of a host to respond to the diverse and highly adaptable organisms colonising oral and respiratory mucosa.

Biochem J, 2004 Feb 1, 377(Pt 3), 579 - 87
Functional reconstitution, gene isolation and topology modelling of porins from Burkholderia pseudomallei and Burkholderia thailandensis; Siritapetawee J et al.; The sequences for Omp38 from Burkholderia pseudomallei and Burkholderia thailandensis have been deposited in the DDBJ, EMBL, GenBank(R) and GSDB Nucleotide Sequence Databases under the accession numbers AY312416 and AY312417 respectively . The intracellular pathogen Burkholderia pseudomallei is the causative agent of tropical melioidosis, and Burkholderia thailandensis is a closely-related Gram-negative bacterium that does not cause serious disease . Like other bacteria, the major outer membrane (OM) porins of Burkholderia strains, Bps Omp38 and Bth Omp38 may have roles in antibiotic resistance and immunity . We purified both proteins and found them to be immunologically related, SDS-resistant, heat-sensitive trimers with M (r) of approx . 110000 . In functional liposome-swelling assays, both proteins showed similar permeabilities for small sugar molecules, compatible with a pore diameter of between 1.2 and 1.6 nm . Secondary structure analysis by FTIR (Fourier-transform infrared) spectroscopy revealed almost identical spectra with predominantly beta-sheet structures, typical of bacterial porins . MALDI-TOF (matrix-assisted laser-desorption ionization-time of flight) MS and ESI/MS (electrospray ionization MS) analysis of each protein showed extensive sequence similarities to the OpcP1 porin from Burkholderia cepacia (later found to be 76.5% identical) . Based on information from the incomplete B . pseudomallei genome-sequencing project, the genes encoding Omp38 were identified and amplified by PCR from B . pseudomallei and B . thailandensis genomic DNA . The nucleotide sequences are 99.7% identical, and the predicted processed proteins are 100% identical . Topology prediction and molecular modelling suggest that this newly-isolated and cloned porin is a 16-stranded beta-barrel and the external loops of the protein could be important determinants of the immune response to infection.

Jpn J Antibiot, 2003 Aug, 56(4), 309 - 35
{Yearly changes in antibacterial activities of cefozopran against various clinical isolates between 1996 and 2001--II . Gram-negative bacteria}; Suzuki Y et al.; The in vitro antibacterial activities of cefozopran (CZOP), an agent of cephems, against various clinical isolates obtained between 1996 and 2001 were yearly evaluated and compared with those of other cephems, oxacephems and carbapenems . A total of 3,245 strains in 32 species of Gram-negative bacteria were isolated from the clinical materials annually collected from January to December, and consisted of Moraxella subgenus Branhamella catarrhalis, Escherichia coli, Citrobacter freundii, Citrobacter koseri, Klebsiella pneumoniae, Klebsiella oxytoca, Enterobacter aerogenes, Enterobacter cloacae, Serratia marcescens, Proteus mirabillis, Proteus vulgaris, Morganella morganii, Providencia spp . (P . alcalifaciens, P . rettgeri, P . stuartii), Pseudomonas aeruginosa, Pseudomonas putida, Burkholderia cepacia, Stenotrophomonas maltophilia, Haemophilus influenzae, Acinetobactor baumannii, Acinetobactor lwoffii, Bacteroides fragilis group (B . fragilis, B . vulgatus, B . distasonis, B . ovatus, B . thetaiotaomicron), and Prevotella spp . (P . melaninogenica, P . intermedia, P . bivia, P . oralis, P . denticola) . CZOP possessed stable antibacterial activities against M . (B.) catarrhalis, E . coli, C . freundii, C . koseri, K . pneumoniae, K . oxytoca, E . aerogenes, E . cloacae, S . marcescens, P . mirabilis, P . vulgaris, M . morganii, Providencia spp., P . aeruginosa, and A . lwoffii throughout 6 years . The MIC90 of CZOP against those strains were consistent with those obtained from the studies performed until the new drug application approval . On the other hand, the MIC90 of CZOP against H . influenzae yearly obviously increased with approximately 64-time difference during the study period . The MIC90 of cefpirome, cefepime, and flomoxef against H . influenzae also yearly tended to rise . The present results demonstrated that CZOP had maintained the antibacterial activity against almost Gram-negative strains tested . However, the decrease in antibacterial activities of CZOP against B . cepacia, and H . influenzae was suggested.

J Bacteriol, 2003 Nov, 185(21), 6233 - 40
Identification and characterization of the emhABC efflux system for polycyclic aromatic hydrocarbons in Pseudomonas fluorescens cLP6a; Hearn EM et al.; The hydrocarbon-degrading environmental isolate Pseudomonas fluorescens LP6a possesses an active efflux mechanism for the polycyclic aromatic hydrocarbons phenanthrene, anthracene, and fluoranthene but not for naphthalene or toluene . PCR was used to detect efflux pump genes belonging to the resistance-nodulation-cell division (RND) superfamily in a plasmid-cured derivative, P . fluorescens cLP6a, which is unable to metabolize hydrocarbons . One RND pump, whose gene was identified in P . fluorescens cLP6a and was designated emhB, showed homology to the multidrug and solvent efflux pumps in Pseudomonas aeruginosa and Pseudomonas putida . The emhB gene is located in a gene cluster with the emhA and emhC genes, which encode the membrane fusion protein and outer membrane protein components of the efflux system, respectively . Disruption of emhB by insertion of an antibiotic resistance cassette demonstrated that the corresponding gene product was responsible for the efflux of polycyclic aromatic hydrocarbons . The emhB gene disruption did not affect the resistance of P . fluorescens cLP6a to tetracycline, erythromycin, trimethoprim, or streptomycin, but it did decrease resistance to chloramphenicol and nalidixic acid, indicating that the EmhABC system also functions in the efflux of these compounds and has an unusual selectivity . Phenanthrene efflux was observed in P . aeruginosa, P . putida, and Burkholderia cepacia but not in Azotobacter vinelandii . Polycyclic aromatic hydrocarbons represent a new class of nontoxic, highly hydrophobic compounds that are substrates of RND efflux systems, and the EmhABC system in P . fluorescens cLP6a has a narrow substrate range for these hydrocarbons and certain antibiotics.

Microbes Infect, 2003 Oct, 5(12), 1125 - 31
Characterization of experimental equine glanders; Lopez J et al.; Considerable advances in understanding of the disease caused by Burkholderia mallei have been made employing a combination of tools including genetic techniques and animal infection models . The development of small animal models has allowed us to assess the role of a number of putative virulence determinants in the pathogenesis of disease due to B . mallei . Due to the difficulties in performing active immunization studies in small animals, and due to the fact that the horse is the target mammalian species for glanders, we have initiated experimental studies on glanders in horses . Intratracheal deposition of B . mallei produced clinical glanders with organisms being recovered from tissues of infected horses . The model should prove to be of considerable value in our ongoing studies on the pathogenesis and vaccine development for glanders.

Environ Pollut, 2004, 127(1), 41 - 8
Ability of bacterial biphenyl dioxygenases from Burkholderia sp . LB400 and Comamonas testosteroni B-356 to catalyse oxygenation of ortho-hydroxychlorobiphenyls formed from PCBs by plants; Francova K et al.; Capacity of enzymes of the biphenyl/chlorobiphenyl pathway, especially biphenyl dioxygenase (BPDO) of two polychlorinated biphenyls (PCB) degrading bacteria, Burkholderia sp . LB400 and Comamonas testosteroni B-356, to metabolize ortho-substituted hydroxybiphenyls was tested.,These compounds found among plant products of PCB metabolism, are carrying chlorine atoms on the hydroxyl-substituted ring . The abilities of His-tagged purified LB400 and B-356 BPDOs to catalyze the oxygenation of 2-hydroxy-3-chlorobiphenyl, 2-hydroxy-5-chlorobiphenyl and 2-hydroxy-3,5-dichlorobiphenyl were compared . Both enzyme preparations catalyzed the hydroxylation of the three chloro-hydroxybiphenyls on the non-substituted ring . Neither LB400 BPDO nor B-356 BPDO oxygenated the substituted ring of the ortho-hydroxylated biphenyl . The fact that metabolites generated by both enzymes were identical for all three hydroxychlorobiphenyls tested; exclude any other mode of attack of these compounds by LB400 BPDOs than the ortho-meta oxygenation.

Braz J Infect Dis, 2003 Oct, 7(5), 282 - 9 Epub 2004 Jan 22.
Ability of Latin America laboratories to detect antimicrobial resistance patterns: experience of the SENTRY Antimicrobial Surveillance Program (1997-2000); Mendes RE et al.; The accuracy of antimicrobial susceptibility tests is a crucial step for the clinical management of patients with serious infections . They must be reliable and precise because they will guide antimicrobial therapy . Our main objective was to compare the results of susceptibility testing performed by the SENTRY coordinator laboratory with those reported by the participating Latin American medical centers . A total of 10,277 bacterial isolates were tested by the reference broth microdilution method at the coordinator laboratory in the United States . The tests were performed and interpreted following the National Committee for Clinical Laboratory Standards (NCCLS) recommendations . Ten antimicrobial agent-organism combinations were analyzed . The susceptibility methods utilized in each of the medical centers were also evaluated . Total agreement of the results was obtained in nearly 88% of the antimicrobial agent-organism combinations . "Very major" (false-susceptible results) and "major errors" (false-resistant results) were observed in 12% and 6% of the cases, respectively . The highest disagreements were observed for coagulase-negative Staphylococcus_oxacillin (20% - very major error) and Burkholderia cepacia_imipenem (21% - very major error) . The susceptibility method with the highest agreement rate was Etest (92%) > PASCO (91%) > agar dilution (91%) > MicroScan (90%) > Vitek (87%) . External quality assurance data obtained by surveillance programs such as the SENTRY Antimicrobial Surveillance Program are not only helpful for detecting the emergence of patterns of antimicrobial resistance, but also to monitor the performance of the participating microbiology laboratories.

Arch Pediatr, 2003 Oct, 10(10), 882 - 6
{Nosocomial Burkholderia cepacia outbreak in an intensive pediatric care unit}; Bureau-Chalot F et al.; BACKGROUND: We report an outbreak of Burkholderia cepacia respiratory tract infection and colonization in an intensive pediatric care unit.P PATIENTS AND METHODS: Between February and December 1999, B . cepacia was isolated from five children hospitalized in this unit . We reviewed the charts of the patients, evaluated the antiseptics use and the disinfection practices for reusable patient care equipment . An environmental study was conducted and comparison of B . cepacia was performed with genotypic method (RAPD) . RESULTS: All patients were mechanically ventilated and had received large spectrum antibiotics . The disinfection procedure for reusable equipment was not respected and some single-dose of antiseptics solutions were used for several patients . B . cepacia was not found in 34 environmental samples . The RAPD assay revealed that all five isolates had identical DNA profiles . CONCLUSION: Despite the investigation the source of the B . cepacia clone in this nosocomial outbreak remained unknown, but antiseptics use and disinfection practices were revised . No new B . cepacia infections were identified after control measures were implemented.

J Biochem Mol Biol, 2003 Sep 30, 36(5), 493 - 8
Bacterial expression of the scFv fragment of a recombinant antibody specific for Burkholderia pseudomallei exotoxin; Su YC et al.; The scFv antibody towards the Burkholderia pseudomallei exotoxin was previously constructed by phage display and exhibited good specificity towards the exotoxin . We report here the optimization of the scFv expression in an E . coli expression system . Four different E . coli strains (ER2537, TG1, HB2151, and XL1-Blue) were examined for optimal expression of the scFv protein . Two types of carbon source (i.e . 0.2% glucose and 0.2% glycerol) were also tested for their ability to induce the scFv expression . Cells that carried the scFv construct were grown at 30 degrees C and induced with 0.05 mM IPTG . The expression was then monitored by SDS-PAGE, Western blotting, and indirect ELISA . The Western blot profile showed different levels of the scFv expression among the host strains; XL1-Blue exhibited the highest level of the scFv protein expression . Glycerol at a concentration of 0.2% (v/v) significantly increased the scFv protein expression level when compared to 0.2% (w/v) glucose . Further optimization demonstrated that the scFv protein expression in XL1-Blue was the most optimal with a glycerol concentration as low as 0.05% . However, by indirect ELISA, only the scFv protein that was expressed in 0.2% (v/v) glycerol exhibited high specificity towards the Burkholderia pseudomallei exotoxin.

Folia Microbiol (Praha), 2003, 48(4), 521 - 4
Detection of Burkholderia pseudomallei in rice fields with PCR-based technique; Kao CM et al.; Burkholderia pseudomallei Ara- in rice fields was detected using PCR-based techniques with 16S RNA and flagella gene primer sets . The sensitivity of these PCRs was at least 1 CFU/mL of B . pseudomallei Ara- preincubated into Ashdown's medium for 6 h . B . pseudomallei Ara- DNA from watery soil were more detectable than from dry soil . The distribution of this DNA was mainly found at a depth of 300-600 mm under crop-covered fields, but not detected in the location of soil close to the land surface . The results suggest that PCR based on 16S RNA and flagella gene primer sets can be applied to investigate the presence of B . pseudomallei Ara- in contaminated soil of rice fields.

J Clin Microbiol, 2003 Oct, 41(10), 4812 - 4
Cellular fatty acid profile distinguishes Burkholderia pseudomallei from avirulent Burkholderia thailandensis; Inglis TJ et al.; Burkholderia pseudomallei, the cause of melioidosis, can be distinguished from the closely related but nonpathogenic Burkholderia thailandensis by gas chromatography (GC) analysis of fatty acid derivatives . A 2-hydroxymyristic acid derivative (14:0 2OH) was present in 95% of B . pseudomallei isolates and no B . thailandensis isolates . GC mass spectrophotometry confirmed that 2-hydroxymyristic acid was present in B . pseudomallei . GC-fatty acid methyl ester analysis may be useful in distinguishing these two closely related species.

J Clin Microbiol, 2003 Oct, 41(10), 4647 - 54
Use of 16S rRNA gene sequencing for rapid identification and differentiation of Burkholderia pseudomallei and B . mallei; Gee JE et al.; Burkholderia pseudomallei and B . mallei, the causative agents of melioidosis and glanders, respectively, are designated category B biothreat agents . Current methods for identifying these organisms rely on their phenotypic characteristics and an extensive set of biochemical reactions . We evaluated the use of 16S rRNA gene sequencing to rapidly identify these two species and differentiate them from each other as well as from closely related species and genera such as Pandoraea spp., Ralstonia spp., Burkholderia gladioli, Burkholderia cepacia, Burkholderia thailandensis, and Pseudomonas aeruginosa . We sequenced the 1.5-kb 16S rRNA gene of 56 B . pseudomallei and 23 B . mallei isolates selected to represent a wide range of temporal, geographic, and origin diversity . Among all 79 isolates, a total of 11 16S types were found based on eight positions of difference . Nine 16S types were identified in B . pseudomallei isolates based on six positions of difference, with differences ranging from 0.5 to 1.5 bp . Twenty-two of 23 B . mallei isolates showed 16S rRNA gene sequence identity and were designated 16S type 10, whereas the remaining isolate was designated type 11 . This report provides a basis for rapidly identifying and differentiating B . pseudomallei and B . mallei by molecular methods.

Appl Environ Microbiol, 2003 Oct, 69(10), 6250 - 6
Invasion of spores of the arbuscular mycorrhizal fungus Gigaspora decipiens by Burkholderia spp; Levy A et al.; Burkholderia species are bacterial soil inhabitants that are capable of interacting with a variety of eukaryotes, in some cases occupying intracellular habitats . Pathogenic and nonpathogenic Burkholderia spp., including B . vietnamiensis, B . cepacia, and B . pseudomallei, were grown on germinating spores of the arbuscular mycorrhizal fungus Gigaspora decipiens . Spore lysis assays revealed that all Burkholderia spp . tested were able to colonize the interior of G . decipiens spores . Amplification of specific DNA sequences and transmission electron microscopy confirmed the intracellular presence of B . vietnamiensis . Twelve percent of all spores were invaded by B . vietnamiensis, with an average of 1.5 x 10(6) CFU recovered from individual infected spores . Of those spores inoculated with B . pseudomallei, 7% were invaded, with an average of 5.5 x 10(5) CFU recovered from individual infected spores . Scanning electron and fluorescence microscopy provided insights into the morphology of surfaces of spores and hyphae of G . decipiens and the attachment of bacteria . Burkholderia spp . colonized both hyphae and spores, attaching to surfaces in either an end-on or side-on fashion . Adherence of Burkholderia spp . to eukaryotic surfaces also involved the formation of numerous fibrillar structures.

Appl Environ Microbiol, 2003 Oct, 69(10), 6128 - 32
Formaldehyde fixation contributes to detoxification for growth of a nonmethylotroph, Burkholderia cepacia TM1, on vanillic acid; Mitsui R et al.; During bacterial degradation of methoxylated lignin monomers, such as vanillin and vanillic acid, formaldehyde is released through the reaction catalyzed by vanillic acid demethylase . When Burkholderia cepacia TM1 was grown on vanillin or vanillic acid as the sole carbon source, the enzymes 3-hexulose-6-phosphate synthase (HPS) and 6-phospho-3-hexuloisomerase (PHI) were induced . These enzymes were also expressed during growth on Luria-Bertani medium containing formaldehyde . To understand the roles of these enzymes, the hps and phi genes from a methylotrophic bacterium, Methylomonas aminofaciens 77a, were introduced into B . cepacia TM1 . The transformant strain constitutively expressed the genes for HPS and PHI, and these activities were two- or threefold higher than the activities in the wild strain . Incorporation of {14C}formaldehyde into the cell constituents was increased by overexpression of the genes . Furthermore, the degradation of vanillic acid and the growth yield were significantly improved at a high concentration of vanillic acid (60 mM) in the transformant strain . These results suggest that HPS and PHI play significant roles in the detoxification and assimilation of formaldehyde . This is the first report that enhancement of the HPS/PHI pathway could improve the degradation of vanillic acid in nonmethylotrophic bacteria.

Respirology, 2003 Sep, 8(3), 401 - 3
Fatal pneumonia caused by Burkholderia cepacia 9 months after resection of aspergilloma; Ishizuka T et al.; A 69-year-old man developed an episode of severe community-acquired pneumonia 9 months after resection of aspergilloma . Although Aspergillus fumigatus was also isolated in the pleural cavity, it did not invade the remaining lung parenchyma . The patient developed progressive bilateral pneumonia leading to death from respiratory failure . Burkholderia cepacia was considered as prime pathogen, as it was repeatedly cultured from sputum and tracheal secretions, as well as the autopsy lung . B . cepacia is resistant to most antibiotics, and seldom causes pneumonia in patients without cystic fibrosis or chronic granulomatous disease . The precise reason that this apparently immunocompetent patient developed B . cepacia pneumonia remains unknown.

Microbiology, 2003 Oct, 149(Pt 10), 2879 - 90
Degradation of alkanes and highly chlorinated benzenes, and production of biosurfactants, by a psychrophilic Rhodococcus sp . and genetic characterization of its chlorobenzene dioxygenase; Rapp P et al.; Rhodococcus sp . strain MS11 was isolated from a mixed culture . It displays a diverse range of metabolic capabilities . During growth on 1,2,4-trichlorobenzene, 1,2,4,5-tetrachlorobenzene (1,2,4,5-TeCB) and 3-chlorobenzoate stoichiometric amounts of chloride were released . It also utilized all three isomeric dichlorobenzenes and 1,2,3-trichlorobenzene as the sole carbon and energy source . Furthermore, the bacterium grew well on a great number of n-alkanes ranging from n-heptane to n-triacontane and on the branched alkane 2,6,10,14-tetramethylpentadecane (pristane) and slowly on n-hexane and n-pentatriacontane . It was able to grow at temperatures from 5 to 30 degrees C, with optimal growth at 20 degrees C, and could tolerate 6 % NaCl in mineral salts medium . Genes encoding the initial chlorobenzene dioxygenase were detected by using a primer pair that was designed against the alpha-subunit (TecA1) of the chlorobenzene dioxygenase of Ralstonia (formerly Burkholderia) sp . strain PS12 . The amino acid sequence of the amplified part of the alpha-subunit of the chlorobenzene dioxygenase of Rhodococcus sp . strain MS11 showed >99 % identity to the alpha-subunit of the chlorobenzene dioxygenase from Ralstonia sp . strain PS12 and the parts of both alpha-subunits responsible for substrate specificity were identical . The subsequent enzymes dihydrodiol dehydrogenase and chlorocatechol 1,2-dioxygenase were induced in cells grown on 1,2,4,5-TeCB . During cultivation on medium-chain-length n-alkanes ranging from n-decane to n-heptadecane, including 1-hexadecene, and on the branched alkane pristane, strain MS11 produced biosurfactants lowering the surface tension of the cultures from 72 to </=29 mN m(-1) . Glycolipids were extracted from the supernatant of a culture grown on n-hexadecane and characterized by (1)H- and (13)C-NMR-spectroscopy and mass spectrometry . The two major components consisted of alpha,alpha-trehalose esterified at C-2 or C-4 with a succinic acid and at C-2' with a decanoic acid . They differed from one another in that one 2,3,4,2'-trehalosetetraester, found in higher concentration, was esterified at C-2, C-3 or C-4 with one octanoic and one decanoic acid and the other one, of lower concentration, with two octanoic acids . The results demonstrate that Rhodococcus sp . strain MS11 may be well suited for bioremediation of soils and sediments contaminated for a long time with di-, tri- and tetrachlorobenzenes as well as alkanes.

FEMS Immunol Med Microbiol, 2003 Oct 15, 38(3), 273 - 82
Pro-inflammatory effects of Burkholderia cepacia on cystic fibrosis respiratory epithelium; Fink J et al.; Burkholderia cepacia causes pulmonary infection with high mortality in cystic fibrosis (CF) patients which is likely to involve interaction with respiratory epithelium . In this study the pro-inflammatory properties of B . cepacia were examined using a range of respiratory epithelial cell lines . B . cepacia and cell-free culture supernatants were used to stimulate cell lines with (SigmaCFTE29o- and IB3) and without (A549) the CF transmembrane conductance regulator mutation (CFTR), together with corrected cell lines (C38 and S9) . Interleukin (IL)-6 and IL-8, but not GM-CSF or IL-1beta, were released from all the cell lines whereas PGE(2) (prostaglandin E(2)) was released from the A549, IB3 and S9 cell lines only . Nuclear factor (NF)-kappaB activation preceded cytokine release and suppression of NF-kappaB activity diminished cytokine release . These studies indicated that B . cepacia secretory products are potent pro-inflammatory agents for respiratory epithelium and suggest functional CFTR is not required for cytokine or prostanoid responses.

Biosci Biotechnol Biochem, 2003 Sep, 67(9), 2026 - 9
Isolation and characterization of phenol-catabolizing bacteria from a coking plant; El-Sayed WS et al.; New phenol degrading bacteria with high biodegradation activity and high tolerance were isolated as Burkholderia cepacia PW3 and Pseudomonas aeruginosa AT2 . Both isolates could grow aerobically on phenol as a sole carbon source even at 3 g/l . The whole-cell kinetic properties for phenol degradation by strains PW3 and AT2 showed a Vmax of 0.321 and 0.253 mg/l/min/(mg protein), respectively . The metabolic pathways for phenol biodegradation in both strains were assigned to the meta-cleavage activity of catechol 2,3-dioxygenase.

Ann Pharmacother, 2003 Oct, 37(10), 1424 - 8
Successful meropenem desensitization in a patient with cystic fibrosis; Wilson DL et al.; OBJECTIVE: To report a case of successful meropenem desensitization in a patient with cystic fibrosis (CF) with documented hypersensitivity to multiple antibiotics including carbapenems . CASE SUMMARY: A 20-year-old white man with CF was admitted to the hospital for treatment of an acute pulmonary exacerbation caused by multidrug-resistant Burkholderia cepacia complex and methicillin-resistant Staphylococcus aureus (MRSA) . Past treatments of his CF exacerbations were complicated by urticarial eruptions following administration of beta-lactams, including meropenem, and ototoxicity from aminoglycosides . Skin testing revealed hypersensitivity reactions to beta-lactam antibiotics including penicillin, piperacillin/tazobactam, ceftazidime, and imipenem . A literature review (MEDLINE, January 3, 2002) and communication with the manufacturer of meropenem revealed no specific information on desensitizing patients to this agent . Because of meropenem's activity against B . cepacia complex alone and in combination with other antimicrobials, a desensitization protocol was adapted and applied to meropenem in an effort to provide the most beneficial treatment available . A 12-dose escalation protocol was successfully employed without incident . DISCUSSION: Antimicrobial therapy is limited in CF patients by susceptibility profiles of common infecting organisms (e.g., Pseudomonas spp., B . cepacia complex, MRSA) . Unfortunately, host responses may further reduce the utility of many effective antibiotic classes due to hypersensitivity and/or adverse reactions . Desensitization is a useful alternative that allows the administration of beneficial medications to patients with documented allergy histories . CONCLUSIONS: Meropenem is an important treatment option in the CF population, particularly due to its activity against B . cepacia complex . Successful desensitization using a dose-escalation protocol in patients with a documented carbapenem allergy will allow the most beneficial therapy to continue.

Am J Ophthalmol, 2003 Oct, 136(4), 748 - 9
Polymicrobial keratitis secondary to Burkholderia ambifaria, enterococcus, and staphylococcus aureus in a patient with herpetic stromal keratitis; Matoba AY; PURPOSE: To report polymicrobial keratitis in a patient with herpetic stromal keratitis . The initial infecting organism, Burkholderia ambifaria, has not previously been reported to cause microbial keratitis . METHODS: Clinical evaluation and corneal culture were performed . RESULTS: A 59-year-old-man undergoing topical corticosteroid therapy for herpes simplex stromal keratitis developed corneal infection with B . ambifaria . The organism was reisolated 12 days after initiation of hourly therapy with topical levofloxacin 0.5% . At reculture Staphylococcus aureus and Enterococcus spp . were also isolated . The addition of topical amikacin and vancomycin led to resolution of the microbial keratitis . CONCLUSIONS: Burkholderia ambifaria infected a compromised cornea, exhibited an unusual sensitivity profile, and remained viable after 12 days of therapy with an antibiotic to which it was sensitive by in vitro tests.

Eur Respir J, 2003 Sep, 22(3), 542 - 50
Melioidosis: an important cause of pneumonia in residents of and travellers returned from endemic regions; Currie BJ; Melioidosis is endemic in South East Asia, Asia and northern Australia . Infection usually follows percutaneous inoculation or inhalation of the causative bacterium, Burkholderia pseudomallei, which is present in soil and surface water in the endemic region . While 20-36% of melioidosis cases have no evident predisposing risk factor, the vast majority of fatal cases have an identified risk factor, the most important of which are diabetes, alcoholism and chronic renal disease . Half of all cases present with pneumonia, but there is great clinical diversity, from localised skin ulcers or abscesses without systemic illness to fulminant septic shock with multiple abscesses in the lungs, liver, spleen and kidneys . At least 10% of cases present with a chronic respiratory illness (sick > 2 months) mimicking tuberculosis and often with upper lobe infiltrates and/or cavities on chest radiography . As with tuberculosis, latency with reactivation decades after infection can also occur, although this is rare . Confirmation of diagnosis is by culture of B . pseudomallei from blood, sputum, throat swab or other samples . Microbiology laboratories need to be informed of the possibility of melioidosis, as those not familiar with it can misidentify the organism . Antibiotic therapy is initial intensive therapy with i.v . ceftazidime or meropenem or imipenem +/- cotrimoxazole for > or = 10 days, followed by eradication therapy with cotrimoxazole +/- doxycycline +/- chloramphenicol (first 4 weeks only) for > or = 3 months . Melioidosis has been increasingly recognised in returning travellers in Europe and recently melioidosis and colonisation with B . pseudomallei have been documented in cystic fibrosis patients visiting or resident in endemic areas.

Infect Control Hosp Epidemiol, 2003 Sep, 24(9), 707 - 10
Epidemiologic investigation of Burkholderia cepacia acquisition in two pediatric intensive care units; Loukil C et al.; OBJECTIVES: To investigate and describe an outbreak of Burkholderia cepacia in a neonatal intensive care unit (NICU) and a pediatric intensive care unit (PICU), and to report the interventions leading to the cessation of the outbreak . DESIGN: We conducted an epidemiologic investigation of an outbreak of B . cepacia colonization or infection in two clinical wards during a 35-month period (December 1998 to October 2001) . SETTING: A 500-bed, university hospital-affiliated, tertiary-care pediatric institution in Paris, France, with a 22-bed PICU and 31-bed NICU . METHODS: Ribotyping was used to determine the genotypes of B . cepacia isolates . Procedures for the maintenance and disinfection of respiratory therapy devices were reviewed . RESULTS: Thirty-two children were colonized (n = 14) or infected (n = 18) by B . cepacia in 2 wards (28 in the PICU and 4 in the NICU) . In the PICU, a single ribotype was found among the isolates obtained from all of the patients except 1, and from the 6 isolates obtained from respiratory therapy devices (ie, heated humidifier water) . In the NICU, the isolates obtained from the patients harbored a single ribotype unrelated to that of the epidemic strain isolated in the PICU; no environmental source of infection was found . CONCLUSION: Two different outbreaks appeared to be associated with 2 ribotypes, 1 of which was linked to patient-to-patient transmission via respiratory therapy devices . Complete elimination of the outbreak was achieved only when disposable, sterilizable, or easy-to-disinfect materials were used in the PICU . The source of infection in the NICU was not found.

Int J Med Microbiol, 2003 Aug, 293(4), 309 - 17
Molecular typing of the bacterial flora in sputum of cystic fibrosis patients; Kolak M et al.; Despite recent advances in therapy, lower airway infections remain the major cause of morbidity and mortality in cystic fibrosis (CF) patients . Bacterial colonisation of the lower airways in CF is limited to a few bacterial species, commonly Staphylococcus aureus, Pseudomonas aeruginosa and Haemophilus influenzae . Burkholderia cepacia colonisation is much rarer, but it has been thought to be associated with more advanced lung disease and increased mortality . A rapid characterisation of the bacterial flora in sputum of CF patients is of great importance for proper treatment . The aim of this study was to establish bacterial profiles and to identify pathogenic bacteria in respiratory specimens by means of molecular methods including temporal temperature gradient gel electrophoresis (TTGE) and DNA sequencing of PCR amplicons derived from 16S rDNA variable V3 and V6 regions . Sputa of 13 CF patients (7 males/6 females, age 19-59 years) collected at the Stockholm CF centre were analysed . TTGE revealed the presence of complex bacterial profiles in all samples . The V3 and V6 PCR amplicons were cloned and sequenced by real-time DNA Pyrosequencing . DNA from Staphylococcus aureus, Haemophilus influenzae, and Pseudomonas aeruginosa, respectively, was identified together with sequences from normal oral cavity flora . The results were in reasonable agreement with those obtained by conventional bacterial culture, considering that only known CF pathogens are included in routine reports . However, the methodology seems too elaborate to be introduced into daily routine

Res Microbiol, 2003 Sep, 154(7), 491 - 8
Molecular characterization of Burkholderia cepacia isolates from cystic fibrosis (CF) patients in an Italian CF center; Petrucca A et al.; Bacteria of the Burkholderia cepacia complex consist of a number of closely related genomic species (genomovars) potentially pathogenic for cystic fibrosis (CF) patients, collectively referred to as the B . cepacia complex . The genomovar status and epidemiological relatedness of B . cepacia complex strains recovered from CF patients, attending a CF Center at the University Hospital "Policlinico Umberto I" of Rome, were investigated using 16S rRNA PCR-RFLP, recA PCR-RFLP, genomovar-specific PCR, and RAPD . Forty-seven isolates identified as B . cepacia by commercial systems were repeatedly recovered from 19 CF patients . The taxonomy approach used in this study showed that 17 of the 19 patients were colonized by B . cepacia complex strains . Genomovar III (11 strains) was the most prevalent genomovar . Two strains of genomovar I, one B . stabilis (genomovar IV), one B . multivorans (genomovar II), and 4 strains of B . anthina (genomovar VIII) were also identified . This is the first report of multiple patient colonization by B . anthina in a CF center . The epidemiological and genetic relatedness as well as the presence of molecular markers associated with virulence and transmissibility of the B . cepacia complex strains were determined and probable patient-to-patient spread was observed.

Int J Syst Evol Microbiol, 2003 Sep, 53(Pt 5), 1631 - 6
Burkholderia sordidicola sp . nov., isolated from the white-rot fungus Phanerochaete sordida; Lim YW et al.; Two bacterial strains associated with the white-rot fungus Phanerochaete sordida were subjected to taxonomic investigation . The isolates, designated KCTC 12081(T) and KCTC 12082, were Gram-negative, non-motile, non-spore-forming and ovoid to rod-shaped . The strains contained major amounts of hexadecanoic acid, cyclo-heptadecanoic acid and omega-7-cis-octadecenoic acid in their cell envelopes . Strain KCTC 12081(T) contained ubiquinone-8 as the major isoprenoid quinone and the G+C content of its genomic DNA was 61.3 mol% . Morphological and chemotaxonomic properties of the strains were consistent with classification in the genus BURKHOLDERIA: In a comparison of 16S rDNA sequence, KCTC 12081(T) shared 100 % similarity with KCTC 12082 and both strains formed a distinct phylogenetic lineage within the genus BURKHOLDERIA: The two strains were also differentiated from other species of this genus by fatty acid composition and phenotypic properties . DNA-DNA relatedness data further supported the separation of the new isolates from closely related species . It is therefore proposed that strains KCTC 12081(T) (=JCM 11778(T)) and KCTC 12082 be recognized as a novel species, for which the name Burkholderia sordidicola sp . nov . is proposed.

Electrophoresis, 2003 Sep, 24(17), 3067 - 74
Analysis of N-acyl-L-homoserine lactones produced by Burkholderia cepacia with partial filling micellar electrokinetic chromatography--electrospray ionization-ion trap mass spectrometry; Frommberger M et al.; A method for the analysis of N-acyl-L-homoserine lactones (AHLs) with micellar electrokinetic chromatography coupled to electrospray ionization-ion trap mass spectrometry, combining the flexibility of capillary electrophoresis with the unmatched structural information provided by mass spectrometry is presented . Different surfactants were evaluated, with sodium dodecyl sulfate (SDS) yielding the best results considering sensitivity and flexibility . We examined the interaction of AHLs with the SDS micelles at different analysis conditions and applied the optimized method to the analysis of a real bacterial sample . Two AHLs from Burkholderia cepacia colonizing the rhizosphere of traditional Indian rice cultivars could be unambiguously determined in an ethyl acetate extract with high resolution flexibility.

Mol Gen Mikrobiol Virusol, 2003, (3), 18 - 22
{Identification of the causative agents of glanders and melioidosis by polymerase chain reaction}; Tkachenko GA et al.; Burkholderia mallei and B . pseudomallei are causative agents of glanders and melioidosis, respectively, i.e . severe and fatal infection diseases of man and animal . The computer-based analysis of the 23S rRNA gene sites was used for selecting the primers . Two pairs of primers were chosen for the identification of B . mallei and Bpseudomallei . DNAs from 48 B . pseudomallei and 15 strains of B . mallei, unlike from other geterological bacteria, were positively amplified . Therefore, the method of polymerase chain reaction can be used in laboratory diagnosis of glanders and melioidosis.

Proc Natl Acad Sci U S A, 2003 Sep 16, 100(19), 10948 - 53 Epub 2003 Sep 05.
Bacterial pathogens modulate an apoptosis differentiation program in human neutrophils; Kobayashi SD et al.; Human polymorphonuclear leukocytes (PMNs or neutrophils) are essential to the innate immune response against bacterial pathogens . Recent evidence suggests that PMN apoptosis facilitates resolution of inflammation during bacterial infection . Although progress has been made toward understanding apoptosis in neutrophils, very little is known about transcriptional regulation of this process during bacterial infection . To gain insight into the molecular processes that facilitate resolution of infection, we measured global changes in PMN gene expression during phagocytosis of a diverse group of bacterial pathogens . Genes encoding key effectors of apoptosis were up-regulated, and receptors critical to innate immune function were down-regulated during apoptosis induced by phagocytosis of Burkholderia cepacia, Borrelia hermsii, Listeria monocytogenes, Staphylococcus aureus, and Streptococcus pyogenes . Importantly, we identified genes that comprise a common apoptosis differentiation program in human PMNs after phagocytosis of pathogenic bacteria . Unexpectedly, phagocytosis of Str . pyogenes induced changes in neutrophil gene expression not observed with other pathogens tested, including down-regulation of 21 genes involved in responses to IFN . Compared with other bacteria, PMN apoptosis was significantly accelerated by Str . pyogenes and was followed by necrosis . Thus, we hypothesize that there are two fundamental outcomes for the interaction of bacterial pathogens with neutrophils: (i) phagocytosis of bacteria induces an apoptosis differentiation program in human PMNs that contributes to resolution of bacterial infection, or (ii) phagocytosis of microorganisms such as Str . pyogenes alters the apoptosis differentiation program in neutrophils, resulting in pathogen survival and disease.

J Clin Microbiol, 2003 Sep, 41(9), 4148 - 53
Molecular typing of, and distribution of genetic markers among, Burkholderia cepacia complex isolates from Brazil; Detsika MG et al.; PCR tests were used to assign genomovar status to 39 non-cystic fibrosis (non-CF) and 11 CF Burkholderia cepacia complex isolates from patients in hospitals in Recife, Brazil . Non-CF isolates were assigned to genomovar IIIA (71.8%), genomovar I (15.4%), B . vietnamiensis (7.7%), and B . multivorans (5.1%) . CF isolates were assigned to genomovar IIIA (18.2%), B . vietnamiensis (18.2%), and genomovar I (9.1%) . Six CF isolates sharing recA PCR-restriction fragment length polymorphism (RFLP) and randomly amplified polymorphic DNA (RAPD) patterns could not be assigned to a genomovar . 16S rDNA sequence obtained from these isolates indicated a closest relationship to B . anthina, but the recA sequence was equally divergent from several genomovars . PCR screening indicated the presence of cblA in only two isolates, whereas the B . cepacia epidemic strain marker was found in 22 of 28 genomovar IIIA isolates . A type III secretion gene was detected in all but genomovar I isolates . RAPD and PCR-RFLP assays, targeting both recA and fliC, indicated a large amount of genetic variability among the isolates, with many novel patterns being observed . Nine genomovar IIIA isolates from different non-CF patients and clinical sources had identical genotypes, indicating the presence of a common clone.

J Clin Microbiol, 2003 Sep, 41(9), 4113 - 20
Molecular analysis of Burkholderia cepacia complex isolates from a Portuguese cystic fibrosis center: a 7-year study; Cunha MV et al.; This work reports results of a systematic molecular analysis involving 113 Burkholderia cepacia complex isolates obtained from 23 cystic fibrosis (CF) patients under surveillance over a 7-year period at the major Portuguese CF center, the Santa Maria Hospital in Lisbon . The majority of the isolates were serial isolates from persistently infected patients (more than one-half of the population examined) . In agreement with previous studies, B . cenocepacia (formerly genomovar III) was the most prevalent species; it was isolated from 52% of the patients infected with B . cepacia complex isolates . Contrasting with previous studies, a very significant percentage of the Portuguese CF subpopulation examined was infected with B . cepacia genomovar I (36%) and B . stabilis (18%) . B . multivorans was recovered from two of the infected patients . All four of the species or genomovars were associated with poor clinical outcome, including the cepacia syndrome, and gave rise to chronic and transient infections, with the clinical condition depending on the patient and other still-unidentified factors . The B . cepacia epidemic strain marker region was found exclusively in genomovar III strains, while cblA was detected in genomovars I and III, only . There was no clear relation between the presence of these markers and transmissibility . Altogether, our results indicate that the use of these markers or the genomovar status in identifying patients at higher risk for infection is uncertain.

Appl Environ Microbiol, 2003 Sep, 69(9), 5410 - 3
The metabolic pathway of 4-aminophenol in Burkholderia sp . strain AK-5 differs from that of aniline and aniline with C-4 substituents; Takenaka S et al.; Burkholderia sp . strain AK-5 utilized 4-aminophenol as the sole carbon, nitrogen, and energy source . A pathway for the metabolism of 4-aminophenol in strain AK-5 was proposed based on the identification of three key metabolites by gas chromatography-mass spectrometry analysis . Strain AK-5 converted 4-aminophenol to 1,2,4-trihydroxybenzene via 1,4-benzenediol . 1,2,4-Trihydroxybenzene 1,2-dioxygenase cleaved the benzene ring of 1,2,4-trihydroxybenzene to form maleylacetic acid . The enzyme showed a high dioxygenase activity only for 1,2,4-trihydroxybenzene, with K(m) and V(max) values of 9.6 micro M and 6.8 micro mol min(-1) mg of protein(-1), respectively.

Appl Environ Microbiol, 2003 Sep, 69(9), 5045 - 50
Biotransformation of natural and synthetic isoflavonoids by two recombinant microbial enzymes; Seeger M et al.; Isolation and synthesis of isoflavonoids has become a frequent endeavor, due to their interesting biological activities . The introduction of hydroxyl groups into isoflavonoids by the use of enzymes represents an attractive alternative to conventional chemical synthesis . In this study, the capabilities of biphenyl-2,3-dioxygenase (BphA) and biphenyl-2,3-dihydrodiol 2,3-dehydrogenase (BphB) of Burkholderia sp . strain LB400 to biotransform 14 isoflavonoids synthesized in the laboratory were investigated by using recombinant Escherichia coli strains containing plasmid vectors expressing the bphA1A2A3A4 or bphA1A2A3A4B genes of strain LB400 . The use of BphA and BphB allowed us to biotransform 7-hydroxy-8-methylisoflavone and 7-hydroxyisoflavone into 7,2',3'-trihydroxy-8-methylisoflavone and 7,3',4'-trihydroxyisoflavone, respectively . The compound 2'-fluoro-7-hydroxy-8-methylisoflavone was dihydroxylated by BphA at ortho-fluorinated and meta positions of ring B, with concomitant dehalogenation leading to 7,2',3',-trihydroxy-8-methylisoflavone . Daidzein (7,4'-dihydroxyisoflavone) was biotransformed by BphA, generating 7,2',4'-trihydroxyisoflavone after dehydration . Biotransformation products were analyzed by gas chromatography-mass spectrometry and nuclear magnetic resonance techniques.

Clin Infect Dis, 2003 Sep 15, 37(6), 780 - 5 Epub 2003 Aug 23.
Burkholderia cepacia complex in cystic fibrosis: frequency of strain replacement during chronic infection; Bernhardt SA et al.; Persons with cystic fibrosis (CF) are susceptible to chronic pulmonary infection due to certain Burkholderia species, but it is not clear whether this typically involves persistent infection with the same strain or sequential infection with distinct strains . We analyzed 1095 Burkholderia isolates recovered from serial sputum cultures from 379 patients with CF receiving care in 112 CF treatment centers in the United States . Genotyping was performed by random amplified polymorphic DNA typing or pulsed-field gel electrophoresis . Overall, a change in infecting strain was found in 24 (6.9%) of 347 patients infected with Burkholderia cepacia complex and in 3 (9%) of 32 patients infected with Burkholderia gladioli . Several patients were likely coinfected, at least transiently, with >1 B . cepacia complex strain . The potential for strain replacement during chronic infection may confound studies of the relationship between strain and clinical outcome and must be considered in designing effective infection-control practices.

Pediatr Pulmonol, 2003 Oct, 36(4), 348 - 52
Pseudomonas aeruginosa and Burkholderia cepacia cannot be detected by PCR in the breath condensate of patients with cystic fibrosis; Vogelberg C et al.; The collection of sputum for microbiological examination in young cystic fibrosis patients can be very difficult . However, a knowledge of bacterial flora colonizing the patient's airways is of paramount importance for proper antimicrobial therapy . It is also known that cystic fibrosis patients colonized by Pseudomonas species have a poorer prognosis than Pseudomonas-negative patients . Noninvasive ways of diagnosing airway inflammation that require only minimal cooperation of the patient might yield new possibilities for early detection of airway colonisation . The breath condensate method as a noninvasive diagnostic technique seems especially appropriate for use in children . Therefore, the aim of this study was to evaluate whether the breath condensate method could be used for detection of Pseudomonas species in children with cystic fibrosis . In total, 32 breath condensate and seven sputum samples were obtained from 13 cystic fibrosis patients with Pseudomonas aeruginosa- or Burkholderia cepacia-positive sputum culture (20 samples were obtained during forced expiration) . PCR for combined detection of Pseudomonas aeruginosa, Stenotrophomonas maltophilia, and Burkholderia cepacia was performed . PCR results of all breath condensate samples were negative for Pseudomonas aeruginosa, Stenotrophomonas maltophilia, or Burkholderia cepacia, while all sputum sample results were positive . A minimum DNA quantity of 10 fg could be detected in dilution series of the positive control group . We conclude that the breath condensate method cannot be used as a tool for detection of Pseudomonas species .

Blood, 2004 Jan 15, 103(2), 673 - 5 Epub 2003 Aug 28.
Naturally occurring anti-IFN-gamma autoantibody and severe infections with Mycobacterium cheloneae and Burkholderia cocovenenans; Hoflich C et al.; Recently various genetic defects in immunity mediated by interferon gamma (IFN-gamma) have been described, including mutations in the IFN-gamma receptor 1 (IFN-gammaR1) and receptor 2 (IFN-gammaR2), signal transducer and activator of transcription 1 (STAT 1), and interleukin 12 receptor beta 1 (IL-12Rbeta1), and IL-12 p40 genes . These mutations are associated with the occurrence of severe infections with intracellular pathogens especially nontuberculous mycobacteria and vaccine-associated bacilli Calmette-Guerin (BCG) . Here we report data on a previously healthy adult patient primarily presenting with severe infections with Burkholderia cocovenenans and subsequently Mycobacterium cheloneae . We found a strong inhibitory anti-IFN-gamma activity in the patient's plasma and identified a high-affinity neutralizing anti-IFN-gamma autoantibody . Unfortunately, the patient died due to severe sepsis before we knew the nature of the inhibitory activity . The application of alternative therapeutic approaches such as intravenous immunoglobulin or immunoadsorption may have been beneficial in this case . Screening for neutralizing anti-IFN-gamma autoantibodies should supplement testing for IFN-gamma and IL-12 pathway defects in patients with recurrent infections with intracellular pathogens, especially with nontuberculous mycobacteria.

Infect Control Hosp Epidemiol, 2003 Aug, 24(8), 624 - 6
Use of pulsed-field gel electrophoresis in infection control issues concerning Burkholderia cepacia; Jenney A et al.; There was concern that nosocomial person-to-person transmission of Burkholderia cepacia had occurred when two patients with cystic fibrosis shared a bathroom . Pulsed-field gel electrophoresis demonstrated that the two isolates were unrelated . Subsequent testing of 34 stored isolates of B . cepacia demonstrated that no particular clone predominated in our hospital.

Antimicrob Agents Chemother, 2003 Sep, 47(9), 2962 - 5
Gentamicin delivery to Burkholderia cepacia group IIIa strains via membrane vesicles from Pseudomonas aeruginosa PAO1; Allan ND et al.; When Pseudomonas aeruginosa PAO1 is treated with gentamicin, it releases membrane vesicles containing gentamicin (g-MVs) and peptidoglycan hydrolase, which makes the MVs bactericidal . We evaluate the ability of g-MVs to deliver gentamicin past the intrinsic permeability barrier of group IIIa Burkholderia cepacia and show that strain CEP0248 with low resistance to gentamicin is killed but the highly resistant strain C5424 is not . Immunoelectron microscopy revealed that gentamicin was delivered into both strains, suggesting that there might be another mechanism of resistance in C5424.

J Endotoxin Res, 2003, 9(4), 201 - 13
Lipopolysaccharide of Burkholderia cepacia complex; Vinion-Dubiel AD et al.; Burkholderia cepacia complex (Bcc) is a group of phenotypically similar, genetically distinct bacteria that are beneficial to the environment but can also cause severe human infections . Bcc are being exploited for use as bioremediation agents and as a way to combat agricultural plant diseases . However, Bcc can cause lung infections in patients with chronic granulomatous disease or cystic fibrosis often resulting in mortality of these patients . Since it is unclear what bacterial components are necessary for causing human infections, studies of Bcc have focused on identifying putative virulence factors . As in other Gram-negative bacteria, the lipopolysaccharide (LPS) of Bcc induces a strong immune response that can contribute to host cell damage . The unusual structure of Bcc LPS lowers the anionic charge of the Bcc cell surface, which inhibits the binding and subsequent effects of cationic antibiotics . These distinguishing features include the substitution of a Ko for a Kdo residue in the inner core oligosaccharide and Ara4N residues bound to phosphates of the lipid A backbone . The structures of O antigen subunits and the consequent serotypes will also be discussed, with particular reference to the O antigen biosynthetic loci of two Bcc strains.

Infect Immun, 2003 Sep, 71(9), 5306 - 13
Comparative analysis of plant and animal models for characterization of Burkholderia cepacia virulence; Bernier SP et al.; A simple alfalfa model was developed as an alternative infection model for virulence studies of the Burkholderia cepacia complex . Symptoms of disease were observed in wounded alfalfa seedlings within 7 days following inoculation of 10(1) to 10(5) CFU of most strains of the B . cepacia complex . Strains from seven genomovars of the B . cepacia complex were tested for virulence in the alfalfa model, and the degree of virulence was generally similar in strains belonging to the same genomovar . Strains of Burkholderia multivorans and some strains of Burkholderia stabilis did not cause symptoms of disease in alfalfa seedlings . Representative strains were also tested for virulence using the rat agar bead model . Most of the strains tested were able to establish chronic lung infections; B . stabilis strains were the exception . Most of the strains that were virulent in the alfalfa infection model were also virulent in the lung infection model . The B . cepacia genomovar III mutants K56pvdA::tp and K56-H15 were significantly less virulent in the alfalfa infection model than their parent strain . Therefore, this alfalfa infection model may be a useful tool for assessing virulence of strains of the B . cepacia complex and identifying new virulence-associated genes.

Infect Immun, 2003 Sep, 71(9), 5225 - 30
Unusual interaction of a lipopolysaccharide isolated from Burkholderia cepacia with polymyxin B; Shimomura H et al.; We have demonstrated that lipopolysaccharide (LPS) obtained from Burkholderia cepacia, an important opportunistic pathogen, has unique characteristics in both structure and activity . One of the structural characteristics is that the B . cepacia LPS has 4-amino-4-deoxy-L-arabinose (Ara4N) in its inner core region . Polymyxin B (PmxB) is known to act as an LPS antagonist, but LPS with Ara4N is suggested to be PmxB resistant by decreasing the binding capability of PmxB . Interaction of B . cepacia LPS with PmxB was investigated and compared with that of a reference LPS of Salmonella enterica serovar Abortusequi, referred to hereafter as the reference LPS . B . cepacia LPS suffered no suppressive effect of PmxB in its activity to stimulate murine peritoneal macrophages for induction of tumor necrosis factor alpha (TNF-alpha) and IL-6 even when the activity of the reference LPS was completely suppressed . A characteristic of B . cepacia LPS is that it has selectively weak interleukin-1 beta (IL-1 beta)-inducing activity while activity to induce TNF-alpha and IL-6 has been shown to be as strong as that of the reference LPS . Remarkably, PmxB augmented the IL-1 beta-inducing activity of B . cepacia LPS to the level of that of the reference LPS and, in contrast, completely suppressed the strong activity of the reference LPS . Using PmxB-immobilized beads, the adsorbances of these LPSs to the beads were compared, and it was found that B . cepacia LPS bound to PmxB with a high affinity similar to that of the reference LPS . These results indicate an unusual interaction of B . cepacia LPS with PmxB whereby B . cepacia LPS not only allows the binding of PmxB with high affinity, even though it contains Ara4N, but also suffers no suppressive effect of PmxB on its activity . Moreover, a remarkable increase in its IL-1 beta-inducing activity was also observed.

Carbohydr Res, 2003 Sep 1, 338(18), 1861 - 7
Macromolecular and solution properties of Cepacian: the exopolysaccharide produced by a strain of Burkholderia cepacia isolated from a cystic fibrosis patient; Sist P et al.; Light scattering and viscosity measurements were carried out on the previously chemically characterised exopolysaccharide produced by a strain of Burkholderia cepacia isolated from a cystic fibrosis patient . The same exopolysaccharide was also produced by other clinical strains in different laboratories . Therefore, the name Cepacian is now proposed for this exopolysaccharide . Experiments performed as a function of the ionic strength on the native polymer revealed a change in the overall shape of the polymer at low ionic strength . This behaviour was absent in the de-acetylated sample . Potentiometric titrations and light scattering experiments carried out on the acidic form of the native polymer revealed the formation of macromolecular aggregates with a stoichiometry n and 2n stabilised by interactions involving the uronic acid residues.

Commun Dis Intell, 2003, 27(2), 272 - 7
Melioidosis in northern Australia, 2001-02; Cheng AC et al.; Melioidosis, caused by the gram negative bacterium Burkholderia pseudomallei, is endemic in northern Australia . Using data collated from centres in Western Australia, the Northern Territory and Queensland, this report describes the epidemiology of this disease between 1 November, 2001 and 31 October, 2002 . There were 47 cases seen during this period with an average annual incidence of 5.8 cases per 100,000 population . In Indigenous Australians, an incidence of 25.5 cases per 100,000 population was seen . The timing and location of cases was generally correlated with rainfall across northern Australia . A case-cluster in a Queensland community was associated with post-cyclonic flooding . Risk factors included diabetes, alcohol-related problems and renal disease . Pneumonia (51%) was the most common clinical diagnosis . The mortality rate attributable to melioidosis was 21 per cent, although a number of other patients died of underlying disease . Despite improvements in recognition and treatment, melioidosis is still associated with a high morbidity and mortality, particularly in Indigenous Australians.

Environ Microbiol, 2003 Sep, 5(9), 719 - 29
Diversity and significance of Burkholderia species occupying diverse ecological niches; Coenye T et al.; Members of the genus Burkholderia are versatile organisms that occupy a surprisingly wide range of ecological niches . These bacteria are exploited for biocontrol, bioremediation and plant growth promotion purposes, but safety issues regarding human infections, especially in cystic fibrosis patients, have not been solved . This minireview gives an overview of the taxonomic and ecological diversity of the genus with particular emphasis on strains belonging to the Burkholderia cepacia complex and addresses the important question whether 'good' and 'bad' strains are actually the same.

Genet Mol Res, 2003 Mar 31, 2(1), 48 - 62
Efflux as a mechanism of resistance to antimicrobials in Pseudomonas aeruginosa and related bacteria: unanswered questions; Schweizer HP; Pseudomonas aeruginosa is an opportunistic human pathogen exhibiting innate resistance to multiple antimicrobial agents . This intrinsic multidrug resistance is caused by synergy between a low-permeability outer membrane and expression of a number of broadly-specific multidrug efflux (Mex) systems, including MexAB-OprM and MexXY-OprM . In addition to this intrinsic resistance, these and three additional systems, MexCD-OprJ, MexEF-OprN and MexJK-OprM promote acquired multidrug resistance as a consequence of hyper-expression of the efflux genes by mutational events . In addition to antibiotics, these pumps export biocides, dyes, detergents, metabolic inhibitors, organic solvents and molecules involved in bacterial cell-cell communication . Homologues of the resistance-nodulation-division systems of P . aeruginosa have been found in Burkholderia cepacia, B . pseudomallei, Stenotrophomonas maltophilia, and the nonpathogen P . putida, where they play roles in resistance to antimicrobials and/or organic solvents . Despite intensive studies of these multidrug efflux systems over the past several years, their precise molecular architectures, their modes of regulation of expression and their natural functions remain largely unknown.

Microbiology, 2003 Aug, 149(Pt 8), 2263 - 71
An extracellular zinc metalloprotease gene of Burkholderia cepacia; Corbett CR et al.; Burkholderia cepacia produces at least one extracellular zinc metalloprotease that may be involved in virulence . A B . cepacia zinc metalloprotease gene was cloned using a Burkholderia pseudomallei zinc metalloprotease gene as a probe . The predicted amino acid sequences of these B . cepacia and a B . pseudomallei extracellular zinc metalloproteases indicate that they are similar to the thermolysin-like family of metalloproteases (M4 family of metalloendopeptidases) and they are likely to be secreted via the general secretory pathway . zmpA isogenic mutants were constructed in B . cepacia genomovar III strains Pc715j and K56-2 by insertional inactivation of the zmpA genes . The zmpA mutants produced less protease than the parent strains . The B . cepacia strain K56-2 zmpA mutant was significantly less virulent than its parent strain in a chronic respiratory infection model; however, there was no difference between the virulence of B . cepacia strain Pc715j and a Pc715j zmpA mutant . The results indicate that this extracellular zinc metalloprotease may play a greater role in virulence in some strains of B . cepacia.

J Clin Microbiol, 2003 Aug, 41(8), 3973 - 7
Seronegative bacteremic melioidosis caused by Burkholderia pseudomallei with ambiguous biochemical profile: clinical importance of accurate identification by 16S rRNA gene and groEL gene sequencing; Woo PC et al.; An aerobic gram-negative bacterium was isolated from the blood and sputum of an 84-year-old, chair-bound nursing home resident with acute bacteremic pneumonia . Although the phenotypic characteristics suggested that the bacterium could be Burkholderia pseudomallei, the Vitek 1 system (GNI+), which can successfully identify 99% of B . pseudomallei strains, showed that the bacterium was "unidentified." Immunoglobulin G against the lipopolysaccharide (LPS) of B . pseudomallei, as detected by an LPS-based enzyme-linked immunosorbent assay with 95% sensitivity, was negative in both the acute-phase and convalescent-phase sera . Sequencing of the groEL gene showed that the isolate was B . pseudomallei . Proper identification of the bacterium in this study is crucial, since there would be a radical difference in the duration of antimicrobial therapy.

J Clin Microbiol, 2003 Aug, 41(8), 3548 - 58
Bacterial diversity in cases of lung infection in cystic fibrosis patients: 16S ribosomal DNA (rDNA) length heterogeneity PCR and 16S rDNA terminal restriction fragment length polymorphism profiling; Rogers GB et al.; The leading cause of morbidity and mortality in cystic fibrosis (CF) patients stems from repeated bacterial respiratory infections . Many bacterial species have been cultured from CF specimens and so are associated with lung disease . Despite this, much remains to be determined . In the present study, we characterized without prior cultivation the total bacterial community present in specimens taken from adult CF patients, extracting DNA directly from 14 bronchoscopy or sputum samples . Bacterial 16S ribosomal DNA (rRNA) gene PCR products were amplified from extracted nucleic acids, with analyses by terminal restriction fragment length polymorphism (T-RFLP), length heterogeneity PCR (LH-PCR), and sequencing of individual cloned PCR products to characterize these communities . Using the same loading of PCR products, 12 distinct T-RFLP profiles were identified that had between 3 and 32 T-RFLP bands . Nine distinct LH-PCR profiles were identified containing between one and four bands . T-RFLP bands were detected in certain samples at positions that corresponded to pathogens cultured from CF samples, e.g., Burkholderia cepacia and Haemophilus influenzae . In every sample studied, one T-RFLP band was identified that corresponded to that produced by Pseudomonas aeruginosa . A total of 103 16S rRNA gene clones were examined from five patients . P . aeruginosa was the most commonly identified species (59% of clones) . Stenotrophomonas species were also common, with eight other (typically anaerobic) bacterial species identified within the remaining 17 clones . In conclusion, T-RFLP analysis coupled with 16S rRNA gene sequencing is a powerful means of analyzing the composition and diversity of the bacterial community in specimens sampled from CF patients.

Expert Rev Vaccines, 2002 Dec, 1(4), 477 - 82
Melioidosis vaccines; Warawa J et al.; Melioidosis is a disease caused by the facultative intracellular pathogen Burkholderia pseudomallei iand is associated with a high mortality rate . Melioidosis is endemic in the tropics of southeast Asia and northern Australia and is of worldwide concern, particularly as it is a potential agent of bioterrorism or biological warfare . Also of concern is the lack of a fully effective antibiotic regime, as cases of bacteremia have unacceptably high mortality rates and relapse of melioidosis is common . This review focuses on the approaches that have been undertaken towards the development of an effective vaccine against this disease and highlights current strategies being used to move towards finalizing such a vaccine.

J Mol Biol, 2003 Aug 15, 331(3), 585 - 92
Inverting enantioselectivity of Burkholderia cepacia KWI-56 lipase by combinatorial mutation and high-throughput screening using single-molecule PCR and in vitro expression; Koga Y et al.; The enantioselectivity of lipase from Burkhorderia cepacia KWI-56 has been inverted using a novel in vitro technique for construction and screening of a protein library by single-molecule DNA amplification by PCR followed by in vitro coupled transcription/translation system termed single-molecule-PCR-linked in vitro expression (SIMPLEX) . Four amino acid residues (L17, F119, L167, and L266) in the hydrophobic substrate-binding pocket of the lipase were selected for mutation based on a structural model of a substrate-enzyme complex, and a combinatorial mutation library was constructed by SIMPLEX and screened for (R) and (S)-configurations of p-nitrophenyl 3-phenylbutyrate . Some combinations of amino acid substitutions in the four positions of the lipase were found as effective for changing the enantiopreference from the (S)-form substrate to the (R)-form . Two variants were expressed in the original host cells and purified to homogeneity, showing completely reversed enantioselectivity for the (R)-form of ethyl 3-phenylbutyrate (selectivity factor E(R)=38 or 33), whereas the wild-type lipase was (S)-selective (selectivity factor E(S)=33) . Thus the semi-rational and semi-random combinatorial design of a mutant library followed by a high-throughput screening based on their enzymatic activity should be a powerful tool to engineer the enantioselectivity of enzymes.

Eur J Clin Microbiol Infect Dis, 2003 Aug, 22(8), 496 - 500 Epub 2003 Jul 25.
Evaluation of the Merlin, Micronaut system for automated antimicrobial susceptibility testing of Pseudomonas aeruginosa and Burkholderia species isolated from cystic fibrosis patients; Haussler S et al.; Since accurate antimicrobial susceptibility testing of bacterial cystic fibrosis isolates is known to be problematic and an optimal in vitro testing method has not yet been evaluated, the study presented here was conducted to compare the performance of the reference agar dilution method and broth microdilution with a commercially available automated susceptibility test system (Merlin; Micronaut, Germany) . In this pilot study, the susceptibility of 70 clinical strains of Pseudomonas aeruginosa and Burkholderia cepacia-like organisms to nine antimicrobial agents was tested using these methods . Susceptibility results generated by broth microdilution (both automated and according to the National Committee for Clinical Laboratory Standards recommendations) were demonstrated to be of good reproducibility, and they compared favourably to the time- and material-consuming standard agar dilution reference method, especially after a prolonged incubation time (48 h).

J Bacteriol, 2003 Aug, 185(16), 4992 - 6
A Burkholderia pseudomallei type III secreted protein, BopE, facilitates bacterial invasion of epithelial cells and exhibits guanine nucleotide exchange factor activity; Stevens MP et al.; We report the characterization of BopE, a type III secreted protein that is encoded adjacent to the Burkholderia pseudomallei bsa locus and is homologous to Salmonella enterica SopE/SopE2 . Inactivation of bopE impaired bacterial entry into HeLa cells, indicating that BopE facilitates invasion . Consistent with this notion, BopE expressed in eukaryotic cells induced rearrangements in the subcortical actin cytoskeleton, and purified BopE exhibited guanine nucleotide exchange factor activity for Cdc42 and Rac1 in vitro.

FEMS Microbiol Lett, 2003 Jul 29, 224(2), 315 - 8
G-CSF immunotherapy for treatment of acute disseminated murine melioidosis; Powell K et al.; Burkholderia pseudomallei, the causative agent of melioidosis, is an important pathogen in tropical regions of Australia and Southeast Asia . Antibiotic therapy can be ineffective in patients with acute septicaemic melioidosis . It has been proposed that adjunctive immunotherapy using granulocyte colony stimulating factor (G-CSF) combined with antibiotics may provide an alternative approach to antibiotics alone . We have developed a murine model for melioidosis that allows novel treatment approaches to be investigated . This study looked at the potential for murine G-CSF therapy both alone and as an adjunct in the treatment of acute disseminated B . pseudomallei infection in BALB/c mice . A number of therapeutic variables involving ceftazidime and recombinant murine G-CSF were studied . Surviving mice were sacrificed and splenic bacterial loads were determined . Combining recombinant murine G-CSF with ceftazidime offered no advantage over ceftazidime alone . Pre-treatment with recombinant murine G-CSF did not demonstrate a significant benefit . This would suggest that adjunct immunotherapy using G-CSF is of limited benefit.

Res Microbiol, 2003 Jul-Aug, 154(6), 451 - 5
A new mini-transposon for in vivo protein epitope tagging: application to Burkholderia multivorans; Kholti A et al.; A short amino acid sequence coding for the mature Pseudomonas aeruginosa OprI lipoprotein was fused to a mini-Tn5 plasposon (mini-transposon with an origin of replication) with tetracycline resistance in order to generate in-frame fusion proteins after transposition . After conjugative transfer to Burkholderia multivorans, clones reacting with an anti-OprI mab were selected . In-frame OprI-tagged proteins were detected and identified for six clones . The six C-tagged proteins were detected by immunoblot . The different mutants had insertions into a histone H1-like coding gene, cspD, encoding a cold-shock protein, dsbC, encoding a putative outer membrane lipoprotein involved in thiol-disulfide exchange, paaE, a ferredoxin-NADPH reductase gene, a gene for the catabolism of propionate, and one encoding an unknown protein.

Biotechnol Bioeng, 2003 Sep 30, 83(7), 798 - 809
Substrate inhibition kinetics for toluene and benzene degrading pure cultures and a method for collection and analysis of respirometric data for strongly inhibited cultures; Alagappan G et al.; We present an evaluation of the qualitative and quantitative effects that high concentrations of benzene and toluene have on the growth rate of several pure cultures that use these compounds as their sole carbon and energy source . The cultures employed were five widely studied environmental isolates: Pseudomonas putida F1, P . putida mt2, P . mendocina KR, Ralstonia pickettii PKO1, and Burkholderia cepacia G4 . Three cultures degraded toluene following a pattern consistent with the kinetic model of Wayman and Tseng (1976) while the other two followed a modification of this model introduced by Alagappan and Cowan (2001) . The pattern followed for benzene degradation was different than that for toluene degradation for all four capable pure cultures and consistent with that described by the model of Luong (1987) . Mechanisms of substrate inhibition and solvent toxicity are discussed, used to conceptually evaluate the reasons for the differences in inhibition behavior, and used to support a call for more widespread use of the empirical, terminal substrate concentration inhibition models employed here . We also present the methodology developed to overcome a limitation commonly encountered when attempting to collect oxygen uptake data for use in quantifying substrate inhibition kinetics . The experimental method was effective for use in the collection of high quality data and the substrate inhibition models most useful in representing the growth of bacteria on these solvents are those that show a complete loss of activity at high concentration rather than the more popular asymptotic inhibition models .

Zh Mikrobiol Epidemiol Immunobiol, 2003 Mar-Apr, (2), 3 - 7
{Effect of cultivation temperature on the extrachromosomal hereditary factors of the causative agent of melioidosis}; Antonov VA et al.; The cultivation temperature of Burkholderia pseudomallei has been shown to determine both the direction of morphological dissociation and the prophage induction rate . Inheriting plasmid replicons was found to depend on the temperature conditions during the growth of these bacteria . No influence of B . pseudomallei plasmids pPM1 and pCM2 on the lysogenic state of the bacterial cell and the formation of different B . pseudomallei variants was noted.

Eur J Clin Microbiol Infect Dis, 2003 Jul, 22(7), 434 - 7 Epub 2003 Jun 27.
Genomovar diversity amongst Burkholderia cepacia complex isolates from an Australian adult cystic fibrosis unit; Kidd TJ et al.; In this study, a combination of recA-based PCR assays and 16S rDNA restriction fragment length polymorphism (RFLP) analysis was used to determine the genomovar diversity of clinical Burkholderia cepacia complex isolates . Twenty-eight isolates were prospectively collected from patients attending a large Australian adult cystic fibrosis (CF) unit, 22 isolates were referred from other Australian CF units and a further eight isolates originated from patients without CF . The 28 prospectively collected isolates were distributed amongst the following genomovars: Burkholderia cepacia genomovar I (28.6%), Burkholderia multivorans (21.4%), Burkholderia cepacia genomovar III (39.3%), Burkholderia vietnamiensis(3.6%) and Burkholderia ambifaria (7.1%) . The results of this study highlight the usefulness of 16S rDNA RFLP typing for the identification of other Burkholderia spp . and non-fermenting gram-negative bacteria.

Folia Microbiol (Praha), 2003, 48(3), 329 - 37
Application of selectively acylated glycosides for the alpha-galactosidase-catalyzed synthesis of disaccharides; Simerska P et al.; 4-Nitrophenyl alpha-D-galactopyranosyl-(1-->3)-6-O-acetyl-alpha-D-galactopyranoside was prepared in a transglycosylation reaction catalyzed by alpha-D-galactosidase from Talaromyces flavus using 4-nitrophenyl alpha-D-galactopyranoside as a glycosyl donor and 4-nitrophenyl 6-O-acetyl-alpha-D-galactopyranoside as an acceptor . 4-Nitrophenyl 6-O-acetyl-alpha-D-galactopyranoside and 4-nitrophenyl 6-O-acetyl-beta-D-galactopyranoside were prepared in a regioselective enzymic transesterification in pyridine-acetone catalyzed by the lipase PS from Burkholderia cepacia . A series of water-miscible organic solvents (acetone, acetonitrile, dimethylformamide, dimethyl sulfoxide, 1,4-dioxane, 2-methoxyethanol, pyridine, 2-methylpropan-2-ol, tetrahydrofuran, propargyl alcohol) were used as co-solvents in this enzymic reaction . Their influence on the activity and stability of the alpha-galactosidase from T . flavus was established . 2-Methylpropan-2-ol and acetone (increasing the solubility of the modified substrate acceptors and displaying the minimum impairment of the activity and stability of the enzyme) were used as co-solvents in transglycosylation reactions.

Biopolymers, 2003 Aug, 69(4), 480 - 97
Conformational analysis of the exopolysaccharide from Burkholderia caribensis strain MWAP71: impact on the interaction with soils; Vanhaverbeke C et al.; The strain MWAP71 of Burkholderia caribensis produces a branched charged exopolysaccharide (EPS) that is responsible for soil aggregation . Understanding the conformational properties of the isolated polysaccharide is a prerequisite for proper investigation of the interactions between the polysaccharide and the soil at the atomic level . The aim of this study is first to have an overall view of the flexibility of the backbone and then to ascertain the role played by side groups in the conformational properties of the main chain . Conformational analysis of each oligomeric segment of the polysaccharide has been performed by means of adiabatic mapping of the backbone glycosidic torsion angles using the MM3(92) force field . Substitution by an acetyl group or by a Kdo unit has only a slight effect on the potential energy surfaces of the fragment model compounds . Calculated partition functions, however, indicate that the overall flexibility is slightly larger for the substituted oligomers than for the unsubstituted ones . Prediction of selected average interproton distances from the AB and BC potential energy surfaces allows comparison between modeling results and NMR measurements performed on the ABC fragment . Agreement between the experimental and the predicted data suggests that the established surfaces correctly reflect the observed conformational behavior of such fragments and validate the modeling protocol . The above results have been extended to regular and disordered long polymer chains, differing in Kdo content . It is found that Kdo affects the helical conformations of the polysaccharide . The number of stable helices is considerably larger with Kdo than without Kdo . On the contrary, Kdo has only a moderate effect on unperturbed disordered conformations of the polysaccharide . Predicted persistence length of 70 A suggests that the polymer is semirigid with moderate extension . A further validation of the modeling results is obtained by the good concordance between this predicted value and the experimental one of 95 A, measured from light scattering and viscosity experiments . The results lead to an understanding of the interactions of this polysaccharide with soils .

J Heart Lung Transplant, 2003 Jul, 22(7), 764 - 9
Pulmonary transplantation for cystic fibrosis: pre-transplant recipient characteristics in patients dying of peri-operative sepsis; De Soyza A et al.; BACKGROUND: Pulmonary transplantation has emerged as a successful treatment for end-stage cystic fibrosis . Despite the chronic bronchial sepsis and often multi-resistant organisms seen in this group of recipients, death due to post-operative sepsis is relatively scarce . Identifying potential recipient risk factors for poor outcome may further improve the utilization of a scarce donor pool . METHODS: We assessed the role of pre-operative clinical measures of sepsis, microbial characteristics and recipient characteristics on post-transplant outcome in 85 cystic fibrosis patients who underwent pulmonary transplantation . Ten percent of patients died in the early post-operative period due to sepsis . The prognostic role of recipient factors including markers of sepsis, such as white cells and C-reactive protein (CRP), and the influence of multi-resistant organisms, in particular organisms from the Burkholderia cepacia complex, on outcomes were investigated . RESULTS: We found no prognostic effect of gender, pre-transplant CRP, forced expiratory volume in 1 second (FEV(1)), weight, diabetic status or infection with multi-resistant Pseudomonas organisms . A raised white cell count or temperature or a pre-transplant infection with B cepacia was, however, associated with a significantly poorer prognosis at p = 0.03, 0.03 and 0.001, respectively . CONCLUSIONS: Pre-operative B cepacia complex infection, leukocytosis and pyrexia, but not CRP, weight, diabetes or lung function, were found to be associated with poorer post-transplant outcome . The most clinically relevant of these to the subsequent risk of post-operative death from sepsis appear to be B cepacia infection and pyrexia.

J Med Assoc Thai, 2003 May, 86(5), 436 - 41
Splenic abscess: clinical features, microbiologic finding, treatment and outcome; Sangchan A et al.; Splenic abscess is a rare clinical entity but may be underreported . A retrospective study at Srinagarind Hospital revealed 60 cases of splenic abscess between 1992 and 2001 . The causative organisms were identified in 41 cases (68.3%) . Gram negative bacilli were commonly isolated and Burkholderia pseudomallei was the most predominant . Diabetes mellitus and leukemia were common underlying diseases found in 46.3 per cent and 9.7 per cent of culture confirmed cases, respectively . The patients usually presented with fever, left upper quadrant pain, tenderness and splenomegaly . Multiple abscesses were more commonly found in the melioidosis than in the non-melioidosis group (p = 0.032), but a single abscess was more commonly found in the non-melioidosis than in the melioidosis group (p = 0.032) . Concurrent liver abscesses, often multiple, were not different in both groups . Antimicrobials alone were given in 66.7 per cent of cases with melioidosis and 64.7 per cent of non-melioidosis group . Splenectomy and percutaneous aspiration were performed only in 29.3 per cent and 4.9 per cent of cases with splenic abscess . The overall mortality rate of splenic abscess was only 4.9 per cent in the present series . In conclusion, splenic abscess is not uncommon . Burkholderia pseudomalleli is the most common causative agent found in the present series . Therefore, it should be targeted in the initial empirical antibiotic therapy before the culture results are available especially when multiple lesions in the spleen and concurrent multiple liver abscesses are seen . Prolonged treatment with appropriate antimicrobials alone is usually effective . Splenectomy and/or aspiration may be useful in selected patients.

FEMS Microbiol Lett, 2003 Jul 15, 224(1), 133 - 8
Development of a species-specific recA-based PCR test for Burkholderia fungorum; Chan CH et al.; The genus Burkholderia comprises over 28 species and species-specific, recA-based polymerase chain reaction (PCR) tests are available for several species, but not for some soil-inhabiting species including B . fungorum . Previous analysis of several novel rhizospheric, environmental isolates belonging to the B . cepacia complex suggested they may be closely related to B . fungorum . To discover any relationship between these isolates and B . fungorum we set out to clone and sequence a portion of the B . fungorum recA gene in order to design species-specific primer pairs for use in a recA-based PCR assay . Using a similar procedure we extended the recA-based PCR assay to identify B . sacchari and B . caledonica, two additional soil-inhabiting Burkholderia spp.

Crit Care Med, 2003 Jul, 31(7), 1908 - 14
Strategy of antibiotic rotation: long-term effect on incidence and susceptibilities of Gram-negative bacilli responsible for ventilator-associated pneumonia; Gruson D et al.; OBJECTIVE: To evaluate the long-term effect of a program of rotating antibiotics on the incidence of ventilator-associated pneumonia and the susceptibilities of Gram-negative bacilli responsible for ventilator-associated pneumonia . DESIGN: Prospective program for the surveillance of antibiotic susceptibilities of microorganisms responsible for ventilator-associated pneumonia . SETTING: Academic, university-based, medical intensive care unit (16 beds) . SUBJECTS: 2,856 mechanically ventilated patients . INTERVENTIONS: A new program of antibiotic use was introduced at the end of 1996 that involved the rotation of antibiotics in empirical and therapeutic use of the treatment of ventilator-associated pneumonia . The rotation concerned the beta-lactam and aminoglycoside classes, with a rotation interval of 1 month . The use of antibiotics was monitored monthly . No preference was given to any particular antibiotic . In a previous study, the period before the introduction of this protocol (1995-1996) was compared with the period 2 yrs after (1997-1998): The results indicated a decreased incidence of ventilator-associated pneumonia, a lower incidence of potentially resistant Gram-negative bacilli, and increased sensitivities of Gram-negative bacilli, especially Pseudomonas aeruginosa and Burkholderia cepacia . After 1998, we decided to continue a routine for this rotation . The long-term effect of this program was studied by comparing the incidence of Gram-negative bacilli responsible for ventilator-associated pneumonia and their susceptibilities obtained in a third period: 1999-2001 . The long-term effect (5 yrs) of such a strategy-2-yr protocol period (1997-1998) and 3-yr routine period (1999-2001)-could be evaluated . MEASUREMENTS AND MAIN RESULTS: During the 7-yr study period, 2,856 patients were mechanically ventilated for >48 hrs . The incidence of ventilator-associated pneumonia remained significantly lower in period 3 (1999-2001): 23% (period 1, 1995-1996) vs . 15.7% (period 2, 1997-1998) vs . 16.3% (period 3, 1999-2001; p =.002) . Late-onset ventilator-associated pneumonia occurred in 86.6% and 94% of cases, respectively, in periods 1 and 3 (p =.02) . The decrease of the incidence of early-onset ventilator-associated pneumonia was statistically significant during the 7-yr study period: 13% vs . 9% vs . 5.9% (p =.02) . Combined with a higher incidence of late-onset ventilator-associated pneumonia, the incidence of potentially resistant Gram-negative bacilli increased in period 3: 42.2% vs . 34.5% vs . 41.7% (nonsignificant), except for B . cepacia: 11.7% vs . 7.4% vs . 3.7% (p =.005) . Nevertheless, the potential antibiotic-resistant Gram-negative bacilli were more sensitive to most of the beta-lactams, especially piperacillin-tazobactam and cefepime . CONCLUSIONS: Rotation of antibiotics could help to avoid ventilator-associated pneumonia . It could greatly improve the susceptibilities of the potentially antibiotic-resistant Gram-negative bacilli responsible for late-onset ventilator-associated pneumonia . This program could be applied in routine with good results 5 yrs after its introduction . Further studies, especially multiple-center trials, are necessary to confirm this result and better define the rotation type and intervals.

J Clin Microbiol, 2003 Jul, 41(7), 3415 - 8
Use of amplified ribosomal DNA restriction analysis for identification of Ralstonia and Pandoraea species: interest in determination of the respiratory bacterial flora in patients with cystic fibrosis; Segonds C et al.; The recovery of Ralstonia and Pandoraea species from respiratory tract cultures of patients with cystic fibrosis has recently been reported . These species are difficult to identify, and especially to differentiate from Burkholderia cepacia complex organisms, with classical methods . The discriminatory power of amplified ribosomal DNA restriction analysis (ARDRA) within the two genera was assessed by comparing the restriction profiles of reference strains of each species by using a panel of six enzymes already proven suitable for the identification of Burkholderia species . ARDRA provided differentiation of all the Ralstonia species tested and of Pandoraea norimbergensis . Pandoraea species P . pnomenusa, P . sputorum, P . pulmonicola, and P . apista were not discriminated to the species level . This method allowed the identification of five clinical isolates recovered from French cystic fibrosis patients as Ralstonia mannitolilytica.

J Clin Microbiol, 2003 Jul, 41(7), 3312 - 6
Novel selective medium for isolation of Burkholderia pseudomallei; Howard K et al.; Isolation of Burkholderia pseudomallei currently relies on the use of Ashdown's selective agar (ASA) . We designed a new selective agar (Burkholderia pseudomallei selective agar {BPSA}) to improve recovery of the more easily inhibited strains of B . pseudomallei . B . pseudomallei, Burkholderia cepacia, and Pseudomonas aeruginosa were used to determine the selectivity and sensitivity of BPSA . BPSA was more inhibitory to P . aeruginosa and B . cepacia and should make recognition of Burkholderia species easier due to distinctive colony morphology . BPSA also inhibited Enterococcus, Escherichia, Staphylococcus, and Streptococcus: These results indicate that BPSA is a potential replacement for ASA.

J Bacteriol, 2003 Jul, 185(14), 4038 - 49
Crystal structure of D-Hydantoinase from Burkholderia pickettii at a resolution of 2.7 Angstroms: insights into the molecular basis of enzyme thermostability; Xu Z et al.; D-Hydantoinase (D-HYD) is an industrial enzyme that is widely used in the production of D-amino acids which are precursors for semisynthesis of antibiotics, peptides, and pesticides . This report describes the crystal structure of D-hydantoinase from Burkholderia pickettii (HYD(Bp)) at a 2.7-A resolution . The structure of HYD(Bp) consists of a core (alpha/beta)(8) triose phosphate isomerase barrel fold and a beta-sheet domain, and the catalytic active site consists of two metal ions and six highly conserved amino acid residues . Although HYD(Bp) shares only moderate sequence similarity with D-HYDs from Thermus sp . (HYD(Tsp)) and Bacillus stearothermophilus (HYD(Bst)), whose structures have recently been solved, the overall structure and the structure of the catalytic active site are strikingly similar . Nevertheless, the amino acids that compose the substrate-binding site are less conserved and have different properties, which might dictate the substrate specificity . Structural comparison has revealed insights into the molecular basis of the differential thermostability of D-HYDs . The more thermostable HYD(Tsp) contains more aromatic residues in the interior of the structure than HYD(Bp) and HYD(Bst) . Changes of large aromatic residues in HYD(Tsp) to smaller residues in HYD(Bp) or HYD(Bst) decrease the hydrophobicity and create cavities inside the structure . HYD(Tsp) has more salt bridges and hydrogen-bonding interactions and less oxidation susceptible Met and Cys residues on the protein surface than HYD(Bp) and HYD(Bst) . Besides, HYD(Tsp) also contains more rigid Pro residues . These factors are likely to make major contributions to the varying thermostability of these enzymes . This information could be exploited in helping to engineer more thermostable mesophilic enzymes.

Eur J Clin Microbiol Infect Dis . 2003 Jun 27; {Epub ahead of print}
Genomovar Diversity Amongst Burkholderia cepacia Complex Isolates From an Australian Adult Cystic Fibrosis Unit; Kidd TJ et al.; In this study, a combination of recA-based PCR assays and 16S rDNA restriction fragment length polymorphism (RFLP) analysis was used to determine the genomovar diversity of clinical Burkholderia cepacia complex isolates . Twenty-eight isolates were prospectively collected from patients attending a large Australian adult cystic fibrosis (CF) unit, 22 isolates were referred from other Australian CF units and a further eight isolates originated from patients without CF . The 28 prospectively collected isolates were distributed amongst the following genomovars: Burkholderia cepacia genomovar I (28.6%), Burkholderia multivorans (21.4%), Burkholderia cepacia genomovar III (39.3%), Burkholderia vietnamiensis(3.6%) and Burkholderia ambifaria (7.1%) . The results of this study highlight the usefulness of 16S rDNA RFLP typing for the identification of other Burkholderia spp . and non-fermenting gram-negative bacteria.

J Biol Chem, 2003 Sep 12, 278(37), 35687 - 92 Epub 2003 Jun 29.
Characterization of the catalase-peroxidase KatG from Burkholderia pseudomallei by mass spectrometry; Donald LJ et al.; The electron density maps of the catalase-peroxidase from Burkholderia pseudomallei (BpKatG) presented two unusual covalent modifications . A covalent structure linked the active site Trp111 with Tyr238 and Tyr238 with Met264, and the heme was modified, likely by a perhydroxy group added to the vinyl group on ring I . Mass spectrometry analysis of tryptic digests of BpKatG revealed a cluster of ions at m/z 6585, consistent with the fusion of three peptides through Trp111, Tyr238, and Met264, and a cluster at m/z approximately 4525, consistent with the fusion of two peptides linked through Trp111 and Tyr238 . MS/MS analysis of the major ions at m/z 4524 and 4540 confirmed the expected sequence and suggested that the multiple ions in the cluster were the result of multiple oxidation events and transfer of CH3-S to the tyrosine . Neither cluster of ions at m/z 4525 or 6585 was present in the spectrum of a tryptic digest of the W111F variant of BpKatG . The spectrum of the tryptic digest of native BpKatG also contained a major ion for a peptide in which Met264 had been converted to homoserine, consistent with the covalent bond between Tyr238 and Met264 being susceptible to hydrolysis, including the loss of the CH3-S from the methionine . Analysis of the tryptic digests of hydroperoxidase I (KatG) from Escherichia coli provided direct evidence for the covalent linkage between Trp105 and Tyr226 and indirect evidence for a covalent linkage between Tyr226 and Met252 . Tryptic peptide analysis and N-terminal sequencing revealed that the N-terminal residue of BpKatG is Ser22.

Antimicrob Agents Chemother, 2003 Jul, 47(7), 2082 - 7
Burkholderia pseudomallei class a beta-lactamase mutations that confer selective resistance against ceftazidime or clavulanic acid inhibition; Tribuddharat C et al.; Burkholderia pseudomallei, the causative agent of melioidosis, is inherently resistant to a variety of antibiotics including aminoglycosides, macrolides, polymyxins, and beta-lactam antibiotics . Despite resistance to many beta-lactams, ceftazidime and beta-lactamase inhibitor-beta-lactam combinations are commonly used for treatment of melioidosis . Here, we examine the enzyme kinetics of beta-lactamase isolated from mutants resistant to ceftazidime and clavulanic acid inhibition and describe specific mutations within conserved motifs of the beta-lactamase enzyme which account for these resistance patterns . Sequence analysis of regions flanking the B . pseudomallei penA gene revealed a putative regulator gene located downstream of penA . We have cloned and sequenced the penA gene from B . mallei and found it to be identical to penA from B . pseudomallei.

J Hosp Infect, 2003 Jun, 54(2), 120 - 3
Polyclonal outbreak of Burkholderia cepacia complex bacteraemia in haemodialysis patients; Magalhaes M et al.; We report a polyclonal outbreak of bacteraemia involving 24 patients at a haemodialysis facility in Recife (Brazil) . During the outbreak period (4 June to 11 July, 2001), three Burkholderia cepacia complex strains were isolated from human blood and from various water samples collected at different sites in the haemodialysis unit and from dialysate fluids . Out of 14 patients with positive blood cultures, six were infected by Burkholderia cepacia complex bacteria: three with Burkholderia cepacia genomovar III, two with a first strain of Burkholderia vietnamiensis, and one with the Burkholderia cepacia genomovar III strain and a second B . vietnamiensis strain.

Int J Syst Evol Microbiol, 2003 May, 53(Pt 3), 739 - 46
DNA-DNA hybridization study of Burkholderia species using genomic DNA macro-array analysis coupled to reverse genome probing; Ramisse V et al.; The present study was aimed at simplifying procedures to delineate species and identify isolates based on DNA-DNA reassociation . DNA macro-arrays harbouring genomic DNA of reference strains of several Burkholderia species were produced . Labelled genomic DNA, hybridized to such an array, allowed multiple relative pairwise comparisons . Based on the relative DNA-DNA relatedness values, a complete data matrix was constructed and the ability of the method to discriminate strains belonging to different species was assessed . This simple approach led successfully to the discrimination of Burkholderia mallei from Burkholderia pseudomallei, but also discriminated Burkholderia cepacia genomovars I and III, Burkholderia multivorans, Burkholderia pyrrocinia, Burkholderia stabilis and Burkholderia vietnamiensis . Present data showed a sufficient degree of congruence with previous DNA-DNA reassociation techniques . As part of a polyphasic taxonomic scheme, this straightforward approach is proposed to improve species definition, especially for application in the rapid screening necessary for large numbers of clinical or environmental isolates.

J Laryngol Otol, 2003 May, 117(5), 417 - 8
Disseminated septicaemic melioidosis: an unusual presentation of masticator space infection; Srirompotong S et al.; Melioidosis is an infectious disease caused by a saprophytic bacterium, Burkholderia pseudomallei . It is endemic to Southeast Asia and Northern Australia . The spectrum of melioidosis in humans varies from sub-clinical to overwhelming protean manifestations resembling other acute and chronic bacterial infections . Disseminated septicaemia melioidosis presenting as a masticator space infection is reported here . This is germane to those treating diabetic patients with deep neck infections living in, or having visited, areas endemic for B . pseudomallei.

Biodegradation, 2003, 14(1), 19 - 29
Microbial consortia that degrade 2,4-DNT by interspecies metabolism: isolation and characterisation; Snellinx Z et al.; Two consortia, isolated by selective enrichment from a soil sample of a nitroaromatic-contaminated site, degraded 2,4-DNT as their sole nitrogen source without accumulating one or more detectable intermediates . Though originating from the same sample, the optimised consortia had no common members, indicating that selective enrichment resulted in different end points . Consortium 1 and consortium 2 contained four and six bacterial species respectively, but both had two members that were able to collectively degrade 2,4-DNT . Variovorax paradoxus VM685 (consortium 1) and Pseudomonas sp . VM908 (consortium 2) initiate the catabolism of 2,4-DNT by an oxidation step, thereby releasing nitrite and forming 4-methyl-5-nitrocatechol (4M5NC) . Both strains contained a gene similar to the dntAa gene encoding 2,4-DNT dioxygenase . They subsequently metabolised 4M5NC to 2-hydroxy-5-methylquinone (2H5MQ) and nitrite, indicative of DntB or 4M5NC monooxygenase activity . A second consortium member, Pseudomonas marginalis VM683 (consortium 1) and P . aeruginosa VM903, Sphingomonas sp . VM904, Stenotrophomonas maltophilia VM905 or P . viridiflava VM907 (consortium 2), was found to be indispensable for efficient growth of the consortia on 2,4-DNT and for efficient metabolisation of the intermediates 4M5NC and 2H5MQ . Knowledge about the interactions in this step of the degradation pathway is rather limited . In addition, both consortia can use 2,4-DNT as sole nitrogen and carbon source . A gene similar to the dntD gene of Burkholderia sp . strain DNT that catalyses ring fission was demonstrated by DNA hybridisation in the second member strains . To our knowledge, this is the first time that consortia are shown to be necessary for 2,4-DNT degradation.

Mol Gen Mikrobiol Virusol, 2003, (2), 3 - 10
{Bacteria of the Burkholderia cepacia complex: specific features of diagnostics, genome organization and metabolism}; Shaginian IA et al.; Modern data, related with the identification and typing of the complex B . cepacia bacteria, are analyzed in the article by using the poly-phase taxonomic approach . An optimal scheme for identifying and typing the complex B . cepacia bacteria, involving the microbiological and molecular-biological methods of laboratory diagnostics, is presented . The key and assumed factors of pathogenicity of the discussed bacteria are described . The possible phylogenetic relations of the complex B . cepacia bacteria with phytopathgens as well as with pathogenic bacteria of species Burkholderia, Pseudomonas, Escherichia, B . mallei, B . pdeudomallei, P . seruginosa and E . coli are described . A possible role of genome alterations and mutations in the genome of the complex B . cepacia bacteria (with the latter genome having unusual properties, i.e . a big size, and a considerable quantity of insertion sequences) in creating the conditions for the "pulsing" evolution "jerks", i.e . for a rapid change-over from saprophytism in the soil to a pathogenic causative agent of a viral-and-bacteriological infection . Such mechanism can be regarded as a rapid and radical adaptation of a microorganism under the conditions of changing ecological niches.

Folia Microbiol (Praha), 2003, 48(2), 253 - 6
Effects of soil pH, temperature and water content on the growth of Burkholderia pseudomallei; Chen YS et al.; Optimum conditions were determined for the growth of Burkholderia pseudomallei in natural soils or waters . It grows better in paddy soil, crop-covered and fallow field than in fresh and salty water . Although the optimal temperature and pH for the growth were 37 or 42 degrees C, and 6.5 or 7.5 in an environmental-mimicking soil medium, this bacterium can still grow at 4 degrees C, which was suggested to be related with the occurrence of melioidosis in some cold areas . In soil media with water content < 15 . B . pseudomallei did not grow until 60 d of incubation, suggesting that water contents of soils in which it dwelled would be one important factor in determining the growth rate.

J Burn Care Rehabil, 2003 May-Jun, 24(3), 154 - 7
Silver-sulfadiazine eschar pigmentation mimics invasive wound infection: a case report; Eldad A et al.; A 3-year-old girl with 52% TBSA scalds, mostly partial thickness, was treated topically with 5% mafenide acetate solution and 1% silver sulfadiazine cream . All blood cultures and wound swabs were negative for the first 5 days . On day 6 gram-negative bacteria and yeast forms were isolated from her wounds . High fever and leukocytosis were present and the child was treated with intravenous ampicillin and gentamicin according to sensitivity bacteriogram . The bacteria were identified as Pseudomonas aeruginosa and the yeast was Candida tropicalis . On day 7, Escherichia coli was identified in blood cultures and intravenous cefixime was added . Amphotericin B was added on day 9 when blood cultures grew Candida tropicalis and Burkholderia cepacia . On day 13 dark pigmentation foci developed on some areas of partial-thickness burns in the back, resembling invasive wound infection . White blood cell count was 14,300 cells/mm3, and her body temperature reached 39.7 degrees C . Cultures from the pigmented areas were negative, and biopsies revealed deposits of silver . Most of the areas healed uneventfully, and only about 8% TBSA needed grafting, including some of the pigmented areas . No residual pigmentation remained on discharge.

J Clin Microbiol, 2003 Jun, 41(6), 2471 - 6
Identification by subtractive hybridization of a novel insertion element specific for two widespread Burkholderia cepacia genomovar III strains; Liu L et al.; Species of the Burkholderia cepacia complex cause chronic and life-threatening infections in persons with cystic fibrosis . Epidemic strains infect multiple patients, reside primarily in genomovar III, and have an apparent enhanced capacity for human infection and/or interpatient transmission . By using subtractive hybridization, a novel insertion element, designated IS1363, was identified in epidemic strain PHDC, known to infect many cystic fibrosis patients in the mid-Atlantic region of the United States . IS1363 was also found in most isolates of the ET12 lineage, responsible for infecting large numbers of patients in Ontario, Canada, and the United Kingdom . Southern blot analysis demonstrated that whereas multiple copies of IS1363 were present in strain PHDC, only one copy was present in ET12 isolates . IS1363 was used to probe a collection of 943 B . cepacia complex isolates, representing all nine genomovars, recovered from 761 cystic fibrosis patients or the natural environment . IS1363 was not found in other genomovar III strains and, with the exception of B . ambifaria, was absent from other B . cepacia complex species . Genotyping analyses of all IS1363-positive isolates demonstrated that strain PHDC was more widely distributed in the United States than previously appreciated; 212 cystic fibrosis patients in 24 states were identified as being infected with PHDC.

Transpl Infect Dis, 2003 Mar, 5(1), 59 - 61
Burkholderia urinary tract infection after renal transplantation; Li FK et al.; Urinary tract infection is a common complication after renal transplantation . The etiologies are diverse and the bacterial agents may sometimes be acquired during the hospital stay . We report a patient who developed Burkholderia cepacia urinary tract infection after renal transplantation . The bacteria showed in vivo resistance to all of the available antibiotics . A graft nephrectomy was eventually required to clear the infection . The consequence of some fastidious infection may be catastrophic and early recognition and treatment is necessary to optimize the treatment.

Biosci Biotechnol Biochem, 2003 Apr, 67(4), 889 - 92
Isolation and characterization of aromatics-degrading microorganisms from the gut of the lower termite Coptotermes formosanus; Harazono K et al.; We isolated aromatics-degrading bacteria from the gut of a lower termite, Coptotermes formosanus, using a mineral salt medium containing various aromatic compounds as the sole carbon source . Two species, Burkholderia sp . strain VE22 and Citrobacter sp . strain VA53, were isolated by aerobic enrichment culture with veratraldehyde and vanillin, respectively . Strain VA53 could also grow and metabolize vanillin anaerobically.

J Bone Joint Surg Am, 2003 Jun, 85-A(6), 1058 - 61
Melioidotic septic arthritis and its risk factors; Kosuwon W et al.; BACKGROUND: Melioidotic septic arthritis is an infection caused by the gram-negative bacillus Burkholderia pseudomallei . It is commonly found in Northeast Thailand . The goal of our study was to identify specific characteristics of patients with melioidotic septic arthritis by comparing them with patients with non-melioidotic septic arthritis and to describe the results of treatment of melioidotic septic arthritis . METHODS: We conducted a retrospective study of seventy-seven patients with septic arthritis who were treated in our hospital over a period of four years . Twenty-five of the patients had melioidotic septic arthritis, and fifty-two had non-melioidotic septic arthritis . Univariate and multivariate analyses were conducted to identify the risk factors for melioidotic septic arthritis, and the clinical course of the twenty-five patients with melioidotic septic arthritis was followed until the infection resolved . RESULTS: Patients with melioidotic septic arthritis differed significantly (p = 0.002 ) from those with non-melioidotic septic arthritis with regard to the frequency of diabetes mellitus and of involvement of an upper-extremity joint . The odds ratio that melioidosis was the cause of the infection was 15.7 (95% confidence interval, 4.5 to 55.6) in a patient with diabetes mellitus and 4.51 (95% confidence interval, 1.04 to 19.65) in a patient with involvement of an upper-extremity joint . Twenty-two of the twenty-five patients with melioidotic septic arthritis responded to treatment, which consisted of six months of antibiotic therapy combined with needle aspiration, as well as surgical drainage of the affected joint when necessary (sixteen patients) . CONCLUSIONS: A diagnosis of melioidotic septic arthritis should be considered when septic arthritis is seen in an individual who is indigenous to or has recently visited Southeast Asia . The infection is more likely to be melioidotic septic arthritis if it involves an upper-extremity joint and if the patient has diabetes mellitus.

Cell Microbiol, 2003 Jun, 5(6), 385 - 93
Actin-based motility of Burkholderia pseudomallei involves the Arp 2/3 complex, but not N-WASP and Ena/VASP proteins; Breitbach K et al.; The facultative intracellular bacterium Burkholderia pseudomallei induces actin rearrangement within infected host cells leading to formation of actin tails and membrane protrusions . To investigate the underlying mechanism we analysed the contribution of cytoskeletal proteins to B . pseudomallei-induced actin tail assembly . By using green fluorescent protein (GFP)-fusion constructs, the recruitment of the Arp2/3 complex, vasodilator-stimulated phosphoprotein (VASP), Neural Wiskott-Aldrich syndrome protein (N-WASP), zyxin, vinculin, paxillin and alpha-actinin to the surface of B . pseudomallei and into corresponding actin tails was studied . In addition, antibodies against the same panel of proteins were used for immunolocalization . Whereas the Arp2/3 complex and alpha-actinin were incorporated into B . pseudomallei-induced actin tails, none of the other proteins were detected in these structures . The overexpression of an Arp2/3 binding fragment of the Scar1 protein, shown previously to block actin-based motility of Listeria, had no effect on B . pseudomallei tail formation . Infections of either N-WASP- or Ena/VASP-defective cells showed that these proteins are not essential for B . pseudomallei-induced actin polymerization . In conclusion, our results suggest that B . pseudomallei induces actin polymerization through a mechanism that differs from those evolved by Listeria, Shigella, Rickettsia or vaccinia virus.

Microbiology, 2003 Jun, 149(Pt 6), 1559 - 67
Site-directed mutagenesis of an extradiol dioxygenase involved in tetralin biodegradation identifies residues important for activity or substrate specificity; Andujar E et al.; The sequence of the extradiol dioxygenase ThnC, involved in tetralin biodegradation, was aligned with other extradiol dioxygenases involved in biodegradation of polycyclic compounds, and a three-dimensional model of ThnC, based on the structure of the previously crystallized 2,3-dihydroxybiphenyl dioxygenase from Burkholderia fungorum LB400, was built . In order to assess the functional importance of some non-active-site residues whose relevance could not be established by structural information, a number of positions surrounding the substrate-binding site were mutated in ThnC . Ten mutant proteins were purified and their activity towards 1,2-dihydroxytetralin, 1,2-dihydroxynaphthalene and 2,3-dihydroxybiphenyl was characterized . N213H, Q198H, G206M, A282R and A282G mutants increased k(cat)/K(m) at least twofold using 1,2-dihydroxytetralin as the substrate, thus showing that activity of ThnC is not maximized for this substrate . N213H and Q198H mutants increased k(cat)/K(m) using any of the substrates tested, thus showing the relevance for activity of these two histidines, which are highly conserved in dihydroxybiphenyl dioxygenases, but not present in dihydroxynaphthalene dioxygenases . Different substitutions in position 282 had different effects on general activity or substrate specificity, thus showing the functional importance of the most C-terminal beta-sheet of the protein . A251M and G206M mutants showed increased activity specifically for a particular substrate . N213H, G206M, A282R, A282G and Y177I substitutions resulted in enzymes more tolerant to acidic pH, the most striking effect being observed in mutant Y177I, which showed maximal activity at pH 5.5 . In addition, Q198D and V175D mutants, which had altered K(m), also showed altered sensitivity to substrate inhibition, thus indicating that inhibition is exerted through the same binding site . This mutational analysis, therefore, identified conserved residues important for activity or substrate specificity, and also shed some light on the mechanism of substrate inhibition exhibited by extradiol dioxygenases.

Microbiology, 2003 Jun, 149(Pt 6), 1475 - 81
A genetic system for the rapid isolation of aromatic-ring-hydroxylating dioxygenase activities; Kahl S et al.; Aromatic-ring-hydroxylating dioxygenases (ARHDOs) are key enzymes in the aerobic bacterial metabolism of aromatic compounds . They are of biotechnological importance as they function as biocatalysts in the stereospecific synthesis of chiral synthons and the degradation of aromatic pollutants . This report describes the development and validation of a system for the rapid isolation and characterization of specific ARHDO activities . The system is based on the identification of ARHDO gene segments that encode the enzymes' major functional determinants, on consensus primers for the direct amplification of such partial genes and on a 'recipient' ARHDO gene cluster for the insertion of the amplified segments . Previously, it has been shown that neither the N- nor the C-terminal portions but only the core region of the large or alpha-subunit of a class II ARHDO significantly influence substrate and product spectra . On the basis of these observations, consensus primers were designed for the amplification of the gene segment encoding the catalytic core of the large subunit . These primers were tested on 11 bacterial isolates known to metabolize aromatic compounds . In 10 cases, a gene fragment of expected length was amplified . DNA sequencing confirmed similarity to ARHDO alpha-subunit gene cores . The heterologously well-expressible bphA gene cluster of Burkholderia sp . strain LB400 was modified to facilitate the in-frame insertion of amplified segments . It was used successfully to express the resulting hybrid gene clusters and to form catalytically active chimaeric ARHDOs . The metabolic properties of these enzymes differed significantly from each other and from the parental ARHDO of strain LB400 . These results indicate that the system described here can be used to rapidly isolate and functionally characterize ARHDO activities, starting from isolated strains, mixtures of organisms or samples of nucleic acids . Applications of the system range from the recruitment of novel ARHDO activities to an improved characterization of natural ARHDO diversity.

FEMS Microbiol Lett, 2003 May 28, 222(2), 251 - 5
BphK shows dechlorination activity against 4-chlorobenzoate, an end product of bph-promoted degradation of PCBs; Gilmartin N et al.; A bphK gene encoding glutathione S-transferase (GST) activity is located in the bph operon in Burkholderia sp . strain LB400 but its role in polychlorinated biphenyl (PCB) metabolism is unknown . This gene was over-expressed in Escherichia coli and an in vivo assay based on growth of E . coli containing GST activity was used to identify potential novel substrates for this enzyme . Using this assay, 4-chlorobenzoate (4-CBA) was identified as a substrate for the BphK enzyme . High pressure liquid chromatography analysis and chloride ion detection showed removal of 4-CBA and an equivalent increase of chloride in cell extracts when incubated with this enzyme . These results would indicate that this BphK enzyme has dechlorination activity in relation to 4-CBA and may have a role in protection of other Bph enzymes against certain chlorinated metabolites of PCB degradation.

Mol Genet Genomics, 2003 Jul, 269(4), 499 - 507 Epub 2003 May 24.
ABC transporters of the wheat pathogen Mycosphaerella graminicola function as protectants against biotic and xenobiotic toxic compounds; Zwiers LH et al.; We have studied the role of five ABC transporter genes (MgAtr to MgAtr5) from the wheat pathogen Mycosphaerella graminicola in multidrug resistance (MDR) . Complementation of Saccharomyces cerevisiae mutants with the ABC transporter genes from M . graminicola showed that all the genes tested encode proteins that provide protection against chemically unrelated compounds, indicating that their products function as multidrug transporters with distinct but overlapping substrate specificities . Their substrate range in yeast includes fungicides, plant metabolites, antibiotics, and a mycotoxin derived from Fusarium graminearum (diacetoxyscirpenol) . Transformants of M . graminicola in which individual ABC transporter genes were deleted or disrupted did not exhibit clear-cut phenotypes, probably due to the functional redundancy of transporters with overlapping substrate specificity . Independently generated MgAtr5 deletion mutants of M . graminicola showed an increase in sensitivity to the putative wheat defence compound resorcinol and to the grape phytoalexin resveratrol, suggesting a role for this transporter in protecting the fungus against plant defence compounds . Bioassays with antagonistic bacteria indicated that MgAtr2 provides protection against metabolites produced by Pseudomonas fluorescens and Burkholderia cepacia . In summary, our results show that ABC transporters from M . graminicola play a role in protection against toxic compounds of natural and artificial origin.

Lancet, 2003 May 17, 361(9370), 1715 - 22
Melioidosis; White NJ; Melioidosis, which is infection with the gram-negative bacterium Burkholderia pseudomallei, is an important cause of sepsis in east Asia and northern Australia . In northeastern Thailand, melioidosis accounts for 20% of all community-acquired septicaemias, and causes death in 40% of treated patients . B pseudomallei is an environmental saprophyte found in wet soils . It mostly infects adults with an underlying predisposing condition, mainly diabetes mellitus . Melioidosis is characterised by formation of abscesses, especially in the lungs, liver, spleen, skeletal muscle, and prostate . In a third of paediatric cases in southeast Asia, the disease presents as parotid abscess . In northern Australia, 4% of patients present with brain stem encephalitis . Ceftazidime is the treatment of choice for severe melioidosis, but response to high dose parenteral treatment is slow (median time to abatement of fever 9 days) . Maintenance antibiotic treatment is with a four-drug regimen of chloramphenicol, doxycycline, and trimethoprim-sulfamethoxazole, or with amoxicillin-clavulanate in children and pregnant women . However, even with 20 weeks' antibiotic treatment, 10% of patients relapse . With improvements in health care and diagnostic microbiology in endemic areas of Asia, and increased travel, melioidosis will probably be recognised increasingly during the next decade.

Pediatr Res, 2003 Sep, 54(3), 297 - 305 Epub 2003 May 21.
Burkholderia cepacia-induced IL-8 gene expression in an alveolar epithelial cell line: signaling through CD14 and mitogen-activated protein kinase; Reddi K et al.; Burkholderia cepacia is a prevalent pulmonary pathogen in patients with cystic fibrosis (CF) . The lung pathology observed in patients with CF is postulated to be due to an overexpression of chemokines . This study investigated the induction of the neutrophil chemoattractant chemokine IL-8 and the signaling pathways activated by B . cepacia-infected human lung epithelial A549 (HLE) cells . Cells were infected with B . cepacia (genomovar III of the B . cepacia complex), and reverse transcriptase-PCR and ELISA for the cytokines were performed . B . cepacia (multiplicity of infection > or =4:1) induced HLE cells to significantly secrete IL-8 in a more potent manner than the predominant CF pathogen Pseudomonas aeruginosa (multiplicity of infection > or =64:1) . IL-8 secretion by B . cepacia-infected HLE cells was abrogated by the gene transcription inhibitor actinomycin D and the protein translation inhibitor cycloheximide, confirming that B . cepacia-induced IL-8 secretion was mediated through de novo protein synthesis . Treatment of B . cepacia with proteinase K failed to down-regulate IL-8 secretion; furthermore, IL-8 secretion by B . cepacia-infected HLE cells was abrogated by > or =80% in the presence of anti-CD14 {specific lipopolysaccharide (LPS) receptor} antibody, thus suggesting that the IL-8-inducing component of B . cepacia was LPS and therefore dependent on CD14 . The p38 mitogen-activated protein kinase (MAPK) inhibitor and the extracellular signal-regulated kinase MAPK inhibitor significantly abrogated IL-8 secretion by B . cepacia-infected HLE cells (SB203580, > or =80% inhibition; PD98059, > or =30% inhibition) . In conclusion, B . cepacia-induced IL-8 secretion in A549 airway epithelial cells is more potent than P . aeruginosa; is mediated through LPS, which is CD14 dependent; and involves activation of the p38 and ERK MAPK pathways.

Infect Immun, 2003 Jun, 71(6), 3053 - 7
Involvement of beta interferon in enhancing inducible nitric oxide synthase production and antimicrobial activity of Burkholderia pseudomallei-infected macrophages; Utaisincharoen P et al.; Burkholderia pseudomallei is the causative agent of melioidosis, a life-threatening disease that affects both humans and animals . This bacterium is able to survive and multiply inside both phagocytic and nonphagocytic cells . We recently reported that mouse macrophages infected with B . pseudomallei fail to produce a significant level of inducible nitric oxide synthase (iNOS), a crucial enzyme needed for the cells to control the intracellular growth of this bacterium . In the present study, we extended our investigation to demonstrate that, unlike other gram-negative bacteria that have been investigated, B . pseudomallei only minimally activates beta interferon (IFN-beta) production; this minimal activation leads to a low level of interferon regulating factor 1 (IRF-1) in the macrophages, in parallel with poor iNOS expression . Adding exogenous IFN-beta to the system could upregulate IRF-1 production, which in turn could enhance iNOS expression in the B . pseudomallei-infected macrophages and lead to suppression of the intracellular growth of this bacterium . Taken together, these results imply that the failure of macrophages to successfully control the growth and survival of intracellular B . pseudomallei is related, at least in part, to the defective production of IFN-beta, which modulates the ability of macrophages to synthesize iNOS.

Southeast Asian J Trop Med Public Health, 2002 Dec, 33(4), 739 - 41
Seroprevalence of melioidosis in dairy cattle in Chiang Mai Province, northern Thailand; Srikitjakarn L et al.; The seroprevalence of melioidosis in dairy cattle in Chiang Mai Province was investigated using of the indirect hemagglutination antibody (IHA) method . Two hundred and fifty-three samples were tested for serum antibodies to Burkholderia pseudomallei . The samples were from a total population of 8,688 dairy cattle in the province; random sampling, stratified by the location of cattle, was used . The seroprevalence was determined as 2% at 1:40 cut-off value, which was estimated to equate to 0.3% to 3.7% (95% CI) . This report of relatively low disease prevalence in the animal population corresponds to other prevalence studies of the agent in the environment and the human population in the region . The prevalence is markedly different to that reported from northeastern Thailand, where the disease is highly endemic.

J Bacteriol, 2003 Jun, 185(11), 3333 - 43
Distribution and organization of auxotrophic genes on the multichromosomal genome of Burkholderia multivorans ATCC 17616; Komatsu H et al.; The Burkholderia multivorans strain ATCC 17616 carries three circular chromosomes with sizes of 3.4, 2.5, and 0.9 Mb . To determine the distribution and organization of the amino acid biosynthetic genes on the genome of this beta-proteobacterium, various auxotrophic mutations were isolated using a Tn5 derivative that was convenient not only for the determination of its insertion site on the genome map but also for the structural analysis of the flanking regions . Analysis by pulsed-field gel electrophoresis revealed that 20 out of 23 insertion mutations were distributed on the 3.4-Mb chromosome . More detailed analysis of the his, trp, arg, and lys mutations and their flanking regions revealed the following properties of these auxotrophic genes: (i) all nine his genes were clustered on the 3.4-Mb chromosome; (ii) seven trp genes were organized within two distinct regions, i.e., a trpEGDC cluster on the 3.4-Mb chromosome and a trpFBA cluster on the 2.5-Mb chromosome; (iii) the leu gene cluster, leuCDB, was also located close to the trpFBA cluster; and (iv) lysA and argG genes were located on the 2.5-Mb chromosome, in contrast to the argH gene, which was located on the 3.4-Mb chromosome . Southern hybridization analysis, allelic exchange mutagenesis of ATCC 17616, and complementation tests demonstrated that all of the genes examined were functional and existed as a single copy within the genome . The present findings also indicated that the 2.5-Mb chromosome carried various auxotrophic genes with no structural or functional counterparts on the remaining two chromosomes.

J Med Microbiol, 2003 Jun, 52(Pt 6), 483 - 90
Lysogeny and bacteriophage host range within the Burkholderia cepacia complex; Langley R et al.; The Burkholderia cepacia complex comprises a group of nine closely related species that have emerged as life-threatening pulmonary pathogens in immunocompromised patients, particularly individuals with cystic fibrosis or chronic granulomatous disease . Attempts to explain the genomic plasticity, adaptability and virulence of the complex have paid little attention to bacteriophages, particularly the potential contribution of lysogenic conversion and transduction . In this study, lysogeny was observed in 10 of 20 representative strains of the B . cepacia complex . Three temperate phages and five lytic phages isolated from soils, river sediments or the plant rhizosphere were chosen for further study . Six phages exhibited T-even morphology and two were lambda-like . The host range of individual phages, when tested against 66 strains of the B . cepacia complex and a representative panel of other pseudomonads, was not species-specific within the B . cepacia complex and, in some phages, included Burkholderia gladioli and Pseudomonas aeruginosa . These new data indicate a potential role for phages of the B . cepacia complex in the evolution of these soil bacteria as pathogens of plants, humans and animals, and as novel therapeutic agents.

J Ind Microbiol Biotechnol, 2003 Jun, 30(6), 362 - 8 Epub 2003 May 13.
Effects of Vitreoscilla hemoglobin on the 2,4-dinitrotoluene (2,4-DNT) dioxygenase activity of Burkholderia and on 2,4-DNT degradation in two-phase bioreactors; Lin JM et al.; Expression of vgb, encoding Vitreoscilla hemoglobin (VHb), in Burkholderia strain YV1 was previously shown to improve cell growth and enhance 2,4-dinitrotoluene (2,4-DNT) degradation compared with control strain DNT, especially under hypoxic conditions . In the work reported here, the ratio of 2,4-DNT degraded to oxygen uptake was approximately 5-fold larger for strain YV1 than for strain DNT . The addition of purified VHb to cytosolic fractions of strain DNT increased 2,4-DNT degradation 1.5-fold, compared with 1.1-fold for control bovine Hb, but increased the 2,4-DNT degradation 2.7-fold when added to partially purified 2,4-DNT dioxygenase, compared with 1.3-fold for bovine Hb . This suggests a direct transfer of oxygen from VHb to the oxygenase . In a bioreactor at high 2,4-DNT concentration (using 100 ml oleyl alcohol containing 2 g 2,4-DNT as the second phase) with 1.5 l culture, both strains could remove 0.8 g 2,4-DNT by 120 h; and, under the same conditions in a fed-batch reactor, the degradation increased to 1 g for strain YV1 but not for strain DNT.

Curr Genet, 2003 Aug, 43(5), 358 - 63 Epub 2003 May 13.
Sequence analysis and functional characterization of the dialkylglycine decarboxylase gene DGD1 from Mycosphaerella graminicola; Adachi K et al.; Dialkylglycine decarboxylase is a pyridoxal phosphate-dependent enzyme in the aminotransferases class III group of enzymes . The enzyme is unique in terms of catalyzing both decarboxylation and transamination . Although the enzymatic activity is present in some bacteria and fungi, the biological role is unclear . We identified and disrupted the dialkylglycine decarboxylase-encoding gene DGD1 in the wheat blotch fungus Mycosphaerella graminicola by transposon-arrayed gene knockout . The DGD1 gene is highly similar to dialkylglycine decarboxylase from the soil bacterium Burkholderia cepacia . Phylogenetic analysis of various class III aminotransferases showed that dialkylglycine decarboxylases from bacteria and fungi are found in a distinct cluster . Functional analysis revealed that dgd1 disruption mutants display wild-type morphology and pathogenicity to wheat . The dgd1 mutants cannot utilize 2-methylalanine as a sole nitrogen source, as assessed by large-scale nutritional utilization analysis . This is the first description of a mutant phenotype of the fungal dialkylglycine decarboxylase gene.

Clin Diagn Lab Immunol, 2003 May, 10(3), 423 - 5
Recombinant truncated flagellin of Burkholderia pseudomallei as a molecular probe for diagnosis of melioidosis; Chen YS et al.; Current serological tests for melioidosis, using impure or uncharacterized cell antigens from Burkholderia pseudomallei, have problems in detection sensitivity and specificity . Therefore, we designed and expressed the recombinant flagellin (truncated at both the N- and C-terminal ends), and used the antigen to develop an indirect enzyme-linked immunosorbent assay (ELISA) to diagnose melioidosis . Comparison of the immunoreactivities of the full-length and truncated flagellins reveals that the truncated flagellin performed much better in detection specificity and sensitivity . Only the full-length flagellin was recognized by other bacterial causing septicemia and gave a false-positive result in Western analysis, indicating that the cross-reactive epitopes were located on the more highly conserved N- and C-terminal regions of flagellin . The indirect ELISA using recombinant truncated flagellin as the antigen achieved 93.8% sensitivity and 96.3% specificity and offered a more efficient serodiagnosis of melioidosis.

Biol Pharm Bull, 2003 May, 26(5), 667 - 70
Microbial viability in preparations packaged for single use; Obayashi A et al.; We evaluated microbial viability in preparations packaged for single use only which mandate that residual solution be discarded such as albumin and globulin preparations as blood products, preparations containing albumin (such as urokinase and interferon), fat emulsions, and a preparation containing fat emulsions (propofol) . In most preparations, Serratia marcescens and Burkholderia cepacia proliferated rapidly at 30 degrees C . However, in globulin preparations containing 1-2.25% glycine to prevent protein degradation (Gamma-Venin P, Venilon-I, Globulin Injection, and Ahlbulin), no growth of S . marcescens and B . cepacia was detected over 24 h at 30 degrees C . For globulin preparations containing 1-2.25% glycine, the injunction to "Discard residual solution after the package has been used" in the package inserts can be revised to "It is possible to use residual solution within 24 h after the package has been used with storage in a cool place."

J Clin Microbiol, 2003 May, 41(5), 2255 - 7
Sepsis, multiple organ failure, and death due to Pandoraea pnomenusa infection after lung transplantation; Stryjewski ME et al.; A 30-year-old man died with Pandoraea pnomenusa sepsis after lung transplantation . Pandoraea species are gram-negative rods, closely related to, and commonly misidentified as, Burkholderia cepacia complex or Ralstonia species . Heretofore considered soil bacteria and colonizers that infect patients with chronic lung diseases, Pandoraea species can produce severe infections.

J Clin Microbiol, 2003 May, 41(5), 2068 - 79
Multilocus sequence typing and evolutionary relationships among the causative agents of melioidosis and glanders, Burkholderia pseudomallei and Burkholderia mallei; Godoy D et al.; A collection of 147 isolates of Burkholderia pseudomallei, B . mallei, and B . thailandensis was characterized by multilocus sequence typing (MLST) . The 128 isolates of B . pseudomallei, the causative agent of melioidosis, were obtained from diverse geographic locations, from humans and animals with disease, and from the environment and were resolved into 71 sequence types . The utility of the MLST scheme for epidemiological investigations was established by analyzing isolates from captive marine mammals and birds and from humans in Hong Kong with melioidosis . MLST gave a level of resolution similar to that given by pulsed-field gel electrophoresis and identified the same three clones causing disease in animals, each of which was also associated with disease in humans . The average divergence between the alleles of B . thailandensis and B . pseudomallei was 3.2%, and there was no sharing of alleles between these species . Trees constructed from differences in the allelic profiles of the isolates and from the concatenated sequences of the seven loci showed that the B . pseudomallei isolates formed a cluster of closely related lineages that were fully resolved from the cluster of B . thailandensis isolates, confirming their separate species status . However, isolates of B . mallei, the causative agent of glanders, recovered from three continents over a 30-year period had identical allelic profiles, and the B . mallei isolates clustered within the B . pseudomallei group of isolates . Alleles at six of the seven loci in B . mallei were also present within B . pseudomallei isolates, and B . mallei is a clone of B . pseudomallei that, on population genetics grounds, should not be given separate species status.

Med Trop (Mars), 2002, 62(6), 607 - 10
{Suspected epidemic at a hospital in Senegal}; De Pina JJ et al.; Most outbreaks of noscomial infection are detected by means of molecular biological testing . However Euclidian distance is a rapid, reliable alternative if facilities for molecular biological testing are unavailable, as at the Principal Hospital in Dakar, Senegal . Over the 7 month period from 01/10/98 to 31/04/99, 110 of the 1033 blood cultures specimens collected in a group of 2360 children hospitalized in pediatric departments A and B of the Principal Hospital were found to be positive for Burkholderia cepacia . This high incidence suggested the possibility of an epidemic . This likelihood was further increased by evidence showing that all strains exhibited the same biotype and by results of pulsed-field electrophoresis (CHEF Mapper, Spel digestion) indicating that bacteria was of clonal origin . These findings were entirely consistent with those obtained by Euclidian distance but excessive mortality was not observed in the children involved . Testing of environmental specimens demonstrated the presence of the same strains in respirators and catheters . The most feasible hypothesis to account for these findings is environmental contamination resulting in mucocutaneous contamination of patients . Hemoculture containers were probably contaminated during handling.

FEBS Lett, 2003 May 8, 542(1-3), 17 - 21
Regulation of the katG-dpsA operon and the importance of KatG in survival of Burkholderia pseudomallei exposed to oxidative stress; Loprasert S et al.; Homologues of the catalase-peroxidase gene katG and the gene for the non-specific DNA binding protein dpsA were identified downstream of oxyR in Burkholderia pseudomallei . Northern experiments revealed that both katG and dpsA are co-transcribed during oxidative stress . Under conditions where the katG promoter is not highly induced, dpsA is transcribed from a second promoter located within the katG-dpsA intergenic region . A katG insertion mutant was found to be hypersensitive to various oxidants . Analysis of katG expression in the oxyR mutant indicates that OxyR is a dual function regulator that represses the expression of katG during normal growth and activates katG during exposure to oxidative stress . Both reduced and oxidized OxyR were shown to bind to the katG promoter.

Can J Microbiol, 2003 Feb, 49(2), 139 - 44
Degradation of phenanthrene and naphthalene by a Burkholderia species strain; Kang H et al.; Burkholderia sp . TNFYE-5 was isolated from soil for the ability to grow on phenanthrene as sole carbon and energy source . Unlike most other phenanthrene-degrading bacteria, TNFYE-5 was unable to grow on naphthalene . Growth substrate range experiments coupled with the ring-cleavage enzyme assay data suggest that TNFYE-5 initially metabolizes phenanthrene to 1-hydroxy-2-naphthoate with subsequent degradation through the phthalate and protocatechuate and beta-ketoadipate pathway . A metabolite in the degradation of naphthalene by TNFYE-5 was isolated by high-pressure liquid chromatography (HPLC) and was identified as salicylate by UV-visible spectral and gas chromatography-mass spectrometry analyses . Thus, the inability to degrade salicylate is apparently one major reason for the incapability of TNFYE-5 to grow on naphthalene.

Chemotherapy, 2003 May, 49(1-2), 44 - 8
In vitro synergy studies using aztreonam and fluoroquinolone combinations against six species of Gram-negative bacilli; Critchley IA et al.; BACKGROUND: Combination antimicrobial therapy is often necessary to eradicate infections caused by gram-negative bacteria . METHODS: To evaluate the potential benefit of aztreonam and fluoroquinolone combination therapy, the activity of aztreonam in combination with ciprofloxacin, gatifloxacin, levofloxacin and moxifloxacin was assessed using checkerboard testing for four clinical isolates of each of Escherichia coli, Klebsiella pneumoniae, Proteus mirabilis, Enterobacter cloacae, Burkholderia cepacia and Pseudomanas aeruginosa . RESULTS: All aztreonam and fluoroquinolone combinations demonstrated additive activity {fractional inhibitory concentration (FIC) index >0.5-4} or synergy (FIC index <or=0.5) for all 24 isolates tested . Aztreonam plus ciprofloxacin was the most effective combination, demonstrating synergy against 16.7% of isolates (2 P . mirabilis, 1 E . cloacae, 1 B . cepacia) . Aztreonam in combination with moxifloxacin was synergistic against 2 isolates (P . mirabilis, E . cloacae), and in combination with gatifloxacin against 1 isolate (E . cloacae) . Aztreonam and levofloxacin demonstrated only additive activity . CONCLUSION: Additive activity was observed for the majority of the aztreonam and fluoroquinolone combinations tested against six species of gram-negative bacilli . Synergy involving aztreonam and fluoroquinolone combinations was less common than additive activity and was isolate dependent . Although in vivo clinical successes have been documented using aztreonam in combination with other agents, confirmation by laboratory techniques requires further investigation .

Cell Microbiol, 2003 May, 5(5), 343 - 51
Killing of Caenorhabditis elegans by Burkholderia cepacia is controlled by the cep quorum-sensing system; Kothe M et al.; Burkholderia cepacia H111, which was isolated from a cystic fibrosis patient, effectively kills the nematode Caenorhabditis elegans . Depending on the medium used for growth of the bacterium two different killing modes were observed . On high-osmolarity medium the nematodes became paralysed and died within 24 h . Using filter assays we provide evidence that this killing mode involves the production of an extracellular toxin . On nematode growth medium killing occurs over the course of 2-3 days and involves the accumulation of bacteria in the intestinal lumen of C . elegans . We demonstrate that the cep quorum-sensing system of H111 is required for efficient killing of C . elegans under both killing conditions . Using the C . elegans phm-2 mutant that has a non-functional grinder evidence is provided that the cep system is required to enter the intestinal lumen but is dispensable for the colonization of the gut . Furthermore, we demonstrate that the type II secretion machinery is not essential for nematode killing.

Antimicrob Agents Chemother, 2003 May, 47(5), 1739 - 41
Burkholderia is highly resistant to human Beta-defensin 3; Sahly H et al.; The bactericidal activity of the novel beta-defensin hBD-3 against 28 species and 55 strains of gram-positive cocci and gram-negative fermentative and nonfermentative rods was tested . All strains proved to be highly or intermediately susceptible to hBD-3 (minimal bactericidal concentration {MBC}, </=50 micro g/ml), except for Burkholderia cepacia, for all 23 tested strains of which MBCs were >100 micro g/ml.

Infect Immun, 2003 May, 71(5), 2970 - 5
Structural and functional cellular changes induced by Burkholderia pseudomallei rhamnolipid; Haussler S et al.; In this study we report that extracellular Burkholderia pseudomallei rhamnolipid induced cytopathic changes characterized by retraction, rounding up, and, finally, detachment in phagocytic and nonphagocytic cell lines . These changes were due to a progressive reorganization of the F-actin network resulting in impaired cell cycle progression and a reduced phagocytic function of macrophages.

J Bacteriol, 2003 May, 185(9), 2786 - 92
Characterization of chlorophenol 4-monooxygenase (TftD) and NADH:flavin adenine dinucleotide oxidoreductase (TftC) of Burkholderia cepacia AC1100; Gisi MR et al.; Burkholderia cepacia AC1100 uses 2,4,5-trichlorophenoxyacetic acid, an environmental pollutant, as a sole carbon and energy source . Chlorophenol 4-monooxygenase is a key enzyme in the degradation of 2,4,5-trichlorophenoxyacetic acid, and it was originally characterized as a two-component enzyme (TftC and TftD) . Sequence analysis suggests that they are separate enzymes . The two proteins were separately produced in Escherichia coli, purified, and characterized . TftC was an NADH:flavin adenine dinucleotide (FAD) oxidoreductase . A C-terminally His-tagged fusion TftC used NADH to reduce either FAD or flavin mononucleotide (FMN) but did not use NADPH or riboflavin as a substrate . Kinetic and binding property analysis showed that FAD was a better substrate than FMN . TftD was a reduced FAD (FADH(2))-utilizing monooxygenase, and FADH(2) was supplied by TftC . It converted 2,4,5-trichlorophenol to 2,5-dichloro-p-quinol and then to 5-chlorohydroxyquinol but converted 2,4,6-trichlorophenol only to 2,6-dichloro-p-quinol as the final product . TftD interacted with FADH(2) and retarded its rapid oxidation by O(2) . A spectrum of possible TftD-bound FAD-peroxide was identified, indicating that the peroxide is likely the active oxygen species attacking the aromatic substrates . The reclassification of the two enzymes further supports the new discovery of FADH(2)-utilizing enzymes, which have homologues in the domains Bacteria and Archaea.

J Antimicrob Chemother, 2003 May, 51(5), 1203 - 11 Epub 2003 Apr 14.
In vitro activity of gatifloxacin alone and in combination with cefepime, meropenem, piperacillin and gentamicin against multidrug-resistant organisms; Dawis MA et al.; OBJECTIVES: To study the in vitro interaction of gatifloxacin in combination with gentamicin and with the beta-lactams cefepime, meropenem and piperacillin against clinical isolates of Stenotrophomonas maltophilia, Pseudomonas aeruginosa, Burkholderia cepacia, extended-spectrum beta-lactamase (ESBL)-producing Klebsiella pneumoniae, vancomycin-resistant Enterococcus faecium (VRE) and methicillin-resistant Staphylococcus aureus (MRSA) . METHODS: The activity of each drug alone was determined by an agar dilution method . Chequerboard synergy testing was then performed against all the isolates . Time-kill assays were done on selected isolates to assess correlation with the chequerboard results . RESULTS: Synergy was demonstrated with the following combinations at achievable serum concentrations: gatifloxacin/piperacillin for 80% and gatifloxacin/cefepime for 60% of S . maltophilia; gatifloxacin/gentamicin for 60%, and gatifloxacin/cefepime for 50% of ESBL-producing K . pneumoniae, and in all drug combinations for 50-70% of P . aeruginosa . Indifference was noted for the majority of B . cepacia and VRE isolates . Antagonism at therapeutic serum levels was observed with gatifloxacin/piperacillin against a single isolate of B . cepacia . No distinct trend in drug interaction was seen with the different drug combinations against MRSA . Time-kill analyses against selected isolates confirmed the synergic activity of the following drug combinations seen in the chequerboard assays: gatifloxacin/cefepime and gatifloxacin/piperacillin against P . aeruginosa, gatifloxacin/gentamicin against B . cepacia, and gatifloxacin/gentamicin and gatifloxacin/meropenem against ESBL-producing K . pneumoniae . CONCLUSIONS: Gatifloxacin was synergic with the beta-lactams piperacillin, cefepime and meropenem, and with gentamicin against some drug-resistant pathogens . Some of the time-kill analyses against P . aeruginosa, B . cepacia and ESBL-producing K . pneumoniae were in accordance with chequerboard results . Time-kill analyses against S . maltophilia did not confirm the synergy seen in chequerboard testing.

J Appl Microbiol, 2003, 94(5), 936 - 45
Specific detection of Pseudomonas spp . in milk by fluorescence in situ hybridization using ribosomal RNA directed probes; Gunasekera TS et al.; AIMS: Pseudomonas spp . are considered the most important milk spoilage organisms . Here we describe development of a fluorescence in situ hybridization (FISH) probe specific for detection and enumeration of Pseudomonas spp . in milk . METHODS AND RESULTS: 16S rRNA sequences were analysed to develop specific oligonucleotide probe for the genus Pseudomonas . Twenty different Pseudomonas spp . and 23 bacterial species from genera other than Pseudomonas (as negative controls) were tested . All tested Pseudomonas spp . yielded a positive FISH reaction, whereas negative controls showed no FISH reaction except for Burkholderia cepacia that showed a relatively weak FISH reaction . The FISH assay specifically stains Pseudomonas in milk when the milk contains a mixture of other bacterial species . The FISH assay takes 2 h and compares favourably with current culturing methods, which take a minimum of 48 h . Specificity of the probe was validated using polymerase chain reaction to selectively amplifying the Pseudomonas rDNA gene and sequencing the gene products . CONCLUSIONS: The method presented in this study allows simultaneously detection, identification and enumeration of Pseudomonas spp . in milk . SIGNIFICANCE AND IMPACT OF THE STUDY: Rapid and accurate enumeration of Pseudomonas facilitates the identification of specific contamination sources in dairy plants, the accurate validation of pasteurization treatments and the prediction of shelf life of processed milk.

J Cosmet Sci, 2003 Jan-Feb, 54(1), 1 - 7
Evaluation of preservative systems in a sunscreen formula by linear regression method; Bou-Chacra NA et al.; A sunscreen formula with eight different preservative systems was evaluated by linear regression, pharmacopeial, and the CTFA (Cosmetic, Toiletry and Fragrance Association) methods . The preparations were tested against Staphylococcus aureus, Burkholderia cepacia, Shewanella putrefaciens, Escherichia coli, and Bacillus sp . The linear regression method proved to be useful in the selection of the most effective preservative system used in cosmetic formulation.

Eur J Clin Microbiol Infect Dis, 2003 Apr, 22(4), 249 - 53 Epub 2003 Mar 28.
Fatal outcome of lung transplantation in cystic fibrosis patients due to small-colony variants of the Burkholderia cepacia complex; Haussler S et al.; The aim of this study was to investigate the possible role of small-colony variant morphotypes of Burkholderia cepacia-like organisms in infectious complications in cystic fibrosis patients following lung transplantation . Respiratory tract specimens from 470 cystic fibrosis patients were screened over a 22-month period for Burkholderia cepacia-like organisms . Nineteen patients were positive for these organisms, eight of whom harboured small-colony-variant morphotypes . Three patients underwent bilateral lung transplantation during the study, two of whom harboured small-colony variants in addition to clonally identical wildtypes of Burkholderia multivorans and Burkholderia cepacia genomovar III prior to lung transplantation . Both patients developed fatal systemic infections post transplantation due to small-colony variants . In vitro testing revealed that small-colony variants exhibited increased serum resistance in comparison to wildtypes . The results of this study indicate that diagnostic efforts should be undertaken to carefully identify small-colony variants of Burkholderia cepacia complex, since they might be an indicator of poor post-transplantation outcome in patients with cystic fibrosis.

Microbiology, 2003 Apr, 149(Pt 4), 961 - 71
Identification and molecular analysis of cable pilus biosynthesis genes in Burkholderia cepacia; Sajjan US et al.; Burkholderia cepacia is an opportunistic respiratory pathogen in cystic fibrosis patients . One highly transmissible and virulent clone belonging to genomovar IIIa expresses pili with unique cable morphology, which enable the bacterium to bind cytokeratin 13 in epithelial cells . The cblA gene, encoding the major pilin subunit, is often used as a DNA marker to identify potentially virulent isolates . The authors have now cloned and sequenced four additional genes, cblB, cblC, cblD and cblS, in the pilus gene cluster . This work shows that the products of the first four genes of the cbl operon, cblA, cblB, cblC and cblD, are sufficient for pilus assembly on the bacterial surface . Deletion of cblB abrogated pilus assembly and compromised the stability of the CblA protein in the periplasm . In contrast, deletion of cblD resulted in no pili, but there was no effect on expression and stability of the CblA protein subunit . These results, together with protein sequence homologies, predicted structural analyses, and the presence of typical amino acid motifs, are consistent with the assignment of functional roles for CblB as a chaperone that stabilizes the major pilin subunit in the periplasm, and CblD as the initiator of pilus biogenesis . It is also shown that expression of Cbl pili in Escherichia coli is not sufficient to mediate the binding of bacteria to the epithelial cell receptor cytokeratin 13, and that B . cepacia still binds to cytokeratin 13 in the absence of Cbl pili, suggesting that additional bacterial components are required for effective binding.

Microbiology, 2003 Apr, 149(Pt 4), 843 - 53
Transmissible Burkholderia cepacia genomovar IIIa strains bind and convert monomeric iron(III) protoporphyrin IX into the mu-oxo oligomeric form; Smalley JW et al.; Burkholderia cepacia isolates of genomovar III are highly transmissible amongst patients with cystic fibrosis (CF) and express a 97 kDa putative haem-binding protein (HBP) {Smalley, J . W., Charalabous, P., Birss, A . J . & Hart, C . A . (2001) . Clin Diagn Lab Immunol 8, 509-514} . An investigation of the interactions of iron(III) protoporphyrin IX with epidemic and non-epidemic strains of B . cepacia to determine the role of the above protein in haem acquisition and binding is reported herein . Spectrophotometric titrations of cell suspensions of genomovar IIIa strains BC7 and C5424 with iron(III) protoporphyrin IX, at pH 7.0, resulted in the depletion of Fe(III)PPIX.OH monomers and formation of the micro -oxo oligomeric species, {Fe(III)PPIX}(2)O . Difference spectroscopy indicated a continuous conversion of the monomeric iron(III) protoporphyrin IX into micro -oxo oligomers . Incubations with Fe(III)PPIX.OH monomers at pH 6.5 also showed that cells could shift the equilibrium to generate the micro -oxo oligomeric form . Genomovar I strains ATCC 25416 and LMG 17997 were unable to mediate this conversion . SDS-PAGE of genomovar IIIa strains exposed to Fe(III)PPIX.OH at pH 6.5 followed by tetramethylbenzidine/H(2)O(2) staining revealed, in addition to the 97 kDa HBP, two proteins of 77 and 149 kDa located in the outer membrane which bound Fe(III)PPIX.OH monomers . These proteins were absent from the genomovar I strains . Genomovar IIIa strains BC7 and C5424 showed increased cellular binding of {Fe(III)PPIX}(2)O, and as a consequence, displayed increased catalase activities compared to cells of the genomovar I isolates . It is concluded that, in addition to the putative 97 kDa HBP, B . cepacia genomovar IIIa strains express two outer-membrane proteins which function to bind and convert Fe(III)PPIX.OH monomers into the micro -oxo oligomeric form, {Fe(III)PPIX}(2)O . The ability to perform this conversion at both neutral and slightly acidic pHs may enable epidemic strains to withstand attack from neutrophil-derived H(2)O(2) in the inflamed CF lung.

Infect Control Hosp Epidemiol, 2003 Mar, 24(3), 198 - 201
Contamination of trypan blue with Burkholderia cepacia in a cornea bank; Morel PC et al.; OBJECTIVE: To describe Burkholderia cepacia contamination of a cornea bank and the measures taken to identify and eliminate the source of infection . METHODS: Cultures were performed to assess the extent and source of contamination, and pulsed-field gel electrophoresis was used for molecular typing . RESULTS: Routine surveillance cultures identified 5 contaminated corneas during a 10-day period . Additional cultures showed that 28 of 88 samples were positive for this organism . Environmental investigation showed that an open bottle of trypan blue used to assess corneal morphology was contaminated with the epidemic strain . CONCLUSION: Trypan blue played a major role in this contamination of corneas . This episode shows that microbial contamination can affect transplanted corneas despite ongoing culture surveillance and suggests that new methods may be needed to avoid this risk.

J Vasc Surg, 2003 Apr, 37(4), 882 - 5
Relapsing melioidosis as cause of iliac mycotic aneurysm: an indigenous case in Taiwan; Luo CY et al.; Melioidosis, an infectious disease caused by Burkholderia pseudomallei, an aerobic gram-negative bacillus, is normally transmitted through skin wounds and contact with infected human beings and animals . Its primary source is rice paddy soil and stagnant water . Melioidosis manifesting as an arterial mycotic aneurysm is rare, and, to our knowledge, infected true and false aneurysms of the iliac artery have never been reported . We report the case of a patient without contact with the normal sources of infection in whom an iliac mycotic aneurysm was caused by relapsing melioidosis and treated with an extra-anatomic bypass graft.

Mol Cells, 2003 Feb 28, 15(1), 27 - 33
Molecular cloning and complementation analysis of nifV gene from Frankia EuIK1 strain; Oh CJ et al.; The nifV gene from the Frankia EuIK1 strain, a symbiont of Elaeagnus umbellata, was cloned and a complementation test using the Klebsiella pneumoniae nifV mutant was performed to verify its function . The nifV ORF consists of 1245 bp, which encodes 414 amino acids . However, the putative promoter and Shine-Dalgarno sequences were not found in the 5' region of the ORF . The Frankia EuIK1 nifV ORF showed about a 70% nucleotide identity and 80% amino acid similarity with that of Frankia sp . FaC1 . In the upstream region of the nifV, a putative ORF that showed a 51% nucleotide identity with the afcD gene from Burkholderia cepacia BC11 was found . The other partial ORF that showed a 59% identity with the pkaD gene from Streptomyces coelicolor A(3) was found in the downstream region . In this respect, Frankia EuIK1 nifV has an unusual location on the genome, considering the nif gene organization . A phylogenetic analysis revealed that the NifV from Frankia EuIK1 was close to those from two Alnus-infective Frankia species, and they were grouped with those of the alpha-class proteobacteria, supporting the vertical descent of nifV . The transcription and function of Frankia EuIK1 nifV were verified by a RT-PCR analysis and complementation test with the K . pneumoniae mutant, respectively . These results suggested that Frankia EuIK1 nifV is a functional gene.

Chemistry, 2003 Apr 4, 9(7), 1542 - 8
The structure of lipid A of the lipopolysaccharide from Burkholderia caryophylli with a 4-amino-4-deoxy-L-arabinopyranose 1-phosphate residue exclusively in glycosidic linkage; Molinaro A et al.; From the lipopolysaccharides (LPSs) of the plant-pathogenic bacterium Burkholderia caryophylli, the complete structure of lipid A has been characterized . For the first time, a 4-amino-4-deoxy-L-arabinopyranose 1-phosphate residue was proven to be exclusively linked to the reducing end of lipid A from a wild-type LPS . The LPSs of B . caryophylli were degraded by mild acetate buffer hydrolysis at pH 4.4 . The obtained lipid A was analyzed as such, and also after de-O-acylation or dephosphorylation . The structure of lipid A was identified mainly by means of matrix-assisted laser desorption/ionisation mass spectrometry, and by various 1D and 2D (1)H and (13)C NMR spectroscopic measurements.

Cornea, 2003 Apr, 22(3), 221 - 5
Bacterial contamination of a cornea tissue bank: implications for the safety of graft engineering; Morel P et al.; PURPOSE: To analyze the difficulties involved in managing an episode of bacterial contamination in a cornea bank . We describe (1) the circumstances of bacterial contamination discovery, (2) the methods used to investigate the outbreak, (3) the corrective measures adopted, and (4) the method introduced to improve the reaction capacity in case of bacterial contamination . METHODS: All the samples collected were cultured in an attempt to identify the environmental reservoir of the contaminated epidemic clone . Bacteria were identified by Gram stain, oxidase test, and biochemical characteristics . The clonality of the strains was assessed by pulsed-field gel electrophoresis . RESULTS: The bacterial contamination was confirmed for 28 corneas, and 70 additional corneas were discarded . The source of the contamination was identified 17 days after the beginning of the episode . It consisted of a clonal bacterial strain that was found in trypan blue, the dye, used to examine all the tissues . The contaminating bacterium was Burkholderia cepacia, a well-known nosocomial pathogen . A total of 169 grafted corneas had been checked with the contaminated reagent . No cases of post-graft infection were recorded . CONCLUSION: Trypan blue played a major role in this outbreak . The mode and chronology of contamination remain unresolved . This exceptional event emphasizes the risk of bacterial contamination in tissue/cell banks, the necessity to improve methods for its prevention, and procedures to limit its consequences.

Int J Syst Evol Microbiol, 2003 Jan, 53(Pt 1), 121 - 4
'Candidatus glomeribacter gigasporarum' gen . nov., sp . nov., an endosymbiont of arbuscular mycorrhizal fungi; Bianciotto V et al.; Arbuscular mycorrhizal fungi are obligate endosymbionts that colonize the roots of almost 80 % of land plants . The present paper describes morphological and molecular data on a bacterial endosymbiont living in the cytoplasm of dormant or germinating spores and symbiotic mycelia of the fungal species Gigaspora margarita, Scutellospora persica and Scutellospora castanea . PCR amplification of almost the entire 16S rRNA gene of the Gigaspora margarita BEG 34 endosymbiont, using universal bacterial primers, and subsequent sequence analysis demonstrated that this organism occupies a very distinct phylogenetic position within the beta-Proteobacteria, with the genera Burkholderia, Pandoraea and Ralstonia as its closest neighbours . Primers specific to the 16S rDNA of the endosymbiotic bacteria of BEG 34 allowed amplification of spore DNA from endosymbionts of Gigaspora margarita, Gigaspora decipiens, S . persica and S . castanea, but not from the Gigaspora gigantea endosymbiont (which was morphologically different) or from the cytoplasm of Gigaspora rosea (which did not contain endosymbiotic bacteria) . These specific primers were successfully used as a probe for the in-situ hybridization of endobacteria in Gigaspora margarita spores . The overall rod-shaped morphology of the Gigaspora margarita, Gigaspora decipiens, S . persica and S . castanea endosymbionts was similar, and amplification and sequence analysis of the almost-complete 16S rRNA genes of several Gigaspora margarita, S . persica and S . castanea endosymbionts revealed over 98% sequence similarity . These morphological and genomic characteristics were used to assign the endosymbionts of these three species (five isolates) of arbuscular mycorrhizal fungi as 'Candidatus Glomeribacter gigasporarum' gen . nov., sp . nov.

Infect Immun, 2003 Apr, 71(4), 2280 - 2
Flagellum-mediated adhesion by Burkholderia pseudomallei precedes invasion of Acanthamoeba astronyxis; Inglis TJ et al.; In this study we investigated the role of the bacterial flagellum in Burkholderia pseudomallei entry to Acanthamoeba astronyxis trophozoites . B . pseudomallei cells were tethered to the external amoebic surface via their flagella . MM35, the flagellum-lacking fliC knockout derivative of B . pseudomallei NCTC 1026b did not demonstrate flagellum-mediated endocytosis in timed coculture, confirming that an intact flagellar apparatus assists B . pseudomallei entry into A . astronyxis.

Infect Immun, 2003 Apr, 71(4), 1622 - 9
Flagella are virulence determinants of Burkholderia pseudomallei; Chua KL et al.; Burkholderia pseudomallei, a facultatively intracellular pathogen, is a flagellated and motile gram-negative bacterium and is the causative agent of melioidosis in humans . Flagella are commonly recognized as important virulence determinants expressed by bacterial pathogens since the motility phenotype imparted by these organelles often correlates with the ability of an organism to cause disease . We used a virulent isolate of B . pseudomallei, KHW, to construct an isogenic deletion mutant with a mutation in the flagellin gene (fliC) by gene replacement transposon mutagenesis . The KHWDeltafliCKm mutant was aflagellate and nonmotile in semisolid agar . The isogenic KHWDeltafliCKm mutant was not impaired in terms of the ability to invade and replicate in cultured human lung cells compared with the wild type . It was also equally virulent in slow-killing assays involving Caenorhabditis elegans, but it was avirulent during intranasal infection of BALB/c mice . Very few bacteria, if any, were isolated from the lungs and spleens of KHWDeltafliCKm-infected mice . In contrast, the bacterial loads in the lungs and spleens were similar in mice infected with KHW and in mice infected with the complemented mutant, KHWDeltafliCKm/pUCP28TfliC . Unlike the Syrian hamster or diabetic rat models of infection, the B . pseudomallei flagellin was also a virulence factor during intraperitoneal infection of BALB/c mice . In this study, all animals infected with KHWDeltafliCKm remained healthy and did not succumb to disease regardless of the route of infection . The flagellum is therefore an important and necessary virulence determinant of B . pseudomallei during intranasal and intraperitoneal infection of mice.

Clin Exp Immunol, 2003 Apr, 132(1), 70 - 5
CpG ODN enhances uptake of bacteria by mouse macrophages; Utaisincharoen P et al.; Unmethylated CpG motif in synthetic oligodeoxynucleotide (CpG ODN) or bacterial DNA is well recognized for its role in innate immunity, including enhancing production of NO and cytokines by macrophages . In the present study, we demonstrated the effect of CpG ODN on the phagocytic uptake of bacteria by macrophages . Flow cytometric analysis of mouse macrophages (RAW 264.7) incubated with fluorescein isothiocyanate (FITC)-labelled Burkholderia pseudomallei, Salmonella enterica serovar Typhi or Escherichia coli showed that CpG ODN increased the uptake of these bacteria by mouse macrophages . The enhancement of bacterial uptake by CpG ODN was concentration-dependent . The increase of bacterial uptake by CpG ODN-activated macrophages shown above is consistent with the result of bacteria internalization study using a standard antibiotic protection assay . There was also an increase in the rate and degree of multi-nucleated giant cell formation, phenomena which have been shown previously to be unique when the cells were infected with B . pseudomallei . These observations may provide significant insights for future investigation into host cell-pathogen interaction.

Res Microbiol, 2003 Mar, 154(2), 91 - 6
Burkholderia cenocepacia sp . nov.--a new twist to an old story; Vandamme P et al.; DNA-DNA hybridisation experiments between isolates representing Burkholderia cepacia genomovar III recA lineages IIIA and IIIB reinforced the classification of both phylogenetic subgroups as a single genospecies, distinct from B . cepacia (genomovar I) . A formal classification of B . cepacia genomovar III encompassing the recA lineages IIIA and IIIB, and the new recA lineages IIIC and IIID, as B . cenocepacia sp . nov., with LMG 16656 as the type strain, is proposed.

Environ Health Perspect, 2002 Dec, 110 Suppl 6, 1005 - 11
Biodegradation kinetics of aromatic hydrocarbon mixtures by pure and mixed bacterial cultures; Reardon KF et al.; Microbial growth on pollutant mixtures is an important aspect of bioremediation and wastewater treatment . However, efforts to develop mathematical models for mixed substrate kinetics have been limited . Nearly all models group either the microbial population (as "biomass") or the chemical species (e.g., as biological oxygen demand) . When individual chemical species are considered, most models assume either no interaction or that the nature of the interaction is competition for the same rate-limiting enzyme . And when individual microbial species are considered, simple competition for the growth substrate is the only interaction included . Here, we present results using Pseudomonas putida F1 and Burkholderia sp . strain JS150 growing individually and together on benzene, toluene, phenol, and their mixtures and compare mathematical models to describe these results . We demonstrate that the simple models do not accurately predict the outcome of these biodegradation experiments, and we describe the development of a new model for substrate mixtures, the sum kinetics with interaction parameters (SKIP) model . In mixed-culture experiments, the interactions between species were substrate dependent and could not be predicted by simple competition models . Together, this set of experimental and modeling results presents our current state of work in this area and identifies challenges for future modeling efforts.

FEMS Immunol Med Microbiol, 2003 Mar 20, 35(2), 87 - 92
Lack of correlation between O-serotype, bacteriophage susceptibility and genomovar status in the Burkholderia cepacia complex; Kenna DT et al.; The Burkholderia cepacia complex comprises at least nine phylogenetically related genomic species (genomovars) which cause life-threatening infection in immunocompromised humans, particularly individuals with cystic fibrosis or chronic granulomatous disease . Prior to recognition that 'B . cepacia' comprise multiple species, in vitro studies revealed that the lipopolysaccharide (LPS) of these Gram-negative bacteria is strongly endotoxic . In this study, we used 117 B . cepacia complex isolates to determine if there is a correlation between O-antigen serotype and genomovar status . Isolates were also tested for their ability to act as bacterial hosts for the LPS-binding bacteriophages NS1 and NS2 . The absence of genomovar II (Burkholderia multivorans) in 'historical B . cepacia' isolates was notable . Neither O-serotype nor phage susceptibility correlated with genomovar status . We conclude that variability in LPS may contribute to the success of these highly adaptable bacteria as human pathogens.

J Mol Biol, 2003 Mar 21, 327(2), 475 - 89
Catalase-peroxidase KatG of Burkholderia pseudomallei at 1.7A resolution; Carpena X et al.; The catalase-peroxidase encoded by katG of Burkholderia pseudomallei (BpKatG) is 65% identical with KatG of Mycobacterium tuberculosis, the enzyme responsible for the activation of isoniazid as an antibiotic . The structure of a complex of BpKatG with an unidentified ligand, has been solved and refined at 1.7A resolution using X-ray synchrotron data collected from crystals flash-cooled with liquid nitrogen . The crystallographic agreement factors R and R(free) are 15.3% and 18.6%, respectively . The crystallized enzyme is a dimer with one modified heme group and one metal ion, likely sodium, per subunit . The modification on the heme group involves the covalent addition of two or three atoms, likely a perhydroxy group, to the secondary carbon atom of the vinyl group on ring I . The added group can form hydrogen bonds with two water molecules that are also in contact with the active-site residues Trp111 and His112, suggesting that the modification may have a catalytic role . The heme modification is in close proximity to an unusual covalent adduct among the side-chains of Trp111, Tyr238 and Met264 . In addition, Trp111 appears to be oxidized on C(delta1) of the indole ring . The main channel, providing access of substrate hydrogen peroxide to the heme, contains a region of unassigned electron density consistent with the binding of a pyridine nucleotide-like molecule . An interior cavity, containing the sodium ion and an additional region of unassigned density, is evident adjacent to the adduct and is accessible to the outside through a second funnel-shaped channel . A large cleft in the side of the subunit is evident and may be a potential substrate-binding site with a clear pathway for electron transfer to the active-site heme group through the adduct.

Am J Respir Cell Mol Biol, 2003 Aug, 29(2), 206 - 12 Epub 2003 Mar 06.
Lactoperoxidase and human airway host defense; Wijkstrom-Frei C et al.; The lactoperoxidase (LPO) antibiotic system is a well-characterized component of mammary and salivary gland secretions . Because LPO has been shown to function in ovine airways, human airway tissue and secretions were examined for the presence of LPO and its substrate, the anion thiocyanate (SCN-) . In addition, human airway secretions were tested for LPO-mediated antibacterial activity, and LPO's activity was assessed against some human airway pathogens . The data showed that normal human airway secretions contained LPO enzyme activity (0.65 +/- 0.09 microg/mg secreted protein; n = 17), and Western blots of secretions demonstrated bands of the expected sizes for LPO . LPO mRNA was detected in trachea by sequencing PCR-amplified cDNA . SCN-, LPO's substrate, was present in undiluted airway secretions at concentrations sufficient for LPO catalysis (0.46 +/- 0.19 mM; n = 8), and diluted secretions contained antibacterial activity with LPO-like properties . Immunocytochemistry localized LPO to submucosal glands in human bronchi . Finally, as expected based on the known antibacterial spectrum of the LPO system, airway secretions showed LPO-dependent activity against Pseudomonas aeruginosa . In addition, the airway LPO system was shown to be effective against Burkholderia cepacia and Haemophilus influenzae . Thus, a functional LPO system exists in human airways and may contribute to airway host defense against infection.

Appl Environ Microbiol, 2003 Mar, 69(3), 1739 - 47
Quorum-sensing system and stationary-phase sigma factor (rpoS) of the onion pathogen Burkholderia cepacia genomovar I type strain, ATCC 25416; Aguilar C et al.; Bacterial strains belonging to Burkholderia cepacia can be human opportunistic pathogens, plant pathogens, and plant growth promoting and have remarkable catabolic activity . B . cepacia consists of several genomovars comprising what is now known as the B . cepacia complex . Here we report the quorum-sensing system of a genomovar I onion rot type strain ATCC 25416 . Quorum sensing is a cell-density-dependent regulatory response which involves the production of N-acyl homoserine lactone (HSL) signal molecules . The cep locus has been inactivated in the chromosome, and it has been shown that CepI is responsible for the biosynthesis of an N-hexanoyl HSL (C(6)-HSL) and an N-octanoyl HSL (C(8)-HSL) and that the cep locus regulates protease production as well as onion pathogenicity via the expression of a secreted polygalacturonase . A cep-lacZ-based sensor plasmid has been constructed and used to demonstrate that CepR responded to C(6)-HSL with only 15% of the molar efficiency of C(8)-HSL, that a cepR knockout mutant synthesized 70% less HSLs, and that CepR responded best towards long-chain HSLs . In addition, we also report the cloning and characterization of the stationary-phase sigma factor gene rpoS of B . cepacia ATCC 25416 . It was established that quorum sensing in B . cepacia has a negative effect on rpoS expression as determined by using an rpoS-lacZ transcriptional fusion; on the other hand, rpoS-null mutants displayed no difference in the accumulation of HSL signal molecules.

Chembiochem, 2003 Mar 3, 4(2-3), 203 - 10
Behavior of silica aerogel networks as highly porous solid solvent media for lipases in a model transesterification reaction; El Rassy H et al.; Highly porous silica aerogels with differing balances of hydrophobic and hydrophilic functionalities were studied as a new immobilization medium for enzymes . Two types of lipases from Candida rugosa and Burkholderia cepacia were homogeneously dispersed in wet gel precursors before gelation . The materials obtained were compared in a simple model reaction: transesterification of vinyl laurate by 1-octanol . To allow a better comparison of the hydrophobic/hydrophilic action of the solid, very open aerogel networks with traditional organic hydrophobic/hydrophilic liquid solvents, this reaction was studied in mixtures containing different proportions of 2-methyl-2-butanol, isooctane, and water . The results are discussed in relation to the porous and hydrophobic nature of aerogels, characterized by nitrogen adsorption . It was found that silica aerogels can be considered as "solid" solvents for the enzymes, able to provide hydrophobic/hydrophilic characteristics different from those prevailing in the liquid surrounding the aerogels . A simple mechanism of action for these aerogel networks is proposed.

Plant Cell, 2003 Mar, 15(3), 745 - 59
Disease resistance and abiotic stress tolerance in rice are inversely modulated by an abscisic acid-inducible mitogen-activated protein kinase; Xiong L et al.; Mitogen-activated protein kinase (MAPK) cascades play an important role in mediating stress responses in eukaryotic organisms . However, little is known about the role of MAPKs in modulating the interaction of defense pathways activated by biotic and abiotic factors . In this study, we have isolated and functionally characterized a stress-responsive MAPK gene (OsMAPK5) from rice . OsMAPK5 is a single-copy gene but can generate at least two differentially spliced transcripts . The OsMAPK5 gene, its protein, and kinase activity were inducible by abscisic acid as well as various biotic (pathogen infection) and abiotic (wounding, drought, salt, and cold) stresses . To determine its biological function, we generated and analyzed transgenic rice plants with overexpression (using the 35S promoter of Cauliflower mosaic virus) or suppression (using double-stranded RNA interference {dsRNAi}) of OsMAPK5 . Interestingly, suppression of OsMAPK5 expression and its kinase activity resulted in the constitutive expression of pathogenesis-related (PR) genes such as PR1 and PR10 in the dsRNAi transgenic plants and significantly enhanced resistance to fungal (Magnaporthe grisea) and bacterial (Burkholderia glumae) pathogens . However, these same dsRNAi lines had significant reductions in drought, salt, and cold tolerance . By contrast, overexpression lines exhibited increased OsMAPK5 kinase activity and increased tolerance to drought, salt, and cold stresses . These results strongly suggest that OsMAPK5 can positively regulate drought, salt, and cold tolerance and negatively modulate PR gene expression and broad-spectrum disease resistance.

Arch Microbiol, 2003 Mar, 179(3), 214 - 23 Epub 2003 Feb 08.
Suppression-subtractive hybridisation reveals variations in gene distribution amongst the Burkholderia cepacia complex, including the presence in some strains of a genomic island containing putative polysaccharide production genes; Parsons YN et al.; Some strains of the Burkholderia cepacia complex, including the ET12 lineage, have been implicated in epidemic spread amongst cystic fibrosis (CF) patients . Suppression-subtractive hybridisation was used to identify genomic regions within strain J2315 (ET12 lineage; genomovar IIIA) that were absent from a non-transmissible genomovar IIIB strain . Sequence data from 15 subtracted clones were used to interrogate the genome sequence of strain J2315 and identify genomic regions incorporating the subtracted sequences . Many of the genomic regions displayed abnormally low GC content and similarity to sequences implicated in gene transfer . The distribution of three subtracted regions amongst members of the B . cepacia complex varied . A large cluster of genes with strong sequence similarity to capsular production genes from Burkholderia mallei and other bacterial pathogens was identified . This genomic island was detected in some but not all representatives of genomovar IIIA, two out of four genomovar I strains, and one of two strains of Burkholderia multivorans, but was not detected in Burkholderia stabilis, Burkholderia vietnamiensis, genomovar VI or Burkholderia . ambifaria . The polysaccharide production gene cluster of strain J2315 carries an IS 407-like sequence within the gene similar to B . mallei wcbO that is lacking in other ET12 isolates . Genes from this cluster are expressed during exponential growth in broth.

J Infect Dis, 2003 Mar 1, 187(5), 845 - 53 Epub 2003 Feb 24.
Reinfection, rather than persistent infection, in patients with chronic granulomatous disease; Guide SV et al.; Chronic granulomatous disease (CGD) is characterized by severe recurrent infections with Staphylococcus aureus, certain gram-negative rods, Nocardia species, and fungi . When infections with the same species recur, they may represent relapses or new infections . We collected organisms from infections that occurred between 1992 and 2000 in patients with CGD and determined the biochemical phenotypes, in vitro antibiotic susceptibility patterns, and pulsed-field gel electrophoresis (PFGE) patterns of the organisms causing the initial and recurrent infections . Recurrence of infection with Burkholderia cepacia or Serratia marcescens was caused by a new strain in 9 of 10 cases (P=.001) . Recurrent S . aureus infections were caused by new strains in 7 of 8 cases (P=.006) . In patients with CGD, recurrence of infection with the same bacterial species after appropriate antibiotic therapy usually represents new infection.

Infect Immun, 2003 Mar, 71(3), 1405 - 15
Attenuated virulence of a Burkholderia cepacia type III secretion mutant in a murine model of infection; Tomich M et al.; Type III secretion systems are utilized by a number of gram-negative bacterial pathogens to deliver virulence-associated proteins into host cells . Using a PCR-based approach, we identified homologs of type III secretion genes in the gram-negative bacterium Burkholderia cepacia, an important pulmonary pathogen in immunocompromised patients and patients with cystic fibrosis . One of the genes, designated bscN, encodes a member of a family of ATP-binding proteins believed to generate energy driving virulence protein secretion . Genetic dissection of the regions flanking the bscN gene revealed a locus consisting of at least 10 open reading frames, predicted to encode products with significant homology to known type III secretion proteins in other bacteria . A defined null mutation was generated in the bscN gene, and the null strain and wild-type parent strain were examined by use of a murine model of B . cepacia infection . Quantitative bacteriological analysis of the lungs and spleens of infected C57BL/6 mice revealed that the bscN null strain was attenuated in virulence compared to the parent strain, with significantly lower bacterial recovery from the lungs and spleens at 3 days postinfection . Moreover, histopathological changes, including an inflammatory cell infiltrate, were more pronounced in the lungs of mice infected with the wild-type parent strain than in those of mice infected with the isogenic bscN mutant . These results implicate type III secretion as an important determinant in the pathogenesis of B . cepacia.

Gene, 2003 Feb 13, 305(1), 1 - 12
A family history of deoxyribonuclease II: surprises from Trichinella spiralis and Burkholderia pseudomallei; MacLea KS et al.; Deoxyribonuclease IIalpha (DNase IIalpha) is an acidic endonuclease found in lysosomes and nuclei, and it is also secreted . Though its Caenorhabditis elegans homolog, NUC-1, is required for digesting DNA of apoptotic cell corpses and dietary DNA, it is not required for viability . However, DNase IIalpha is required in mice for correct development and viability, because undigested cell corpses lead to lesions throughout the body . Recently, we showed that, in contrast to previous reports, active DNase IIalpha consists of one contiguous polypeptide . To better analyze DNase II protein structure and determine residues important for activity, extensive database searches were conducted to find distantly related family members . We report 29 new partial or complete homologs from 21 species . Four homologs with differences at the purported active site histidine residue were detected in the parasitic nematodes Trichinella spiralis and Trichinella pseudospiralis . When these mutations were reconstructed in human DNase IIalpha, the expressed proteins were inactive . DNase II homologs were also identified in non-metazoan species . In particular, the slime-mold Dictyostelium, the protozoan Trichomonas vaginalis, and the bacterium Burkholderia pseudomallei all contain sequences with significant similarity and identity to previously cloned DNase II family members . We report an analysis of their sequences and implications for DNase II protein structure and evolution.

Infection, 2003 Jan, 31(1), 60 - 2
A case of imported melioidosis presenting as prostatitis; Heyse AM et al.; We report a case of melioidosis in a previously healthy Belgian man . He presented with septicemia and prostatic abscesses 1 week after a trip to Vietnam . Burkholderia pseudomallei was isolated from multiple hemocultures . He was treated successfully with intravenous ceftazidime and trimethoprim-sulfamethoxazole, followed by a per-oral maintenance therapy of amoxicillin-clavulanate with supplementary amoxicillin . There was no need for surgical drainage . This is the second reported case of melioidosis in Belgium.

Infection, 2003 Jan, 31(1), 24 - 30
The humoral immune response in melioidosis patients during therapy; Vasu C et al.; BACKGROUND: This study was undertaken to identify and quantify the class and subclass antibody responses to the culture filtrate antigen (CFA) of Burkholderia pseudomallei in melioidosis patients under long-term maintenance or eradication therapy . MATERIALS AND METHODS: Sequential sera samples from seven melioidosis patients collected between January 1992 and April 1998 were analyzed for immunoglobulin (Ig) types and IgG isotypes by ELISA using B . pseudomallei CFA . RESULTS: Melioidosis patients generated a strong IgG, IgA and IgM response to the CFA of B . pseudomallei throughout the infection and IgG1 and IgG2 were the predominant IgG istotypes produced . Although high levels of these antibodies were detected in all the seven patients, the IgG, IgG1 and IgG2 antibodies showed a consistent response and good correlation with the clinical history in all cases . CONCLUSION: This study suggests that monitoring IgG antibody or IgG1 or IgG2 isotype antibody levels to CFA in patients under maintenance or eradication antibiotic therapy may be useful as a tool to detect the status of infection and as a guideline to determine the duration of maintenance antimicrobial therapy.

FEMS Microbiol Lett, 2003 Jan 28, 218(2), 323 - 8
The wbiA locus is required for the 2-O-acetylation of lipopolysaccharides expressed by Burkholderia pseudomallei and Burkholderia thailandensis; Brett PJ et al.; Burkholderia pseudomallei and Burkholderia thailandensis express similar O-antigens (O-PS II) in which their 6-deoxy-alpha-L-talopyranosyl (L-6dTalp) residues are variably substituted with O-acetyl groups at the O-2 or O-4 positions . In previous studies we demonstrated that the protective monoclonal antibody, Pp-PS-W, reacted with O-PS II expressed by wild-type B . pseudomallei strains but not by a B . pseudomallei wbiA null mutant . In the present study we demonstrate that WbiA activity is required for the acetylation of the L-6dTalp residues at the O-2 position and that structural modification of O-PS II molecules at this site is critical for recognition by Pp-PS-W.

Syst Appl Microbiol, 2002 Dec, 25(4), 611 - 7
Study of the bacterial load in a gelatine production process focussed on Bacillus and related endosporeforming genera; De Clerck E et al.; Gelatine is an animal protein with many industrial applications . Previous studies pointed out that endosporeforming bacteria, belonging to the genus Bacillus or related genera, might contaminate and survive the production process of gelatine, leading to products of low quality and safety . The aim of this study is to determine the bacterial diversity of contaminants isolated from a gelatine production chain with emphasis on aerobic endosporeforming bacteria . Contaminants were isolated from samples taken at five crucial points along two different production lines of a gelatine production process and from water supplies used for extraction and cooling . Gaschromatographic methyl ester analysis of fatty acids was performed to differentiate isolates at the genus level . Apart from members of the genus Bacillus or related endosporeforming genera, also members of Salmonella, Kluyvera, Staphylococcus, Burkholderia, Enterococcus, Pseudomonas, Yersinia, Streptococcus and Brevundimonas could be detected . Isolates identified as belonging to Bacillus and related endosporeforming genera were further characterised by gelatinase tests, rep-PCR and 16S rDNA sequencing . All these isolates showed the ability to liquefy gelatine . Endosporeforming isolates were assigned to Bacillus licheniformis, B . fumarioli, members of the B . cereus group, B . badius, B . coagulans, B . subtilis, Brevibacillus agri, Alicyclobacillus acidocaldarius and a yet undescribed Paenibacillus species.

Syst Appl Microbiol, 2002 Dec, 25(4), 507 - 12
Burkholderia tuberum sp . nov . and Burkholderia phymatum sp . nov., nodulate the roots of tropical legumes; Vandamme P et al.; The taxonomic status of five root nodule isolates from tropical legumes was determined using a polyphasic taxonomic approach . Two isolates were identified as B . caribensis, an organism originally isolated from soil in Martinique (the French West Indies) . One isolate was identified as Burkholderia cepacia genomovar VI, a B . cepacia complex genomovar thus far only isolated from sputum of cystic fibrosis patients . The remaining two isolates were identified as novel Burkholderia species for which we propose the names Burkholderia tuberum sp . nov . and Burkholderia phymatum sp . nov . The type strains are LMG 21444T and LMG 21445T, respectively.

J Assoc Physicians India, 2002 Nov, 50, 1438 - 9
Melioidosis--a report from Pondicherry, South India; Kanungo R et al.; Melioidosis is an acute infectious disease caused by a safety-pin-shaped gram-negative bacteria called Burkholderia pseudomallei . Here, we report the first case of melioidosis in a middle aged male agricultural worker, from Pondicherry . The isolation of this organism from subcutaneous nodules on the extensor aspect of his limbs underlines the diversity of its clinical presentation . Difficulty in identifying the organism which mimics any other non-fementing gram-negative bacilli (NFGNB) on cursory examination, highlights the importance of identification of NFGNB in endemic areas for specific treatment and prevention.

Eur J Clin Microbiol Infect Dis, 2003 Jan, 22(1), 28 - 34 Epub 2003 Jan 25.
Use of the E test to assess synergy of antibiotic combinations against isolates of Burkholderia cepacia-complex from patients with cystic fibrosis; Manno G et al.; Treatment of Burkholderia cepacia-complex infections in cystic fibrosis patients is problematic, since the microorganism is often resistant to most antimicrobial agents . In this study, the Epsilometer test, or E test, was used to assess the activity of antimicrobial combinations against Burkholderia cepacia-complex . In a preliminary evaluation, the E test was compared to the checkerboard method using 10 test organisms . Synergy testing by the E test was then performed on 131 clinical isolates of Burkholderia cepacia-complex using various combinations of antimicrobial agents . Agreement between the E test and the checkerboard method was 90% . The rate of resistance to individual agents ranged from 48% for meropenem to 100% for tobramycin, chloramphenicol, and rifampin . In 71.6%, 15.6%, and 12.6% of the test evaluations performed, the combinations tested resulted in additivity/indifference, synergism, and antagonism, respectively . The highest rates of synergy were observed with combinations of ciprofloxacin-piperacillin (44%), rifampin-ceftazidime (33%), chloramphenicol-ceftazidime (22%), cotrimoxazole-piperacillin/tazobactam (22%), and ciprofloxacin-ceftazidime (21%) . Rates of antagonism for cotrimoxazole and chloramphenicol in combination with beta-lactam agents were higher than those observed for ciprofloxacin plus beta-lactam agents . These results suggest that the E test is a valuable and practical method to be considered for improving the identification of possible therapeutic options in cystic fibrosis patients infected with organisms belonging to the Burkholderia cepacia-complex.

J Appl Anim Welf Sci, 2002, 5(3), 203 - 13
Body condition of feral cats and the effect of neutering; Scott KC et al.; Considerable debate exists regarding the most appropriate methods for controlling feral cat populations, both from humane and logistical points of view . The physical condition of feral cats has not been reported, and it is not known if these cats benefit from neutering . This study investigates the body condition of feral cats by measuring body weight (BW), body condition score (BCS; Burkholder, 2000; Laflamme, Kealy, & Schmidt, 1994), and falciform fat pad . The study includes lateral abdominal radiographs taken at the time of neutering of 105 adult feral cats for measurement of falciform fat pad depth and area . At that time we also assessed BW and BCS . One year later we assessed the effects of neutering on body condition by evaluating a subsample of 14 cats . At the time of surgery, the cats were lean but not emaciated (BW 3.1 +/- 0.9 kg; BCS 4 +/- 1; based on a 1 to 9 scale ranging from 1 {emaciated} to 9 {grossly obese}) . Falciform fat pad depth and area averaged 7.1 mm and 197.4 mm2, respectively, indicating a small amount of fat . Fourteen cats, reevaluated 1 year after neutering, increased 260% + 90% in falciform fat pad depth, 420% +/- 390% in fat pad area, 40% +/- 4% in BW, and 1 level in BCS ranking (1 to 9 scale; all differences p <.001) . Similar to confined socialized cats, feral cats gained significant weight and body fat after neutering.

Microbiology, 2003 Jan, 149(Pt 1), 77 - 88
Population structure analysis of Burkholderia cepacia genomovar III: varying degrees of genetic recombination characterize major clonal complexes; Coenye T et al.; Infection with bacterial species belonging to the Burkholderia cepacia complex contribute significantly to morbidity and mortality in persons with cystic fibrosis (CF) . The majority of isolates recovered from CF patients belong to B . cepacia genomovar III and several distinct 'epidemic' strains have been described . This study examined the population structure of B . cepacia genomovar III by using multilocus restriction typing, indexing allelic variation at five chromosomal genes by restriction analysis of PCR-amplified genes . A collection of 375 isolates, recovered from CF and non-CF patients and natural environments in North America, Europe and Australia, was examined . Among these isolates 144 different restriction types were found . Overall, the population is at linkage disequilibrium, indicating that it has a clonal structure . The majority (86.7 %) of restriction types grouped into three major clonal complexes, comprising the epidemic ET12, PHDC and Midwest clonal lineages . The analysis indicates that these complexes are geographically widespread and demonstrate varying degrees of genetic recombination . These differences in population structure among major clonal complexes within the same species are likely related to differences in evolutionary history and ecology . The observation that genetic recombination is frequent within some B . cepacia genomovar III populations has important implications for the biotechnological use of B . cepacia complex species.

J Clin Microbiol, 2003 Feb, 41(2), 883 - 5
Ubiquity of putative type III secretion genes among clinical and environmental Burkholderia pseudomallei isolates in Northern Australia; Smith-Vaughan HC et al.; Type III secretion (TTSI) genes of an HRP (hypersensitivity response and pathogenicity)-like locus were present in all 116 Northern Australian Burkholderia pseudomallei isolates tested but were not detected in other common environmental Burkholderia species . PCR of TTS1 genes may prove valuable as a diagnostic test {corrected}.

Biochim Biophys Acta, 2003 Feb 21, 1645(2), 133 - 8
Cloning and expression of the gene encoding catalytic subunit of thermostable glucose dehydrogenase from Burkholderia cepacia in Escherichia coli; Inose K et al.; We have cloned a 1620-nucleotide gene encoding the catalytic subunit (alpha subunit) of a thermostable glucose dehydrogenase (GDH) from Burkholderia cepacia . The FAD binding motif was found in the N-terminal region of the alpha subunit . The deduced primary structure of the alpha subunit showed about 48% identity to the catalytic subunits of sorbitol dehydrogenase (SDH) from Gluconobacter oxydans and 2-keto-D-gluconate dehydrogenases (2KGDH) from Erwinia herbicola and Pantoea citrea . The alpha subunit of B . cepacia was expressed in Escherichia coli in its active water-soluble form, showing maximum dye-mediated GDH activity at 70 degrees C, retaining high thermal stability . A putative open reading frame (ORF) of 507 nucleotides was also found upstream of the alpha subunit encoding an 18-kDa peptide, designated as gamma subunit . The deduced primary structure of gamma subunit showed about 30% identity to the small subunits of the SDH from G . oxydans and 2KGDHs from E . herbicola and P . citrea.

J Bacteriol, 2003 Feb, 185(4), 1253 - 60
Characterization of extradiol dioxygenases from a polychlorinated biphenyl-degrading strain that possess higher specificities for chlorinated metabolites; Vaillancourt FH et al.; Recent studies demonstrated that 2,3-dihydroxybiphenyl 1,2-dioxygenase from Burkholderia sp . strain LB400 (DHBDLB400; EC 1.13.11.39) cleaves chlorinated 2,3-dihydroxybiphenyls (DHBs) less specifically than unchlorinated DHB and is competitively inhibited by 2',6'-dichloro-2,3-dihydroxybiphenyl (2',6'-diCl DHB) . To determine whether these are general characteristics of DHBDs, we characterized DHBDP6-I and DHBDP6-III, two evolutionarily divergent isozymes from Rhodococcus globerulus strain P6, another good polychlorinated biphenyl (PCB) degrader . In contrast to DHBDLB400, both rhodococcal enzymes had higher specificities for some chlorinated DHBs in air-saturated buffer . Thus, DHBDP6-I cleaved the DHBs in the following order of specificity: 6-Cl DHB > 3'-Cl DHB approximately DHB approximately 4'-Cl DHB > 2'-Cl DHB > 4-Cl DHB > 5-Cl DHB . It also cleaved its preferred substrate, 6-Cl DHB, three times more specifically than DHB . Interestingly, some of the worst substrates for DHBDP6-I were among the best for DHBDP6-III (4-Cl DHB > 5-Cl DHB approximately 6-Cl DHB approximately 3'-Cl DHB > DHB > 2'-Cl DHB approximately 4'-Cl DHB; DHBDP6-III cleaved 4-Cl DHB two times more specifically than DHB) . Generally, each of the monochlorinated DHBs inactivated the enzymes more rapidly than DHB . The exceptions were 4-Cl DHB for DHBDP6-I and 2'-Cl DHB for DHBDP6-III . As observed in DHBDLB400, chloro substituents influenced the reactivity of the dioxygenases with O2 . For example, the apparent specificities of DHBDP6-I and DHBDP6-III for O2 in the presence of 2'-Cl DHB were lower than those in the presence of DHB by factors of >60 and 4, respectively . DHBDP6-I and DHBDP6-III shared the relative inability of DHBDLB400 to cleave 2',6'-diCl DHB (apparent catalytic constants of 0.088 +/- 0.004 and 0.069 +/- 0.002 s(-1), respectively) . However, these isozymes had remarkably different apparent K(m) values for this compound (0.007 +/- 0.001, 0.14 +/- 0.01, and 3.9 +/- 0.4 micro M for DHBDLB400, DHBDP6-I, and DHBDP6-III, respectively) . The markedly different reactivities of DHBDP6-I and DHBDP6-III with chlorinated DHBs undoubtedly contribute to the PCB-degrading activity of R . globerulus P6.

Wei Sheng Wu Xue Bao, 2001 Feb, 41(1), 70 - 5
{Determination of DNA-DNA homology among Pseudomonas cocovenenans subsp . farinofermentans with microdilution plate hybridization method}; Jiao Z et al.; The DNA-DNA homologies among Pseudomonas cocovenenans subsp . farinofermentans and 11 species in genus Burkholderia were determined with microdilution plate hybridization method . The results showed: the homologies among P . cocovenenans subsp . farinofermentans, B . gladioli and B . cocovenenans were all over 75% . It was advised that these three species were synonym, and should be renamed as Burkholderia gladioli.

Environ Microbiol, 2003 Jan, 5(1), 3 - 12
Adaptation to nickel spiking of bacterial communities in neocaledonian soils; Hery M et al.; Adaptation to nickel of bacterial communities of two extreme neocaledonian soils (an ultramafic soil and an acidic soil) was investigated by nickel spiking and compared with adaptation in a non-neocaledonian soil used as reference . Soil microcosms were amended with nickel chloride (NiCl2), and bacterial community structure was analysed with the ribosomal intergenic spacer analysis (RISA) technique . Then, bacterial populations that respond to nickel stress were identified by cloning and sequencing . In the ultramafic soil, a shift occurred on day zero on the assay profiles and consisted of the emergence of a bacterial group closely related to the Ralstonia/Oxalobacter/Burkholderia group . It is hypothesized that NiCl2 had a physico-chemical impact on soil structure . Fourteen days after nickel spiking, another shift occurred in the two soils that concerned a bacterial group belonging to the Actinomycete group . Only a few changes occurred in the bacterial community structure of the neocaledonian soils compared with those of the reference soil, which is more affected by nickel spiking . These results suggest that neocaledonian soil bacteria are particularly well adapted to nickel.

Infect Immun, 2003 Feb, 71(2), 904 - 9
Colonial morphology of Burkholderia cepacia complex genomovar III: implications in exopolysaccharide production, pilus expression, and persistence in the mouse; Chung JW et al.; The purpose of this study was to determine the role of colonial morphology of Burkholderia cepacia complex (BCC) organisms in pathogenicity in a mouse model of pulmonary infection . BCC strain C1394 was rapidly cleared by leukopenic mice after intranasal challenge, whereas a spontaneous variant (C1394mp2) that was indistinguishable from the parent strain by genetic typing persisted in the lungs and differed in colonial morphology . The parent strain had a matte colonial phenotype, made scant exopolysaccharide (EPS), and was lightly piliated . The variant had a shiny phenotype, produced abundant EPS, and was heavily piliated . Matte to shiny colonial transformation was induced by growth at 42 degrees C . Colonial morphology in the BCC strain variant was associated with persistence after pulmonary challenge and appeared to be correlated with the elaboration of putative virulence determinants.

Lett Appl Microbiol, 2003, 36(2), 77 - 82
Characterization of amplified polymerase chain reaction glnB and nifH gene fragments of nitrogen-fixing Burkholderia species; Marin VA et al.; AIMS: To clone and sequence polymerase chain reaction (PCR)-amplified glnB and nifH genes of the nitrogen-fixing bacteria Burkholderia brasilensis strain M130, B . tropicalis strain PPe8 and B . kururiensis strain KP23 . METHODS AND RESULTS: The glnB and nifH gene fragments were amplified by PCR using universal degenerated primers . A very high percentage of similarity for the nifH (100%) and glnB (96%) genes was observed between strains M130 and KP23 . A similarity of 100% for the nifH gene was also observed between strains M130 and PPe8 . However, the identity for the glnB gene was 98% and the similarity 88% . The phylogenetic tree of the nifH gene showed a very high degree of similarity to the 16S rDNA gene . CONCLUSIONS: The nitrogen-fixing bacteria of the Burkholderia genus formed a cluster separated from the other species of the genus mainly when the nifH rather than the glnB gene was used to construct the phylogenetic tree . Significance and Impact of the Study: Knowledge of the nifH and glnB gene sequences of B . brasilensis, B . tropicalis and B . kururiensis will support new studies on the diversity of these diazotrophs in natural environments.

J Infect Chemother, 2002 Dec, 8(4), 341 - 4
Antimicrobial resistance and DNA-fingerprinting pattern of Burkholderia cepacia blood isolates; Saika T et al.; The minimum inhibitory concentrations (MICs) of six antibiotics against 33 strains of Burkholderia cepacia isolated from 33 patients suspected of having sepsis or bacteremia were determined . All the B . cepacia isolates were resistant to gentamicin (MIC(90), >128 microg/ml), and 21.2% were resistant to imipenem (MIC(90), 16 microg/ml) . They were susceptible to ciprofloxacin, ceftazidime, and minocycline (MIC(90), 2-8 microg/ml) . The 33 blood isolates were classified into 12 types (A-L) by the pulsed-field gel electrophoresis pattern . All 11 of A type, 2/4 of B type, 3/4 of C type, 4/5 of E type, and both G type B . cepacia were isolated from different patients in the same hospital . These results suggest that B . cepacia can spread among patients with sepsis or bacteremia.

Wei Sheng Yan Jiu, 2000 Jul, 29(4), 243 - 5
{Determination of guanine plus cytosine content of Pseudomonas cocovenenans subsp farinofermantans with two methods and the comparative study}; Jiao Z et al.; The guanine plus cytosine content (G + C) mol% of Pseudomonas cocovenenans subsp farinofermantans, Burkholderia gladioli and Burkholderia cocovenenans, which were similar in biochemical characteristics, were determined with Tm values method and HPLC method, and the comparative study of these two methods was discussed . A negative conclusion could not be made from the results . The three bacteria were the same species . The HPLC method was more accurate than the Tm values method, but the repeatability of the latter was better.

Cochrane Database Syst Rev . 2002;(4):CD001263.
Interventions for treating melioidosis; Samuel M et al.; BACKGROUND: Melioidosis is an infectious disease that occurs in tropical regions, particularly in Thailand . It is caused by the bacterium Burkholderia pseudomallei and is a serious condition which can be fatal . Beta-lactam antibiotics have dramatically reduced the risk of death, but mortality still remains high . OBJECTIVES: To summarize reliable evidence on the effects of treatment regimens on death and relapse . SEARCH STRATEGY: We searched the Cochrane Infectious Diseases Group trials register (July 2002), the Cochrane Controlled Trials Register (Issue 3, 2002), MEDLINE (1966 to July 2002), EMBASE (1980 to May 2002), BIOSIS (up to July 2002), Health Star (up to July 2002), and reference lists of articles . We also contacted pharmaceutical companies and researchers in the field . SELECTION CRITERIA: Randomized and quasi-randomized controlled trials comparing antibiotic regimens in people with melioidosis . DATA COLLECTION AND ANALYSIS: We independently assessed the eligibility of studies and the methodological quality of the trials . Adverse effects information was collected from the trials . MAIN RESULTS: Nine trials, all from Thailand, involving a total of 872 participants were included . For intravenous therapy in the acute phase, we identified six trials with a total of 619 participants . Chloramphenicol, doxycycline, and co-trimoxazole (trimethoprim-sulphamethoxazole) combination regimens were associated with a mortality of 50% or more (two studies) . Participants randomized to regimens including ceftazidime were more likely to survive (relative risk {RR} 0.46; 95% confidence interval {CI} 0.30 to 0.71) . When ceftazidime-containing regimens were compared with beta-lactam or alternative beta-lactamase inhibitor regimens such as co-amoxiclav (amoxycillin-clavulanic acid) and cefoperazone-sulbactam, or with imipenem, mortality rates were similar (RR 1.06; 95% CI 0.81 to 1.39) . For oral therapy in the maintenance phase, we found three trials of 253 participants . They compared the conventional regimen (chloramphenicol, doxycycline, and trimethoprim-sulphamethoxazole) with other regimens (amoxycillin-clavulanic acid, ciprofloxacin-azithromycin, and doxycycline alone) . There were fewer deaths with the conventional regimen, but no statistically significant differences demonstrated . REVIEWER'S CONCLUSIONS: Regimens for the acute phase of illness should contain ceftazidime or imipenem . It is not yet clear if combinations of treatments in the early phase reduce relapse . For oral therapy after the acute phase of treatment, trials suggest that conventional four drug regimens can be used for treatment.

An R Acad Nac Med (Madr), 2002, 119(2), 343 - 62; discussion 362-6
{Microbiology and cystic fibrosis}; Piedrola Angulo G; The respiratory infections are the rule in sick persons of cystic fibrosis . After the colonization in one's early childhood by Staphylococcus aureus and Haemophilus influenzae, it is established the colonization produced by the mucous strains of Pseudomonas aeruginosa, the principal etiogenic of this illness, that can become to isolate in more than 95% of sick persons at three years old . That's why the etiogenic factors of this bacterium are studied, as they are toxins, pigments, enzymes, and principally the alginate, hydrocarbonated polymer responsible of the mucous substance that covers these characteristic strains of cystic fibrosis sick persons . All these components are regulated by a complex genome, the biggest until noe sequenced in bacterium, and that explains the importance of the microorganism and its natural resistance or acquired to environmental alterations and to antibiotics and disinfectants . In the evolution of the clinic chart, it can appear others gramnegative bacteriums (among it emphasizes Burkholderia cepacia and Stenotrophomonas maltophilia), mycobacterium and even fungus; all that would explain the chronification of lung charts and the difficulty of these sick persons treatment.

Int J Syst Evol Microbiol, 2002 Nov, 52(Pt 6), 2023 - 7
Identification of the bacterial endosymbionts in leaf galls of Psychotria (Rubiaceae, angiosperms) and proposal of 'Candidatus Burkholderia kirkii' sp . nov; Van Oevelen S et al.; This paper reports the identification of bacterial endosymbionts inhabiting the leaf galls of Psychotria kirkii . A phylogenetic approach was used to reveal the identity of these as yet uncultivable bacterial endophytes . Based on the analysis of 16S rDNA sequences, evolutionary trees were constructed that place the endosymbiont in the genus Burkholderia . Low levels of sequence identity and rather large evolutionary distances to the closest validly named relatives indicate that these symbiotic bacteria represent a novel species . Until cultivation is successful or until more phenotypic data become available the provisional name 'Candidatus Burkholderia kirkii' sp . nov . is proposed.

Zh Mikrobiol Epidemiol Immunobiol, 2002 Nov-Dec, (6), 60 - 4
{Immunobiological properties of Burkholderia pseudomallei and Burkholderia mallei capsular substances}; Popov SF et al.; The biopolymer composition, immunotropic and immunogenic properties of the fractions of B . pseudomallei and B . mallei were under study . The first two capsular fractions of these agents were found to be similar in their biopolymer composition that was indicative of their close relations . At the same time the causative agents of glanders proved to have decreased content of high molecular glycoproteids and LPS fragments . In the causative agents of melioidosis, capsular fractions K3 and K4 were characterized by the domination of proteins with a molecular weight of 42-25 kD . Fraction K4 in B . pseudomallei and fraction K1 in B . mallei had pronounced immunosuppressing properties ensuring the protection of encapsulated microbial cells in the body . The biopolymers forming fractions K1, K2, K3 in B . pseudomallei and fraction K2 in B . mallei were characterized by immunomodulating properties.

J Infect, 2003 Jan, 46(1), 56 - 9
A comparison of pulmonary exacerbations with single and multiple organisms in patients with cystic fibrosis and chronic Burkholderia cepacia infection; Mc Manus TE et al.; STUDY OBJECTIVES: To compare pulmonary exacerbations with single and multiple organisms in patients with chronic Burkholderia cepacia infection . DESIGN: Data was collected over a 23-month period and for each of the patients two episodes of pulmonary exacerbation were identified, one with B . cepacia isolated as the sole respiratory pathogen and the other with one or more coinfecting organisms grown from the sputum culture . SETTING: Regional tertiary referral center for Respiratory Medicine and Adult Cystic Fibrosis Center for Northern Ireland, Belfast City Hospital . PATIENTS: Adult (over 16 years) CF patients with chronic (more than 4 years) B . cepacia complex infection who attended the Belfast City Hospital between January 1997 and November 1998 . MEASUREMENTS: Forced expiratory volume in 1s, forced vital capacity, forced expiratory flow rate 25-75, C-reactive protein, leucocyte count and body mass index were measured before and after two weeks of treatment and analysed for any differences in change between the two episodes . RESULTS: All variables in both groups improved following treatment except for body mass index, which remained unchanged . No significant differences between the measurements were identified on comparison of single (monomicrobial) and multiple (polymicrobial) isolate episodes . CONCLUSION: Patients with chronic B . cepacia infection who develop a pulmonary exacerbation improve, as reflected by clinical and biochemical markers, following IV antibiotic treatment . Pulmonary exacerbations with multiple organisms are no more severe than those where only B . cepacia is isolated.

J Gen Appl Microbiol, 1998 Apr, 44(2), 147 - 152
Screening and identification of a novel lipase from Burkholderia sp . YY62 which hydrolyzes t-butyl esters effectively; Yeo SH et al.; Fatty acid esters composed of sterically hindered alcohol are very poor substrates for known lipases . In order to obtain a novel lipase, t-butyl octanoate (TBO) was selected as a model substrate to screen for bacteria-producing lipase(s) which can preferentially hydrolyze bulky esters . Of 279 strains isolated from 350 soil samples based on the ability to grow with TBO as a sole carbon source, one strain (YY62) was chosen for its strong TBO-hydrolyzing activity . Strain YY62 is a Gram-negative motile rod and was identified as Burkholderia sp . from the taxonomic characters and phylogenetic analysis of 16S rDNA nucleotide sequences . Using the activity ratio between TBO and p-nitrophenyl acetate as a measure for preference to bulky esters, we confirmed that the lipase of strain YY62 was 100-fold superior to commercial lipases in terms of TBO-hydrolyzing activity.

Semin Respir Infect, 2002 Dec, 17(4), 284 - 90
Burkholderia cepacia infection and lung transplantation; Husain S et al.; Burkholderia cepacia has emerged as an important pathogen in patients with cystic fibrosis (CF) undergoing lung transplantation . Taxonomic analyses have divided B . cepacia into 7 closely related species called genomovars . The prevalence of B . cepacia infection ranges from 2% to 13% in CF patients with genomovar III being most prevalent . Risk factors for the acquisition of B . cepacia include greater severity of underlying CF, increasing age, having a sibling colonized with B . cepacia, and previous hospitalizations . One-year survival in patients with CF undergoing lung transplantation usually have been reported to be less than 70% . The antimicrobial resistance pattern of B . cepacia has correlated with inconsistent effect on survival . Presence of genomovar III also has been linked with survival of B . cepacia-infected patients; the survival rates were inferior for genomovar III as compared with non-genomovar III patients . Standard infection control techniques including isolation of both colonized and noncolonized individuals with frequent sputum culture have been recommended to prevent CF patients from acquiring B . cepacia infection . B . cepacia is inherently resistant to antipseudomonal antibiotics, colistin, and polymyxin.The combination of 2 or more antibiotics usually is recommended for the treatment of B . cepacia infections . Studies delineating the epidemiology and influence of B . cepacia infection on lung transplantation are warranted . This is a US government work . There are no restrictions on its use.

Infect Immun, 2003 Jan, 71(1), 584 - 7
Biodefense-driven murine model of pneumonic melioidosis; Jeddeloh JA et al.; A whole-body mouse model of pneumonic melioidosis was established for future evaluation of biodefense vaccine candidates . The aerosol 50% lethal doses of Burkholderia pseudomallei strain 1026b for BALB/c and C57BL/6 mice and the times to death, dissemination in organs, and tissue loads after exposure of the mice to low- and high-dose aerosols are reported . In addition, rpsL mutant backgrounds were attenuated in this acute model of disease.

Infect Immun, 2003 Jan, 71(1), 205 - 10
Phagocyte NADPH oxidase, but not inducible nitric oxide synthase, is essential for early control of Burkholderia cepacia and chromobacterium violaceum infection in mice; Segal BH et al.; Reactive oxygen and nitrogen intermediates have critical, partially overlapping roles in host defense against a variety of pathogens . Using mice deficient in generating phagocyte superoxide (p47(phox)(-/-)) and mice deficient in generating inducible nitric oxide synthase (iNOS(-/-)), we examined the roles of these reactive species in host defense against Burkholderia cepacia and Chromobacterium violaceum, organisms known to have unusual virulence in chronic granulomatous disease . Intraperitoneal B . cepacia challenge (4.0 x 10(3) to 4.0 x 10(5) organisms/mouse) resulted in mortality in all p47(phox)(-/-) mice, with the survival interval being inversely proportionate to the amount of inoculum . Pretreatment with gamma interferon did not affect survival . C . violaceum was strikingly virulent in p47(phox)(-/-) mice (the 50% lethal dose {LD(50)} was <13 organisms) . iNOS(-/-) and wild-type mice were resistant to B . cepacia challenges of at least 10(6) organisms per mouse, and the LD(50) of C . violaceum was between 10(6) and 10(7) organisms per mouse . Consistent with the survival data, numbers of organisms in cultures of B . cepacia from multiple sites were higher for p47(phox)(-/-) mice than for iNOS(-/-) and wild-type mice at day 4 after challenge, but numbers of organisms for different B . cepacia strains varied . The recovery of C . violaceum was strikingly greater at 18 h after challenge for p47(phox)(-/-) mice than for iNOS(-/-) and wild-type mice, in which the organism burdens were virtually nil . In vitro, both B . cepacia and C . violaceum were sensitive to H(2)O(2) and to reactive nitrogen intermediates but the sensitivities of different strains varied significantly . Host defense against B . cepacia and C . violaceum is critically dependent in vivo on reactive oxygen intermediates, and these species are model organisms to further dissect host and pathogen interactions related to the generation and scavenging of microbicidal reactive intermediates.

J Antimicrob Chemother, 2003 Jan, 51(1), 77 - 81
A comparison of antibiotic regimens in the treatment of acute melioidosis in a mouse model; Ulett GC et al.; Melioidosis is caused by the Gram-negative bacillus Burkholderia pseudomallei . Most clinical reports of disease are from south-east Asia and northern Australia . The organism is intrinsically resistant to most commonly available antibiotics . Standard therapy includes ceftazidime either alone or in combination with co-trimoxazole . The clinical advantage in adding co-trimoxazole has never been determined; nor has the activity of newer, fourth-generation cephalosporins, such as cefepime, been studied in the treatment of this condition . BALB/c mice have been shown to represent an animal model of melioidosis . This animal model was used in this study to compare the efficacy of ceftazidime and cefepime alone or with co-trimoxazole, in the therapy of melioidosis . Antibiotic levels in the mice were determined by HPLC, and dosing was modified to keep plasma antibiotic levels at or above the MIC for the organism-antibiotic combination for a significant part of a 12 h period . Bacterial load, as determined by splenic counts, showed that ceftazidime in combination with co-trimoxazole was the most effective therapeutic option . The animal model described in this study can be used as a preliminary evaluation of therapeutic options for melioidosis.

Mikrobiyol Bul, 2002 Jan, 36(1), 1 - 10
{Isolation frequency of Burkholderia cepacia from cystic fibrosis patients}; Ocak F et al.; Microorganisms such as Pseudomonas aeruginosa, Staphylococcus aureus and Haemophilus influenzae frequently cause colonization and infection in airways of patients with cystic fibrosis . Burkholderia cepacia has also been isolated in patients with cystic fibrosis since 1980 . In this study, we aimed to determine the colonization rate of B . cepacia in 286 sputum samples obtained from 129 cystic fibrosis patients . Selective media for B . cepacia were used besides the routine microbiological media in order to increase the isolation rate . The colonies were identified by biochemical tests and the antibiotic susceptibilities of the strains were determined by disc diffusion method . Pathogenic bacteria (S . aureus, P . aeruginosa, Enterobacteriaceae, Streptococcus pneumoniae, H . influenzae) were isolated in 52 of 129 patients (40%) and 66 of 286 sputum samples (23%) . In addition 2 B . cepacia strains were isolated from two different patients (1.55%) . B . cepacia is now being considered as a pathogen isolated from sputum samples of patients with cystic fibrosis with an increasing frequency and causing severe clinical features . According to these results it can be concluded that, the use of selective media for B . cepacia isolation, should be taken into consideration especially by the clinical microbiology laboratories collaborating with the cystic fibrosis centers.

Clin Infect Dis, 2002 Dec 15, 35(12), e138 - 40 Epub 2002 Nov 19.
Cystic Fibrosis and Burkholderia pseudomallei Infection: An Emerging Problem?
Holland DJ, Wesley A, Drinkovic D, Currie BJ.
We recently managed 4 patients with cystic fibrosis who had acquired Burkholderia pseudomallei infection after exposure in a region of endemicity . Person-to-person transmission between 2 siblings may have occurred; otherwise, the evidence suggests that cystic fibrosis may increase the likelihood of infection with this organism, and patients should be warned of this possibility and cautioned to avoid high-risk activities.

J Gen Appl Microbiol, 2002 Feb, 48(1), 25 - 33
Purification and characterization of a novel chitinase from Burkholderia cepacia strain KH2 isolated from the bed log of Lentinus edodes, Shiitake mushroom; Ogawa K et al.; One of the chitinases secreted in the culture filtrate of a gram-negative bacteria, Burkholderia cepacia strain KH2, which was isolated from the bed log of Lentinus edodes, Shiitake mushrooms, was purified by DEAE Sepharose CL-6B chromatography, followed by Sephacryl S-100 HR gel filtration . The purified enzyme was homogenous, determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), with an estimated molecular weight of 34,000 and an isoelectric point (pI) of 5.9 . The enzyme was stable at pH values of 4.0-6.0, and at temperatures up to 50 degrees C; the optimum pH and temperature were 4.5 and 50 degrees C, respectively . The enzyme exhibited higher activities toward chitosan 7B, a 62% deacetylated chitosan, than toward the highly deacetylated chitosan substrates . The enzyme was observed to drastically hydrolyze partially deacetylated chitin substrates, with the subsequent formation of N-acetylchitooligosaccharides {(GlcNAc) (n), n=2-7} . Separation and quantification of the hydrolysis products of (GlcNAc) (n), n52-6, by HPLC showed the splitting into (GlcNAc)(n), n=3-6 . Activity toward N-acetylchitobiose was not detected . Oligomers with a higher number of units than the starting substrate were also detected, which indicate transglycosylation activity.

J Med Microbiol, 2002 Dec, 51(12), 1055 - 62
Passive protection against Burkholderia pseudomallei infection in mice by monoclonal antibodies against capsular polysaccharide, lipopolysaccharide or proteins; Jones SM et al.; Burkholderia pseudomallei, the aetiological agent of melioidosis, is endemic in south-east Asia and northern Australia, where it is an important cause of human disease . There is no vaccine available and antibiotic therapy is associated with high relapse rates . A panel of seven monoclonal antibodies (MAbs) that recognise capsular polysaccharide, lipopolysaccharide or proteins was produced and their ability to protect mice passively against experimental melioidosis was evaluated . The MAbs were capable of protecting mice against intra-peritoneal challenge with 10(4) cfu/250 MLD of a virulent strain of B . pseudomallei (NCTC 4845), when pooled, and four of the MAbs were individually protective . However, at a higher B . pseudomallei challenge level of 10(6) cfu none of the MAbs afforded protection and only the anti-exopolysaccharide MAbs produced a significantly delayed time to death.

J Mol Graph Model, 2002 Dec, 21(3), 241 - 52
A quantitative model for predicting enzyme enantioselectivity: application to Burkholderia cepacia lipase and 3-(aryloxy)-1,2-propanediol derivatives; Tomic S et al.; We describe a new approach for predicting the enantioselectivity of enzymes towards racemic compounds . It is based on comparative binding energy (COMBINE) analysis . The approach is used to rationalise the enantioselectivity of Burkholderia cepacia lipase (BCL) towards thirteen racemic 3-(aryloxy)-1,2-propanediols in the process of acylation . According to our molecular modelling study the two 3-(aryloxy)-1,2-propanediols enantiomers bind in the BCL active site in different orientations . To derive a quantitative structure-activity relationship (QSAR), the difference in the interaction energy between two enantiomers with each amino acid residue was computed . These residue-based energy differences were then subjected to chemometric analysis and 3D QSAR models were derived . The models were able to unambiguously predict the fast-reacting enantiomer and the approximate magnitude of the enantioselectivity . The study enabled identification of interactions between the substrate and the lipase amino acid residues that play key roles in secondary alcohol enantiodifferentiation . From the results, it was possible to propose modifications of both, substrate and protein, which would directionally modify enantioselectivity of BCL towards secondary aryl-alcohols.

Diagn Microbiol Infect Dis, 2002 Oct, 44(2), 143 - 9
Single gene target bacterial identification . groEL gene sequencing for discriminating clinical isolates of Burkholderia pseudomallei and Burkholderia thailandensis; Woo PC et al.; Proper identification of Burkholderia pseudomallei and Burkholderia thailandensis is crucial in guiding clinical management of patients with suspected melioidosis, as more than 99% of cases of melioidosis are caused by B . pseudomallei, whereas B . thailandensis is only responsible for causing less than 1% of the cases . However, the difference between the 16S ribosomal RNA gene sequences of B . pseudomallei and that of B . thailandensis is only 1%, and is therefore not discriminative enough for distinguishing the 2 species confidently . In this study, we amplified and sequenced the groEL genes of 7 strains of B . thailandensis and 6 strains of B . pseudomallei, and compared the sequences with 7 other groEL gene sequences of Burkholderia species . BLAST analysis revealed that the putative protein encoded by the groEL gene of B . thailandensis has 99.6%, 99.5%, 98.4%, 98.5%, and 96.5% amino acid identity with the groEL of B . pseudomallei, B . mallei, B . cepacia, B . vietnamiensis, and B . fungorum respectively . The amino acid sequences of GroEL of the strains of B . thailandensis and B . pseudomallei all showed >99.5% amino acid identity with each other . The nucleotide sequence of the groEL gene of any of the strains of B . thailandensis showed >99.8% nucleotide identity with that of any of the other strains of B . thailandensis, and the nucleotide sequence of the groEL gene of any of the strains of B . pseudomallei showed >99.5% nucleotide identity with that of any of the other strains of B . pseudomallei . However, the nucleotide sequence of any of the strains of B . thailandensis showed <97.6% nucleotide identity with any of the strains of B . pseudomallei . The amino acid sequences of GroEL of the 20 strains of Burkholderia species all showed >96% amino acid identity with each other . Furthermore, the nucleotide sequence of the groEL genes of the 2 strains of B . cepacia showed >99.5% nucleotide identity with each other, and the nucleotide sequence of the groEL gene of B . mallei showed >99.5% nucleotide identity with any of the strains of B . pseudomallei . The groEL gene sequence is therefore good for distinguishing between B . thailandensis and B . pseudomallei, and the GroEL amino acid and groEL nucleotide sequences of this single gene locus may potentially be useful for a 2-tier hierarchical identification of medically important Burkholderia at the genus and species levels respectively.

Mol Pathol, 2002 Dec, 55(6), 398 - 400
Strategies for PCR based detection of Burkholderia pseudomallei DNA in paraffin wax embedded tissues; Hagen RM et al.; Recently, several cases of melioidosis imported to Europe have been reported . The diagnosis of the acute or chronic infection remains challenging . This report describes an optimised protocol for fast and reliable DNA preparation for use in two different polymerase chain reaction (PCR) assays, namely: (1) a seminested PCR assay targeting a genus specific sequence of the ribosomal protein subunit 21 (rpsU) gene and (2) a nested PCR assay targeting the gene encoding the filament forming flagellin (fliC) . Various strains of Burkholderia spp, strains of closely related genera, and spleen tissue samples of experimentally infected mice were investigated . The combination of PCR and sequencing of the amplicons resulted in high sensitivity and specificity . These procedures may allow rapid, sensitive, and reliable detection of B pseudomallei DNA in routinely formalin fixed and paraffin wax embedded samples, thus providing a safe diagnostic tool and avoiding the cultivation of a risk group 3 agent . In addition, this method could be useful for retrospective histopathological investigations.

Front Biosci, 2003 Jan 01, 8, e55 - 67
Molecular epidemiology of Burkholderia species; Coenye T et al.; Although most species in the genus Burkholderia are not pathogenic for healthy persons, a few are capable of causing severe, life threatening infection . B . mallei and B . pseudomallei are the causative agents of glanders and melioidosis, respectively . Interest in these species has increased recently owing to their potential for use as agents of bioterrorism . B . cepacia emerged during the past two decades as an important opportunistic pathogen among persons with certain underlying diseases . Persons with chronic granulomatous disease, a primary immunodeficiency, or cystic fibrosis (CF), the most common lethal inherited disorder in Caucasians, are at particular risk . In CF, respiratory tract infection may be chronic or associated with a rapid deterioration in pulmonary function . Studies in the early 1990s utilized a variety of genotyping techniques to provide compelling evidence of person-to-person transmission of B . cepacia among CF patients . This prompted the institution of rigorous infection control measures that have placed a heavy burden on persons with CF . More recent work has demonstrated that several distinct bacterial species actually exist among bacteria previously identified merely as B . cepacia . How these species, collectively referred to as the B . cepacia complex, differ with respect to their epidemiology, natural history, and pathology in CF is the subject of ongoing investigation.

Acta Crystallogr D Biol Crystallogr, 2002 Dec, 58(Pt 12), 2184 - 6 Epub 2002 Nov 23.
Crystallization and preliminary X-ray analysis of the catalase-peroxidase KatG from Burkholderia pseudomallei; Carpena X et al.; The bifunctional catalase-peroxidase KatG encoded by the katG gene of Burkholderia pseudomallei has a predicted subunit size of 81.6 kDa . It shows high sequence similarity to other catalase-peroxidases of bacterial, archaebacterial and fungal origin, including 64% identity to KatG from Mycobacterium tuberculosis and lesser sequence similarity to members of the plant peroxidase family . Crystals from this protein were grown in 16-20% PEG 4000, 20% 2-methyl-2,4-pentanediol and 0.1 M sodium citrate pH 5.6 by the hanging-drop vapour-diffusion method at 293 K . These crystals diffracted beyond 1.8 A resolution and belong to space group P2(1)2(1)2(1), with unit-cell parameters a = 100.9, b = 115.6, c = 175.2 A . The data are consistent with either a monomer or a dimer in the crystal asymmetric unit.

Acta Crystallogr D Biol Crystallogr, 2002 Dec, 58(Pt 12), 2182 - 3 Epub 2002 Nov 23.
Crystallization and preliminary X-ray crystallographic studies on the class II cholesterol oxidase from Burkholderia cepacia containing bound flavin; Aunpad R et al.; Burkholderia cepacia cholesterol oxidase (ChoS) is a 58.7 kDa molecular-weight flavoenzyme which has been categorized as a 3beta-hydroxysteroid oxidase converting the 3beta-hydroxyl group of a range of hydroxysteroids to the corresponding ketone . Analysis of enzymes with this activity has shown that two classes of cholesterol oxidase can be defined . Enzymes belonging to class I contain non-covalently bound FAD, whereas the class II enzymes contain FAD covalently bound to an active-site histidine . Despite catalysing the same chemical reaction, the class I and class II enzymes show no sequence similarity and have a different molecular architecture . Crystals of a recombinant class II enzyme from B . cepacia have been grown by the hanging-drop vapour-diffusion method using polyethylene glycol as a precipitating agent . The crystals belong to space group P3(1)21, with unit-cell parameters a = b = 119.6, c = 101.1 A, and have one subunit in the asymmetric unit . These crystals diffract to at least 2.0 A resolution at the Daresbury SRS and are suitable for a full structure determination . Ultimately, analysis of the structure of B . cepacia ChoS may allow the characteristics and structural features which contribute to its suitability as a diagnostic reagent for the detection of cholesterol and unresolved mechanistic features of the class II enzymes to be understood.

J Lipid Res, 2002 Dec, 43(12), 2188 - 95
Ammonium hydroxide hydrolysis: a valuable support in the MALDI-TOF mass spectrometry analysis of Lipid A fatty acid distribution; Silipo A et al.; Lipid A is the lipophilic moiety of lipopolysaccharides (LPSs), the major components of the external membrane of almost all gram-negative bacteria . It is responsible for the toxicity of LPS and has a heterogeneous structure composed of a bis-phosphorylated glucosamine disaccharide backbone that is acylated at the positions 2, 3 of the GlcN I (proximal) and GlcN II (distal) residue with O- and N-linked 3-hydroxy fatty acids (primary substitution) . These fatty acids are further acylated by means of their 3-hydroxy groups (secondary substitution) . The toxicity of Lipid A is dependent on its primary structure; the number, the length, and the distribution of the fatty acids on the disaccharide backbone strongly influence the endotoxic activity . In this paper a general and easy methodology to obtain secondary fatty acid distribution, which is one of the most difficult issues in the structural determination of Lipid A, is proposed . The method combines ammonium hydroxide hydrolysis and matrix assisted laser desorption ionization (MALDI)-mass spectrometry analysis and has been successfully proven with five different Lipid A species . The procedure exploits the lower stability under mild alkaline conditions of acyl and acyloxyacyl esters with respect to that of the acyl and acyloxyacyl amides . The partially degraded Lipid A species obtained are analyzed by MALDI-MS . The generality of this approach was tested on five Lipid As, namely those arising from Escherichia coli, Klebsiella pneumoniae, Klebsiella oxytoca, Pseudomonas reactans, and Burkholderia caryophylli.

J Clin Microbiol, 2002 Dec, 40(12), 4625 - 7
Comparison of automated and nonautomated systems for identification of Burkholderia pseudomallei; Lowe P et al.; The identification of Burkholderia pseudomallei, the causative agent of melioidosis, is usually not difficult in laboratories in areas where it is endemic . With the increase in international travel and the threat of bioterrorism, it has become more likely that laboratories in areas where it is not endemic could encounter this organism . The increase in the use of and dependence upon automated identification systems makes accurate identification of uncommonly encountered organisms such as B . pseudomallei critically important . This study compares the manual API 20NE and 20E identification systems with the automated Vitek 1 and 2 systems . A total of 103 B . pseudomallei isolates were tested and correctly identified in 98%, 99%, 99%, and 19% of cases, respectively . The failure of the Vitek 2 to correctly identify B . pseudomallei was largely due to differences in the biochemical reactions achieved compared to expected values in the database . It is suggested that this deficiency in the Vitek 2 may be due to the large number of uncertain results reported for these isolates . These results reduce the discriminating ability of the instrument to distinguish between uncommonly encountered isolates such as those of B . pseudomallei.

J Appl Microbiol, 2002, 93(6), 930 - 6
Statistical medium optimization and production of a hyperthermostable lipase from Burkholderia cepacia in a bioreactor; Rathi P et al.; AIM: Statistical medium optimization for maximum production of a hyperthermostable lipase from Burkholderia cepacia and its validation in a bioreactor . METHODS AND RESULTS: Burkholderia cepacia was grown in shake flasks containing 1% glucose, 0.1% KH2PO4, 0.5% NH4Cl, 0.24% (NH4)2HPO4, 0.01% MgSO4.7H2O and 1% emulsified palm oil, at 45 degrees C and pH 7.0, agitated at 250 rev min(-1) with 6-h-old inoculum (2% v/v) for 20 h . A fourfold enhancement in lipase production (50 U ml(-1)) and an approximately three fold increase in specific activity (160 U mg(-1)) by B . cepacia was obtained in a 14 litre bioreactor within 15 h after statistical optimization following shake flask culture . The statistical model was obtained using face centred central composite design (FCCCD) with five variables: glucose, palm oil, incubation time, inoculum density and agitation . The model suggested no interactive effect of the five factors, although incubation period, inoculum and carbon concentration were the important variables . CONCLUSIONS: The maximum lipase production was 50 U ml(-1), with specific activity 160 U mg(-1) protein, in a 14 litre bioreactor after 15 h in a medium obtained after statistical optimization in shake flasks . Further, the model predicted reduction in time for lipase production with reduction in total carbon supply . SIGNIFICANCE AND IMPACT OF THE STUDY: Statistical optimization allows quick optimization of a large number of variables . It also provides a deep insight into the regulatory role of various parameters involved in enzyme production.

Appl Environ Microbiol, 2002 Dec, 68(12), 5956 - 64
Construction and evaluation of plasmid vectors optimized for constitutive and regulated gene expression in Burkholderia cepacia complex isolates; Lefebre MD et al.; 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.

Biotechniques, 2002 Nov, 33(5), 1062 - 7
Broad-host-range cre-lox system for antibiotic marker recycling in gram-negative bacteria; Marx CJ et al.; Complete genome sequences are now available for many bacterial species that lack sophisticated genetic tools . We describe the development of a broad-host-range cre-lox system that allows antibiotic marker recycling in a variety of gram-negative bacteria . This system consists of an allelic exchange vector bearing a kanamycin cassette flanked by loxP sites and a tetracycline-resistant IncP plasmid that provides expression of the Cre recombinase . We demonstrate this system by generating unmarked deletions of genes in two different bacteria, Methylobacterium extorquens AM1 and Burkholderia fungorum LB400 . This new antibiotic marker recycling system offers the possibility of creating unmarked mutants in a wide variety of gram-negative bacteria . Furthermore, marker recycling allows the generation of strains bearing multiple genetic manipulations in organisms for which few antibiotic markers are currently available.

Antonie Van Leeuwenhoek, 2002 Aug, 81(1-4), 365 - 71
Arbuscular mycorrhizal fungi: a specialised niche for rhizospheric and endocellular bacteria; Bianciotto V et al.; Arbuscular mycorrhizal (AM) fungi produce an extensive hyphal network which develops in the soil, producing a specialised niche for bacteria . The aim of this paper is to review briefly the interactions shown by these symbiotic fungi with two bacterial groups: (i) the plant-growth promoting rhizobacteria (PGPRs) which are usually associated with fungal surfaces in the rhizosphere, and (ii) a group of endocellular bacteria, previously identified as being related to Burkholderia on the basis of their ribosomal sequence strains . The endobacteria have been found in the cytoplasm of some isolates of AM fungi belonging to Gigasporaceae and offer a rare example of bacteria living in symbiosis with fungi.

J Med Microbiol, 2002 Nov, 51(11), 937 - 40
Evaluation of species-specific recA-based PCR tests for genomovar level identification within the Burkholderia cepacia complex; Vermis K et al.; The Burkholderia cepacia complex presently comprises nine genomovars: B . cepacia (genomovar I), B . multivorans (genomovar II), B . cepacia genomovar III, B . stabilis (genomovar IV), B . vietnamiensis (genomovar V), B . cepacia genomovar VI, B . ambifaria (genomovar VII), B . anthina (genomovar VIII) and B . pyrrocinia (genomovar IX) . Strains of each genomovar can colonise the respiratory tract of cystic fibrosis (CF) patients . However, the majority of infections in CF patients are caused by B . multivorans and B . cepacia genomovar III isolates . Accurate genomovar-level identification is best achieved through a polyphasic approach combining phenotypic and genotypic analyses . In the present study, the sensitivity and specificity of recA-based genomovar specific primer pairs were evaluated with a collection of 508 B . cepacia complex isolates representing all nine genomovars . The assays for the identification of B . multivorans (sensitivity and specificity, 100%), B . cepacia genomovar III (sensitivity, 92%; specificity, 100%), and B . ambifaria (sensitivity and specificity, 100%) were the most efficient . However, the B . cepacia genomovar I assay lacked sensitivity (72%) and cross-reacted with all B . pyrrocinia isolates examined . Several new recA RFLP types were also revealed within the B . cepacia complex . One of these profiles was shared by a clinical and an environmental B . cepacia-like isolate and by the B . ubonensis type strain . The latter organism is a recently described soil bacterium . Its relationship to the various B . cepacia complex genomovars needs further study.

Antimicrob Agents Chemother, 2002 Dec, 46(12), 3776 - 81
Pharmacokinetics and efficacies of liposomal and conventional formulations of tobramycin after intratracheal administration in rats with pulmonary Burkholderia cepacia infection; Marier JF et al.; The objective of the present study was to determine the pharmacokinetics and efficacies of liposomal and conventional formulations of tobramycin against Burkholderia cepacia in a model of chronic lung infection . Male Sprague-Dawley rats were inoculated intratracheally with 10(6) CFU of a very resistant strain of B . cepacia (strain BC 1368; MIC, 128 micro g/ml) to establish lung infection . A 1,200- micro g dose of tobramycin was administered intratracheally as a liposomal formulation and as a conventional formulation . Rats were anesthetized and exsanguinated by cardiac puncture at different times over 24 h to assess pulmonary tobramycin concentrations and the number of residual CFU . Pharmacokinetic parameters were calculated by using a two-compartment model with NONMEM . The mean half-life at the beta phase (t(1/2beta)) and the pulmonary exposure (the area under the concentration-time curve {AUC}) of liposomal tobramycin were 19.7 h (coefficient of variation {CV}, 24.2%) and 6,811 micro g . h/lungs (CV, 19.7%), respectively . The pharmacokinetics of conventional tobramycin were statistically different, with a t(1/2beta) and AUC of 12.9 h (CV, 31.4%) and 821 micro g . h/lungs (CV, 15.0%), respectively . Pearson chi-square analyses were performed on residual CFU data distributed in the following categories: <10(3), 10(3) to 10(5), and >10(5) . Differences in CFU data between formulations showed a statistical trend (P < 0.10) when data from all time points were used, and statistically significant differences were found after 12 h (P < 0.05), with greater eradication achieved with the liposomal formulation . In conclusion, intratracheal administration of tobramycin in liposomes was associated with marked changes in the pharmacokinetics of the drug in the lung and an apparent trend for a prolonged efficacy against B . cepacia . These results support the hypothesis that inhalation of liposomal tobramycin may improve the management of chronic pulmonary infections caused by resistant bacteria in patients with cystic fibrosis.

J Heart Lung Transplant, 2002 Nov, 21(11), 1230 - 1
Activity of disinfectants against Gram-negative bacilli isolated from patients undergoing lung transplantation for cystic fibrosis; Perry JD et al.; Lung transplant recipients with cystic fibrosis are frequently colonized with antibiotic-resistant bacteria . We evaluated the in vitro activity of 5 disinfectants frequently used in cardiac surgery against strains of Burkholderia cepacia and Pseudomonas aeruginosa isolated from patients undergoing sequential single lung transplantation . Our results suggest that the activity of Taurolin and Noxyflex is superior to conventional disinfectants.

Microbiology, 2002 Nov, 148(Pt 11), 3477 - 84
Lack of cable pili expression by cblA-containing Burkholderia cepacia complex; Sajjan U et al.; The Burkholderia cepacia complex consists of several closely related bacterial species (or genomovars) which although generally not pathogenic for healthy individuals, contribute significantly to morbidity and mortality among persons with cystic fibrosis (CF) . Certain B . cepacia complex strains are more frequently recovered from CF sputum cultures than are others, and these typically reside in genomovar III . The ET12 clone is a genomovar III strain that predominates among CF patients in Canada and the United Kingdom and is characterized by distinctive cblA-encoded pili that have a cable-like morphology . In a previous survey of B . cepacia complex isolates recovered from 606 CF patients in the US, a single genomovar III ET12 isolate (isolate AU0007) was identified; several cblA-containing genomovar I isolates, however, were also detected . In the study reported here, analysis by PFGE revealed several distinct strain types among these genomovar I isolates, and sequence analysis of their cblA genes demonstrated 87.8-88.4% identity to the ET12 cblA sequence . Southern analysis indicated that the cblA variant from each genomovar I isolate resides on a 4 kbp EcoRI fragment, in contrast to ET12 isolates, in which cblA localizes to a 5 kbp EcoRI fragment . Western blot assay indicated expression of the 16 kDa major pilin subunit by ET12 isolates, including AU0007, but neither whole-cell nor surface-protein extracts of the genomovar I reacted . Electron microscopy revealed the complete absence of pili expression by the genomovar I isolates . In contrast to typical ET12 isolates, AU0007 appeared to be hyperpiliated with rigid pili that lacked the cable morphology and did not bind cytokeratin 13, which has been previously identified as the epithelial cell receptor for the ET12 cable-pili-associated adhesin.

J Biol Chem, 2003 Jan 17, 278(3), 1647 - 55 Epub 2002 Nov 08.
Construction of a deep-rough mutant of Burkholderia cepacia ATCC 25416 and characterization of its chemical and biological properties; Gronow S et al.; Burkholderia cepacia is a bacterium with increasing importance as a pathogen in patients with cystic fibrosis . The deep-rough mutant Ko2b was generated from B . cepacia type strain ATCC 25416 by insertion of a kanamycin resistance cassette into the gene waaC encoding heptosyltransferase I . Mass spectrometric analysis of the de-O-acylated lipopolysaccharide (LPS) of the mutant showed that it consisted of a bisphosphorylated glucosamine backbone with two 3-hydroxyhexadecanoic acids in amide-linkage, 4-amino-4-deoxyarabinose (Ara4N) residues on both phosphates, and a core oligosaccharide of the sequence Ara4N-(1 --> 8) D-glycero-D-talo-oct-2-ulosonic acid (Ko)-(2 --> 4)3-deoxy-D-manno-oct-2-ulosonic acid (Kdo) . The mutant allowed investigations on the biosynthesis of the LPS as well as on its role in human infection . Mutant Ko2b showed no difference in its ability to invade human macrophages as compared with the wild type . Furthermore, isolated LPS of both strains induced the production of tumor necrosis factor alpha from macrophages to the same extent . Thus, the truncation of the LPS did not decrease the biological activity of the mutant or its LPS in these aspects.

FEMS Immunol Med Microbiol, 2002 Nov 15, 34(3), 231 - 6
A possible pitfall in the identification of Burkholderia mallei using molecular identification systems based on the sequence of the flagellin fliC gene; Sprague LD et al.; Amotile Burkholderia mallei and motile Burkholderia pseudomallei display a high similarity with regard to phenotype and clinical syndromes, glanders and melioidosis . The aim of this study was to establish a fast and reliable molecular method for identification and differentiation . Despite amotility, the gene of the filament forming flagellin (fliC) could be completely sequenced in two B . mallei strains . Only one mutation was identified discriminating between B . mallei and B . pseudomallei . A polymerase chain reaction-restriction fragment length polymorphism assay was designed making use of the absence of an AvaII recognition site in B . mallei . All seven B . mallei, 12 out of 15 B . pseudomallei and 36 closely related apathogenic Burkholderia thailandensis strains were identified correctly . However, in three B . pseudomallei strains a point mutation at gene position 798 (G to C) disrupted the AvaII site . Therefore, molecular systems based on the fliC sequence can be used for a reliable proof of strains of the three species but not for the differentiation of B . mallei and B . pseudomallei isolates.

FEMS Immunol Med Microbiol, 2002 Nov 15, 34(3), 173 - 9
The partially degraded lipopolysaccharide of Burkholderia cepacia and ornithine-containing lipids derived from some Gram-negative bacteria are useful complex lipid adjuvants; Kawai Y et al.; The partially degraded lipopolysaccharide of Burkholderia cepacia (LPSdegr) and the ornithine-containing lipids were purified from some bacteria . The substances were developed as complex lipid adjuvants, because they have weak toxicity and are able to activate the immune systems of the living body . After various toxoid antigens such as pertussis toxoid, diphtheria toxoid and tetanus toxoid were mixed with the complex lipid adjuvants, the mixtures were administered to mice subcutaneously . Antitoxoid IgG antibody titers in the serum were measured several times over 3 months . The efficacy of the LPSdegr as adjuvant was almost as high as that of the ornithine-containing lipids, and it was almost equal to that of the aluminum hydroxide adjuvant (Alum), which is generally used as a vaccine adjuvant.

Syst Appl Microbiol, 2002 Oct, 25(3), 376 - 85
Evaluation of tRNA intergenic length polymorphism (tDNA-pCR) for the differentiation of the members of the Burkholderia cepacia complex; Storms V et al.; Nowadays, tentative identification of B . cepacia complex bacteria in routine diagnostic laboratories is based on a combination of selective media, conventional biochemical reactions, commercial test systems and PCR-based assays . Some of these assays have the capacity to discriminate reliably among several members of the B . cepacia complex, however one single method differentiating all B . cepacia-like organisms is not available . In this study, the applicability of tDNA-PCR for the differentiation and rapid identification of the different members of the B . cepacia complex was evaluated . For B . gladioli and most of the B . cepacia genomovars, differentiable patterns were obtained . For some of the members of the B . cepacia complex however, the tDNA-PCR patterns were very similar and sometimes multiple patterns existed within in a single genomovar . No distinction could be made between the tDNA-PCR patterns of B . vietnamiensis and B . pyrrocinia and of B . cepacia genomovars I and VIII respectively . We could conclude that, although tDNA-PCR is not sufficient as a single method to identify all the members of the B . cepacia complex unambiguously or to replace the currently used methods, it is a very fast and easily applicable method that could be a very useful tool for the differentiation and identification of B . cepacia-like organisms.

Syst Appl Microbiol, 2002 Oct, 25(3), 340 - 52
Diversity of transconjugants that acquired plasmid pJP4 or pEMT1 after inoculation of a donor strain in the A- and B-horizon of an agricultural soil and description of Burkholderia hospita sp . nov . and Burkholderia terricola sp . nov; Goris J et al.; We examined the diversity of transconjugants that acquired the catabolic plasmids pJP4 or pEMT1, which encode degradation of 2,4-dichlorophenoxyacetic acid (2,4-D), in microcosms with agricultural soil inoculated with a donor strain (Dejonghe, W., Goris, J., El Fantroussi, S., Hofte, M., De Vos, P., Verstraete, W., and Top, E . M . Appl . Environ . Microbiol . 2000, p . 3297-3304) . Using repetitive element PCR fingerprinting, eight different rep-clusters and six separate isolates could be discriminated among 95 transconjugants tested . Representative isolates were identified using 16S rDNA sequencing, cellular fatty acid analysis, whole-cell protein analysis and/or DNA-DNA hybridisations . Plasmids pJP4 and pEMT1 appeared to have a similar transfer and expression range, and were preferably acquired and expressed in soil by indigenous representatives of Ralstonia and Burkholderia . Two rep-clusters were shown to represent novel Burkholderia species, for which the names Burkholderia hospita sp . nov . and Burkholderia terricola sp . nov . are proposed . When easily degradable carbon sources were added together with the plasmid-bearing donor strain, also a significant proportion of Stenotrophomonas maltophilia isolates were found . The transconjugant collections isolated from A- (0-30 cm depth) and B-horizon (30-60 cm depth) soil were similar, except for B . terricola transconjugants, which were only isolated from the B-horizon.

Anal Biochem, 2002 Sep 15, 308(2), 294 - 9
A nonradioactive method for the assay of polyphosphate kinase activity and its application in the study of polyphosphate metabolism in Burkholderia cepacia; Mullan A et al.; Studies of polyphosphate (polyP) metabolism in microorganisms have been hampered by the lack of a convenient method for the assay in cell extracts of the activity of polyphosphate kinase (PPK), the enzyme principally responsible for microbial polyP biosynthesis . We report the development of such an assay, based on the well-established metachromatic reaction, with toluidine blue, of the polyP formed during the PPK-catalyzed reaction . The method was successfully used in the characterization of PPK activity in crude extracts of an environmental Burkholderia cepacia isolate . The development of a protocol for the physical recovery of polyP from solution is also reported.

Mol Microbiol, 2002 Nov, 46(3), 661 - 76
A novel class of microbial phosphocholine-specific phospholipases C; Stonehouse MJ et al.; In this report we describe the 1,500-fold purification and characterization of the haemolytic phospholipase C (PLC) of Pseudomonas aeruginosa, the paradigm member of a novel PLC/phosphatase superfamily . Members include proteins from Mycobacterium tuberculosis, Bordetella spp., Francisella tularensis and Burkholderia pseudomallei . Purification involved overexpression of the plcHR1,2 operon, ion exchange chromatography and native preparative polyacrylamide gel electrophoresis . Matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometry confirmed the presence of two proteins in the purified sample with sizes of 17,117.2 Da (PlcR2) and 78,417 Da (PlcH) . Additionally, liquid chromatography electrospray mass spectrometry (LCMS) revealed that PlcH and PlcR2 are at a stoichiometry of 1 : 1 . Western blot analysis demonstrated that the enzyme purifies as a heterodimeric complex, PlcHR2 . PlcHR2 is only active on choline-containing phospholipids . It is equally active on phosphatidylcholine (PC) and sphingomyelin (SM) and is able to hydrolyse plasmenylcholine phospholipids (plasmalogens) . Neither PlcHR2 nor the M . tuberculosis homologues are inhibited by D609 a widely used, competitive inhibitor of the Bacillus cereus PLC . PlcH, PlcR2, and the PlcHR2 complex bind calcium . While calcium has no detectable effect on enzymatic activity, it inhibits the haemolytic activity of PlcHR2 . In addition to being required for the secretion of PlcH, the chaperone PlcR2 affects both the enzymatic and haemolytic properties of PlcH . Inclusive in these data is the conclusion that the members of this PC-PLC and phosphatase family possess a novel mechanism for the recognition and hydrolysis of their respective substrates.

Mol Microbiol, 2002 Nov, 46(3), 649 - 59
An Inv/Mxi-Spa-like type III protein secretion system in Burkholderia pseudomallei modulates intracellular behaviour of the pathogen; Stevens MP et al.; Burkholderia pseudomallei is the causative agent of melioidosis, a serious infectious disease of humans and animals that is endemic in subtropical areas . B . pseudomallei is a facultative intracellular pathogen that may invade and survive within eukaryotic cells for prolonged periods . After internalization, the bacteria escape from endocytic vacuoles into the cytoplasm of infected cells and form membrane protrusions by inducing actin polymerization at one pole . It is believed that survival within phagocytic cells and cell-to-cell spread via actin protrusions is required for full virulence . We have studied the role of a putative type III protein secretion apparatus (Bsa) in the interaction between B . pseudomallei and host cells . The Bsa system is very similar to the Inv/Mxi-Spa type III secretion systems of Salmonella and Shigella . Moreover, B . pseudomallei encodes proteins that are very similar to Salmonella and Shigella Inv/Mxi-Spa secreted proteins required for invasion, escape from endocytic vacuoles, intercellular spread and pathogenesis . Antibodies to putative Bsa-secreted proteins were detected in convalescent serum from a melioidosis patient, suggesting that the system is functionally expressed in vivo . B . pseudomallei mutant strains lacking components of the Bsa secretion and translocation apparatus were constructed . The mutant strains exhibited reduced replication in J774.2 murine macrophage-like cells, an inability to escape from endocytic vacuoles and a complete absence of formation of membrane protrusions and actin tails . These findings indicate that the Bsa type III secretion system plays an essential role in modulating the intracellular behaviour of B . pseudomallei.

J Antimicrob Chemother, 2002 Nov, 50(5), 723 - 6
Characterization of a laboratory-generated variant of BPS beta-lactamase from Burkholderia pseudomallei that hydrolyses ceftazidime; Ho PL et al.; Burkholderia pseudomallei produces an Ambler class A beta-lactamase, known as BPS-1 . The beta-lactamase gene from a laboratory-derived, ceftazidime-resistant strain of B . pseudomallei (LH-1-2) was cloned and expressed in Escherichia coli . The beta-lactamase, named BPS-1m, had an identical isoelectric focusing point (pI 7.7) to that of BPS-1, but differed in having a stronger hydrolytic activity against ceftazidime . Susceptibility testing showed that BPS-1m when expressed in E . coli conferred resistance to ceftazidime (MIC >or= 32 mg/L) . The amino acid sequence of BPS-1m differed from that of BPS-1 by a Pro-to-Ser change at position 167 in the omega loop.

Thorax, 2002 Nov, 57(11), 924 - 5
Increased treatment requirements of patients with cystic fibrosis who harbour a highly transmissible strain of Pseudomonas aeruginosa; Jones AM et al.; BACKGROUND: A group of patients who harbour the same highly transmissible strain of Pseudomonas aeruginosa were identified at a cystic fibrosis (CF) centre . Isolates of this strain display a number of unusual phenotypic features including resistance to most typical antipseudomonal antibiotics . A study was undertaken to see if there was a difference in treatment requirements between CF patients with chronic infection with their own unique P aeruginosa strains (group 1) and those who harbour a highly transmissible strain (group 2) . METHODS: Data on treatment requirements for the year 2000 were collected from the case records of CF patients with chronic P aeruginosa infection who had received inpatient treatment . Patients co-infected with Burkholderia cepacia or other highly transmissible strains of P aeruginosa were excluded . RESULTS: There were 2/56 and 3/22 deaths in groups 1 and 2, respectively; these patients were excluded from the analysis . No difference was found between the two groups for mean age, % predicted forced expiratory volume in 1 second (FEV(1)), % predicted forced vital capacity (FVC), and body mass index . Patients in group 2 had a greater median (range) number of intravenous antibiotic days (60 (17-216) v 33 (4-237) days; p=0.01), inpatient days (39 (7-183) v 16 (1-172) days; p<0.01), and inpatient episodes (3 (1-9) v 2 (1-6); p<0.01), and more respiratory exacerbations (mean (SD) 8.2 (3.4) v 6.1 (3.2); p=0.01) . CONCLUSIONS: Patients who harbour the highly transmissible P aeruginosa strain have a greater treatment burden than patients with CF who harbour their own unique strains . These findings support the need for microbiological surveillance for highly transmissible P aeruginosa and the implementation of infection control measures to prevent cross infection.

Curr Microbiol, 2002 Dec, 45(6), 415 - 22
Physiological and cellular responses of the 2,4-D degrading bacterium, Burkholderia cepacia YK-2, to the phenoxyherbicides 2,4-D and 2,4,5-T; Cho YS et al.; Our previous research has demonstrated that novel 43-kDa DnaK and 41-kDa GroEL proteins are synthesized in Burkholderia sp . YK-2 in response to sublethal concentrations of 2,4-D stress {Cho et al . (2000) Curr Microbiol 41:33-38} . In this study, we have extended this work to examine the cellular responses of strain YK-2 to stresses induced in response to the phenoxyherbicides 2,4-D or 2,4,5-T . Strain YK-2 exhibited a more sensitive response to 2,4,5-T stress than to 2,4-D stress, as shown in physiological and morphological changes, suggesting a greater cytotoxic effect of 2,4,5-T . SEM analyses revealed the presence of perforations and irregular rod forms with wrinkled surfaces for cells treated with either herbicide . These irregularities were found more frequently for 2,4,5-T-treated cells than for 2,4-D-treated cells . Analysis of cellular fatty acids showed similar effects in the shifts of total cellular fatty acid composition in response to 2,4-D and 2,4,5-T . Strain YK-2 could degrade 2.25 m M 2,4-D completely during 28 h of incubation with transient production of 2,4-dichlorophenol as a metabolite; however, 2,4,5-T was not catabolized at any of the concentrations tested . BIOLOG and 16S rDNA analyses revealed that strain YK-2 was 98% similar to the Burkholderia cepacia species cluster; therefore, we have designated this strain as B . cepacia YK-2.

Biosci Biotechnol Biochem, 2002 Sep, 66(9), 1792 - 8
Isolation of bacterial strains that produce the endocrine disruptor, octylphenol diethoxylates, in paddy fields; Nishio E et al.; Topsoil samples were collected from 36 different paddy fields in West Japan . Each soil sample was incubated with a basal salt-medium containing 0.2% OPPEO . Twelve samples possessed OPPEO-degrading activity, from which twelve cultures of OPPEO-degrading bacteria were isolated . The isolated bacteria grew on a medium containing 0.2% OPPEO as the sole carbon source, and OP2EO and OP3EO were accumulated in the medium under aerobic conditions . OP1EO and octylphenol, which have often been identified in surface water together with OP2EO, were not observed in this experiment . The bacterial isolates were gram negative and tentatively identified as Pseudomonas putida (10 isolates) and Burkholderia cepacia (one isolate) by BIOLOG and 16S rDNA RFLP analyses.

FEMS Microbiol Lett, 2002 Oct 8, 215(2), 279 - 83
Cloning and characterization of the Burkholderia vietnamiensis norM gene encoding a multi-drug efflux protein; Fehlner-Gardiner CC et al.; Polymyxin B-sensitive mutants in Burkholderia vietnamiensis (Burkholderia cepacia genomovar V) were generated with a mini-Tn5 encoding tetracycline resistance . One of the transposon mutants had an insertion in the norM gene encoding a multi-drug efflux protein . Expression of B . vietnamiensis norM in an Escherichia coli acrAB deletion mutant complemented its norfloxacin hypersensitivity, indicating that the protein functions in drug efflux . However, no effect on antibiotic sensitivity other than sensitivity to polymyxin B was observed in the B . vietnamiensis norM mutant . We demonstrate that increased polymyxin sensitivity in B . vietnamiensis was associated with the presence of tetracycline in the growth medium, a phenotype that was partially suppressed by expression of the norM gene.

Int J Environ Health Res, 2002 Jun, 12(2), 133 - 44
Antibiotic resistance in soil and water environments; Esiobu N et al.; Seven locations were screened for antibiotic-resistant bacteria using a modified agar dilution technique . Isolates resistant to high levels of antibiotics were screened for r plasmids . Low-level resistance (25 micro g x ml(-1)) was widespread for ampicillin, penicillin, tetracycline, vancomycin and streptomycin but not for kanamycin . Resistant populations dropped sharply at high antibiotic levels, suggesting that intrinsic non-emergent mechanisms were responsible for the multiple drug resistance exhibited at low doses . Dairy farm manure contained significantly (P < 0.01) more (%) resistant bacteria than the other sites . Bacteria isolated from a dairy water canal, a lake by a hospital and a residential garden (fertilized by farm manure) displayed resistance frequencies of 77, 75 and 70%, respectively . Incidence of tetracycline resistance was most prevalent at 47-89% of total bacteria . Out of 200 representative isolates analyzed, Pseudomonas, Enterococcus-like bacteria, Enterobacter and Burkholderia species constituted the dominant reservoirs of resistance at high drug levels (50-170 micro g x ml(-1)) . Plasmids were detected in only 29% (58) of these bacteria with tetracycline resistance accounting for 65% of the plasmid pool . Overall, resistance trends correlated to the abundance and type of bacterial species present in the habitat . Environmental reservoirs of resistance include opportunistic pathogens and constitute some public health concern.

Proc R Soc Lond B Biol Sci, 2002 Oct 7, 269(1504), 2023 - 7
Tetraponera ants have gut symbionts related to nitrogen-fixing root-nodule bacteria; van Borm S et al.; Some Tetraponera ants (Formicidae, Pseudomyrmecinae) subsist almost entirely on amino acid deficient honeydew secretions of pseudococcids and harbour a dense aggregation of bacterial symbionts in a unique pouch-shaped organ at the junction of the midgut and the intestine . The organ is surrounded by a network of intruding tracheae and Malpighian tubules, suggesting that these bacteria are involved in the oxidative recycling of nitrogen-rich metabolic waste . We have examined the ultrastructure of these bacteria and have amplified, cloned and sequenced ribosomal RNA-encoding genes, showing that the ant pouch contains a series of close relatives of Flavobacteria and Rhizobium, Methylobacterium, Burkholderia and Pseudomonas nitrogen-fixing root-nodule bacteria . We argue that pouch bacteria have been repeatedly 'domesticated' by the ants as nitrogen-recycling endosymbionts . This ant-associated community of mutualists is, to our knowledge, the first finding of symbionts related to root-nodule bacteria in animals.

Curr Opin Pulm Med, 2002 Nov, 8(6), 535 - 41
Selection of patients with cystic fibrosis for lung transplantation; Liou TG et al.; Lung transplantation is the most aggressive therapy available for end-stage lung disease from cystic fibrosis (CF) . A new predictive survival model of CF uses demographic, FEV1, nutritional, microbiologic, and acute exacerbation data to produce precise estimates of 5-year survival . The model improves the ability to select patients most likely to have survival benefit from transplantation . We discuss potential application of the survival model to four distinct groups of patients with CF: (1) candidates for cadaveric transplantation, (2) potential living donor recipients, (3) patients infected with multiply-resistant organisms such as Burkholderia cepacia, and (4) patients critically ill and dependent on mechanical ventilation . Measuring the impact of transplantation on quality of life remains a difficult task, and further studies are needed to determine whether lung-transplantation-derived survival benefit implies quality-of-life benefit . However, judicious use of the survival model to select patients for transplantation is likely to improve survival outcomes .

Can J Microbiol, 2002 Aug, 48(8), 707 - 16
Interspecies communication between Burkholderia cepacia and Pseudomonas aeruginosa; Lewenza S et al.; Burkholderia cepacia and Pseudomonas aeruginosa are opportunistic pathogens that commonly cause pulmonary infections in cystic fibrosis patients and occasionally co-infect patients' lungs . Both organisms possess quorum-sensing systems dependent on N-acyl homoserine lactone (N-acyl-HSL) . Cross-feeding assays demonstrated that P . aeruginosa and B . cepacia were able to utilize heterologous N-acyl-HSL signaling molecules . The ability of quorum-sensing genes from one species to complement the respective quorum-sensing mutations in the heterologous species was also examined . These studies suggest that B . cepacia CepR can use N-acyl-HSLs synthesized by RhlI and LasI and that P . aeruginosa LasR and RhlR can use N-acyl-HSLs synthesized by CepI . It is possible that a mixed bacterial population of B . cepacia and P . aeruginosa can coordinately regulate some of their virulence factors and influence the progression of lung disease due to infection with these organisms.

J Pediatr, 2002 Oct, 141(4), 512 - 7
Risk factors for Burkholderia cepacia complex colonization and infection among patients with cystic fibrosis; Walsh NM et al.; OBJECTIVES: To determine risk factors for acquiring Burkholderia cepacia complex among patients with cystic fibrosis (CF) . STUDY DESIGN: A case-control study was conducted with active surveillance for B cepacia complex colonization/infection among patients at 21 CF centers from April 1986 to March 1989 (study period) . A case-patient was defined as any CF patient with B cepacia complex colonization for the first time during the study period . Control patients were patients with CF not B cepacia complex colonized during the study period . For each patient, a questionnaire was completed semiannually . RESULTS: In multivariate analyses, hospitalization for pulmonary exacerbations, living with a B cepacia complex-positive person, attending a CF summer camp, and direct contact with a B cepacia complex-colonized CF person outside of camp and home were associated with B cepacia complex acquisition . Receiving antimicrobial aerosol therapy or cleaning and drying a home-used nebulizer between uses were associated with a decrease in B cepacia complex acquisition . CONCLUSIONS: Numerous factors inside and outside the health care setting are associated with person-to-person transmission of B cepacia complex among patients with CF . Prevention programs should reduce direct or indirect contact between noncolonized and B cepacia complex-colonized/infected patients with CF.

Paediatr Respir Rev, 2002 Sep, 3(3), 230 - 5
Advances in Burkholderia cepacia complex; Speert DP; Burkholderia cepacia is an important opportunistic pathogen in certain compromised hosts, particularly those with either cystic fibrosis (CF) or chronic granulomatous disease . The "family" of bacteria known as B . cepacia is highly heterogeneous and is composed of at least nine discrete species or genomovars, constituting the B . cepacia complex . Bacteria from the B . cepacia complex are particularly virulent in susceptible hosts, often causing necrotising invasive infection and death . Whereas the microbial determinants of virulence in B . cepacia complex are currently not defined, the bacteria appear to have features facilitating survival within host cells . Burkholderia cepacia is highly resistant to antibiotics and to neutrophil-mediated non-oxidative killing; infection should be treated with combination antimicrobial therapy . Burkholderia cepacia can spread from one CF patient to another . Transmission appears to be facilitated by close personal contact and by certain bacterial factors .

Environ Toxicol Chem, 2002 Oct, 21(10), 2075 - 9
Effect of pH on cadmium toxicity, speciation, and accumulation during naphthalene biodegradation; Sandrint TR et al.; Lowering pH of a microbiological medium from 7 to 4 decreased cadmium toxicity during naphthalene biodegradation by a Burkholderia sp . Cadmium speciation and cadmium accumulation in the system were studied to explain this effect . Cadmium speciation was determined by direct measurement and by geochemical modeling . Previous studies have implicated the monovalent hydroxylated cadmium (CdOH+) species in the effect of pH on cadmium toxicity . Modeling analysis predicted CdOH+ formation only at very low concentrations (< or = 0.0128 microM), while the measured concentration of divalent ionic cadmium (Cd2+) was at least three orders of magnitude greater, suggesting that Cd2+ is the more significant metal form . With respect to cadmium accumulation, cells contained in media adjusted to pH 4 accumulated only 2.76 +/- 0.76 mg Cd/g cells, whereas cells in media adjusted to pH 7 accumulated 8.52 +/- 0.71 mg Cd/g cells . These data suggest that cadmium toxicity is correlated with increased cadmium accumulation rather than the formation of CdOH as pH is increased . At low pH, the decrease in cadmium accumulation may be caused by increased competition between hydrogen and cadmium ions for binding sites on the cell surface or by an increase in metal efflux pump activity due to an increase in the proton gradient that drives the efflux pump.

J Antimicrob Chemother, 2002 Oct, 50(4), 445 - 55
Cloning of the class D beta-lactamase gene from Burkholderia pseudomallei and studies on its expression in ceftazidime-susceptible and -resistant strains; Niumsup P et al.; Ceftazidime is the antibiotic of choice for the treatment of melioidosis . Ceftazidime-resistant Burkholderia pseudomallei have been identified and beta-lactamase production implicated in resistance . In this study, 25 strains of B . pseudomallei (15 clinical and 10 environmental strains) were examined for their ability to yield mutants that overexpress beta-lactamase . Ceftazidime-resistant mutants were selected readily at high frequency and displayed four- to eight-fold increases in the MICs of ceftazidime . beta-Lactamase activities in both parent and mutant B . pseudomallei strains were examined by a spectrophotometric method . Twelve mutants (48%) showed approximately two- to 31-fold higher ceftazidimase activity compared with their parent strains and 10 (40%) demonstrated more than two-fold increases in imipenemase activity . A class D beta-lactamase gene from B . pseudomallei was cloned and sequenced . The encoded enzyme is an oxacillinase and is homologous to oxacillinases from Ralstonia pickettii and members of the genus Aeromonas . Reverse transcriptase PCR showed that transcription of the class D beta-lactamase gene is increased in ceftazidime-resistant mutants.

Chembiochem, 2002 Jun 3, 3(6), 566 - 71
Factors governing the activity of lyophilised and immobilised lipase preparations in organic solvents; Persson M et al.; Active site titration and activity measurements were performed in hexane on lyophilised lipase preparations containing different amounts of phosphate buffer and lipase immobilised on porous polypropylene . Lyophilisation of Thermomyces lanuginosus lipase with large quantities of phosphate salts (200 mM) increased the specific activity fourfold, and the number of rapidly titratable active sites increased to 50 % from the 13 % observed when smaller amounts of phosphate buffer were used (20 mM) during lyophilisation . The phosphate buffer worked as an immobilisation matrix for the lipase, and the increase in specific activity was at least partly due to decreased mass transfer limitations . When lipase was immobilised on porous polypropylene, the specific activity was 770 times higher than that of the best freeze-dried preparation . At optimal enzyme loading, 93 % of the enzyme molecules were titrated at a high rate; this indicates that this adsorption on a hydrophobic surface was a very efficient means of reducing mass transfer limitations and of immobilising the enzyme in its active conformation for use in organic solvents . The variation in specific activity with water activity was found to correlate very well with the variation in titratable active sites when lipases from Burkholderia cepacia and Thermomyces lanuginosus were immobilised on porous polypropylene . The catalytic activity per competent active site was thus constant over the whole range of water activities.

Eur J Cardiothorac Surg, 2002 Oct, 22(4), 602 - 9
Long term results of lung transplantation for cystic fibrosis; Egan TM et al.; OBJECTIVES: We reviewed our experience with lung transplant for cystic fibrosis (CF) over a 10-year period to identify factors influencing long-term survival . METHODS: One hundred and twenty-three patients with CF have undergone 131 lung transplant procedures at our institution; 114 have had bilateral sequential lung transplants (DLTX) and nine have had bilateral lower lobe transplants from living donors . Three patients had retransplant for acute graft failure, and five had late retransplant for bronchiolitis obliterans syndrome (BOS) . Kaplan-Meier survival was calculated for the entire cohort and for subsets at higher risk of death to determine factors predicting a better outcome . RESULTS: Actuarial survival for the entire group of DLTX CF patients was 81% at 1 year, 59% at 5 years, and 38% at 10 years . Lobar transplant was associated with a poorer survival (37.5% at 1 and 5 years) . Among DLTX patients, colonization with Burkholderia cepacia was present in 22 patients and was associated with poorer outcome (1- and 5-year survival 60 and 36% in B . cepacia patients vs . 86 and 64% in non-cepacia patients) . DLTX patients younger than age 20 (n=22) had a similar survival to patients age 20 or older (n=90) . Being on a ventilator at the time of transplant was not associated with poorer survival (n=8) . BOS affects increasing numbers of survivors with time . Five CF patients have been retransplanted due to BOS with one operative death and 1-year survival of 60% . CONCLUSIONS: DLTX has acceptable long term survival in CF adults and children with end stage disease . CF patients colonized with B . cepacia have a worse outcome but transplantation is still warranted.

J Bacteriol, 2002 Oct, 184(20), 5714 - 22
Differential expression of two catechol 1,2-dioxygenases in Burkholderia sp . strain TH2; Suzuki K et al.; Burkholderia sp . strain TH2, a 2-chlorobenzoate (2CB)-degrading bacterium, metabolizes benzoate (BA) and 2CB via catechol . Two different gene clusters for the catechol ortho-cleavage pathway (cat1 and cat2) were cloned from TH2 and analyzed . Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blot analysis showed that while both catechol dioxygenases (CatA1 and CatA2) were produced in BA-grown cells, CatA1 was undetectable when strain TH2 was grown on 2CB or cis,cis-muconate (CCM), an intermediate of catechol degradation . However, production of CatA1 during growth on 2CB or CCM was observed when cat2 genes were disrupted . The difference in the production of CatA1 and CatA2 was apparently due to a difference in inducer recognition by the regulators of the gene clusters . The inducer of CatA1 was found to be BA, not 2CB, by using a 2-halobenzoate dioxygenase gene (cbd) disruptant, which is incapable of transforming (chloro)benzoate . It was also found that CCM or its metabolite acts as an inducer for CatA2 . When cat2 genes were disrupted, the growth rate in 2CB culture was reduced while that in BA culture was not . These results suggest that although cat2 genes are not indispensable for growth of TH2 on 2CB, they are advantageous.

Southeast Asian J Trop Med Public Health, 2002 Jun, 33(2), 355 - 61
Clinical features of community-acquired pneumonia treated at Srinagarind Hospital, Khon Kaen, Thailand; Reechaipichitkul W et al.; Pneumonia is a serious illness associated with significant morbidity and mortality . The interpretation guidelines for pneumonia management requires knowledge of both the clinical presentation of the disease and local epidemiology . We studied the clinical features, initial laboratory results, antibiotic sensitivities, and outcomes of patients diagnosed with acute community-acquired pneumonia between January 1999 and December 2000 at Srinagarind Hospital . The causative organisms were identified in only 52.2% patients; Streptococcus pneumoniae accounted for 23.1% of infections . Other common causes included Klebsiellapneumoniae (19.2%), Burkholderia pseudomallei (15.4%), Hemophilus influenzae (11.5%), Mycoplasma pneumoniae (6.2%), and Staphylococcus aureus (4.6%) . Younger patients were more likely to be infected with M . pneumoniae, while the mean age of those with other types of infections was 50 . Healthy adults were infected with M . pneumoniae and S . pneumoniae; specific pathogens attacked patients with certain co-morbidity : i) diabetes mellitus and ageing, ii) diabetes mellitus and renal disease, iii) cardiovascular diseases, and iv) connective tissue diseases and steroid-use; these patients were vulnerable to i) K . pneumoniae, ii) B . pseudomallei, iii) H . influenzae, and iv) S . aureus respectively . White blood cell counts were normal in M . pneumoniae infection . Gram-stained sputum had some limitations, especially when determining Gram-negative infections; chest x-rays could not differentiate pathogens . Bactermia was found in one half of patients infected with B . pseudomallei and S . aureus . Antibiotic-resistant organisms were not common in our study . Because morbidity and mortality were high among patients infected with S . aureus and B . pseudomallei, empirical antibiotic treatment should be considered in suspected cases, especially when patients present with acute severe community-acquired pneumonia.

Antimicrob Agents Chemother, 2002 Oct, 46(10), 3215 - 22
Biochemical-genetic analysis and distribution of DES-1, an Ambler class A extended-spectrum beta-lactamase from Desulfovibrio desulfuricans; Morin AS et al.; Desulfovibrio spp . are gram-negative anaerobes phylogenetically related to Bacteroides spp., which are rarely isolated and which are mostly isolated from intra-abdominal abscesses . Desulfovibrio desulfuricans clinical isolate D3 had a clavulanic acid-inhibited beta-lactam resistance profile and was resistant to some expanded-spectrum cephalosporins . A beta-lactamase gene, bla(DES-1), was cloned from whole-cell DNA of isolate D3 and expressed in Escherichia coli . Purified beta-lactamase DES-1, with a pI value of 9.1, had a relative molecular mass of ca . 31 kDa and a mature protein of 288 amino acids . DES-1 was distantly related to Ambler class A beta-lactamases and most closely related to PenA from Burkholderia pseudomallei (48% amino acid identity) . It was weakly related to class A beta-lactamases CblA, CepA, CfxA, and CfxA2 from other anaerobic species, Bacteroides spp . and Prevotella intermedia . Its hydrolysis spectrum included amino- and ureidopenicillins, narrow-spectrum cephalosporins, ceftriaxone, and cefoperazone . bla(DES-1)-like genes were not identified in phylogenetically related Desulfovibrio fairfieldensis isolates . However, they were found in some but not all D . desulfuricans strains, thus suggesting that these genes may be present in a given D . desulfuricans subspecies.

J Appl Microbiol, 2002, 93(4), 616 - 30
Diversity of Burkholderia isolates from woodland rhizosphere environments; Richardson J et al.; AIMS: Determination of genetic diversity among UK Burkholderia cepacia isolates from various environmental niches, principally woodland tree rhizospheres and onions . METHODS AND RESULTS: Genus determination was made using polymerase chain reaction (PCR) amplification and fatty acid methyl ester profiling . Genetic diversity was investigated by repetitive sequence genetic PCR fingerprinting . Several onion isolates were similar to clinical isolates but others were diverse . Some environmental isolates were possibly synonymous with B . cepacia and B . gladioli but most from woodland rhizospheres were distinct and clustered together . The 16S rRNA genes of representatives from these clusters were PCR amplified, sequenced and phylogenetically compared with all known Burkholderia and related species . This revealed that the rhizospheric isolates had closest affinity with Burkholderia spp . with known bioremediative and biocontrol capabilities and were unrelated to taxa comprising plant or human pathogenic strains . CONCLUSIONS: All of the analyses investigated revealed that environmental and onion isolates of B . cepacia complex bacteria are genetically diverse but that woodland rhizospheric isolates are related to each other and unrelated to plant or human pathogenic strains . SIGNIFICANCE AND IMPACT OF THE STUDY: Woodland rhizospheric isolates of B . cepacia are potentially good candidates for use in bioremediation and biocontrol, as they appear distinct from plant or human pathogenic strains.

Mamm Genome, 2002 Aug, 13(8), 445 - 51
Identification and characterization of a novel murine beta-defensin-related gene; Morrison GM et al.; Beta-defensins comprise a family of cationic peptides, which are predominately expressed at epithelial surfaces and have a broad-range antimicrobial activity . We have assembled two BAC-based contigs from the chromosomal region 8A4 that contain the murine defensins, and we have mapped six reported beta-defensin genes . In addition, we have isolated and functionally characterized a novel beta-defensin gene that deviates from the canonical six cysteine motif present in the mature functional peptide of all other beta-defensins . This defensin-related gene (Defr1) is most highly expressed in testis and heart . The genomic organization is highly similar to Defb3, 4, 5, and 6, and the exon 1 sequence is very highly conserved . A synthetic Defr1 peptide displayed antimicrobial activity against Staphylococcus aureus, Pseudomonas aeruginosa, Escherichia coli, and Burkholderia cepacia . The antimicrobial activity of Defr1 against S . aureus, E.coli, and B . cepacia was found to be reduced in raised concentration of NaCl, but its action against P . aeruginosa was independent of NaCl concentration . This is the first report of a functional beta defensin that lacks one of the conserved cysteine residues in its predicted mature peptide . This study has major implications for the structure and functions of these important host defense molecules.

Am J Trop Med Hyg, 2002 Jun, 66(6), 759 - 61
Short report: a rapid method for the differentiation of Burkholderia pseudomallei and Burkholderia thailandensis; Wuthiekanun V et al.; A rapid method for the identification and differentiation of Burkholderia pseudomallei and Burkholderia thailandensis colonies is described . It consists of simultaneous use of 2 monoclonal antibody-based latex agglutination test systems . The anti-lipopolysaccharide test reacts with both species, whereas the anti-exopolysaccharide reacts only with B . pseudomallei . Compared with classical biochemical tests, the method is highly reproducible and accurate . It is particularly useful for the identification of the organisms in environmental specimens, which may contain both of these Burkholderia species.

Can J Microbiol, 2002 Jul, 48(7), 611 - 25
Bacterial diversity associated with subalpine fir (Abies lasiocarpa) ectomycorrhizae following wildfire and salvage-logging in central British Columbia; Khetmalas MB et al.; To assess the effect of fire and salvage logging on the diversity of mycorrhizal-bacterial communities, bacteria associated with Cenococcum, Thelephora, Tomentella, Russulaceae, and E-strain ectomycorrhizae (ECM) of Abies lasiocarpa seedlings were characterized using two approaches . First, bacteria were isolated and characterized by Biolog, gas chromatography fatty acid methyl ester (GC-FAME), and amplified 16S rDNA restriction analysis (ARDRA) . The bacterial communities retrieved from ECM from both sites were dominated by Proteobacteria (groups gamma and beta) . Pseudomonas was the most common genus isolated, followed by Variovorax, Burkholderia, and Xanthomonas . Gram-positive isolates (mostly high-G+C Gram-positive bacteria) were more frequently retrieved on the burned-salvaged site, many commonly associated with the two ascomycete ECM, Cenococcum and E-strain . Pseudomonas species were retrieved more frequently from Thelephora . Although actinomycetes were isolated from all sites, almost no actinomycetes or other Gram-positive bacteria were isolated from either Thelephora or Tomentella . Second, amplified 16S rRNA gene sequences were amplified directly from root tips and then cloned into the plasmid vector pAMP1, followed by restriction analysis . This technique distinguished more genotypes than isolates retrieved by culturing methods, but generally, results were similar in that the largest proportion of the bacteria were putatively Gram-negative; putative Gram-positive bacteria were fewer and most were from the burned-salvaged site . Direct cloning resulted in many patterns that did not match any identified isolates, suggesting that a large proportion of clones were unique or not culturable by the methods used . Analysis for both protocols showed no significant difference in bacterial diversity between the burned-salvaged and unburned sites.

Plasmid, 2002 Jul, 48(1), 1 - 12
Complete characterisation of Tn5530 from Burkholderia cepacia strain 2a (pIJB1) and studies of 2,4-dichlorophenoxyacetate uptake by the organism; Poh RP et al.; The complete genetic characterisation of Tn5530 in Burkholderia cepacia strain 2a (pIJB1) has been accomplished, indicating that it is a Tn3-like transposon with a complex structure bearing operons for the catabolism of 2,4-dichlorophenoxyacetate (2,4-D) and malonate . Tn5530 is terminated at both ends by the IS1071::IS1471 element and the 2,4-D- and malonate-dissimilatory operons are separated by a region encoding a putA and lrp gene and a gene encoding a chloride channel protein . The chloride channel protein may have a role in the expulsion of chloride ions liberated by the dissimilation of 2,4-D . In addition, a putative transposase with a high level of sequence similarity to those of plasmid pGH1 from Xanthomonas campestris pv . vesicatoria and Pseudomonas syringae pv . glycinea, and a transcription factor similar to those of the TetR family with low but significant levels of sequence similarity to those identified in a number of other organisms was observed . The entire Tn5530 sequence length, including the IS1071::IS1471 elements, was found to be 40,956bp, and pIJB1 was replicon-typed and otherwise characterised as being of the IncP-1beta subgroup, bearing merA and merD genes conferring resistance to mercuric chloride . The rate of uptake of 2,4-D by B . cepacia strain 2a was observed to proceed more readily at acid pH, suggesting involvement of the undissociated form of the compound . Uptake did not show saturation kinetics, was concentration-dependent, and appeared to occur in two stages; an initial accumulation followed by a linear second phase . Uptake could be inhibited by sodium azide but not by arsenate, N,N(')-dicyclohexylcarbodi-imide (DCCD) or carbonylcyanide m-chlorophenyl-hydrazone (CCCP) suggesting that it is not energy-dependent.

Rev Inst Med Trop Sao Paulo, 2002 Jul-Aug, 44(4), 203 - 8
Use of selective medium for Burkholderia cepacia isolation in respiratory samples from cystic fibrosis patients; da Silva Filho LV et al.; Burkholderia cepacia colonizes cystic fibrosis (CF) patients . We evaluated the impact of the use of a selective medium in the rate of B . cepacia recovery from respiratory samples of CF patients . During a 6-month period, respiratory samples were collected from 106 CF patients and cultivated on selective media including a B . cepacia selective medium . Confirmation of the identity of B . cepacia isolates was carried out by species specific PCR and determination of genomovar status performed by a sequential PCR approach . Results of B . cepacia isolation during this period were compared to the preceding two years, when the sample processing was identical except for the lack of the B . cepacia selective medium . B . cepacia was isolated in 11/257 (4.2%) of the samples using the selective medium, in contrast with the preceding two years, when it was isolated in 6/1029 samples (0.58%), p < 0.0001 . Identity of all 11 isolates was confirmed by PCR and genomovar determination was accomplished in all but one isolate . These results suggest that the use of a selective medium increases recovery rate of B . cepacia from respiratory samples.

J Postgrad Med, 2002 Apr-Jun, 48(2), 124 - 6
Burkholderia pseudomallei: abscess in an unusual site; Kiertiburanakul S et al.; Melioidosis is an infection caused by Burkholderia pseudomallei . It is an important human pathogen in tropical area . The clinical manifestations are protean and multisystem involvement . We report an unusual case of melioidosis with abscess at root of mesentery in an elderly, non-insulin dependent diabetic Thai women . She presented with prolonged fever and chronic abdominal pain . The early clinical diagnosis was carcinomatous mass with peritonitis . Diagnosis of melioidosis arose from the surgical finding and pus culture . Treatment with surgical drainage and ceftazidime followed by co-trimoxazole plus doxycycline had a good clinical outcome.

FEMS Microbiol Lett, 2002 Aug 27, 214(1), 1 - 5
Evaluation of restriction fragment length polymorphism analysis of 16S rDNA as a tool for genomovar characterisation within the Burkholderia cepacia complex; Vermis K et al.; A total of 154 Burkholderia cepacia complex strains, isolated from cystic fibrosis and non-cystic fibrosis patients and the environment, representing all nine genomovars and a putative tenth, were analysed by 16S rDNA-restriction fragment length polymorphism using the restriction enzymes AluI, CfoI and DdeI . Examining this diverse strain collection resulted in very diverse restriction patterns . Only B . cepacia genomovar VI could be identified unambiguously . The same restriction patterns were observed for B . cepacia genomovars I and III and approximately half of the Burkholderia ambifaria, B . anthina and B . pyrrocinia strains . Burkholderia vietnamiensis and B . ubonensis, a putative tenth B . cepacia complex genomovar, shared identical restriction profiles . The majority of Burkholderia multivorans and B . stabilis isolates generated a unique restriction pattern, but two strains of each showed divergent restriction profiles which were also observed in other genomovars.

J Clin Microbiol, 2002 Sep, 40(9), 3485 - 8
Direct PCR detection of Burkholderia cepacia complex and identification of its genomovars by using sputum as source of DNA; Drevinek P et al.; We developed a nested PCR assay that detects the recA gene of the Burkholderia cepacia complex in sputum . The product of the first PCR round is also used to identify the genomovar of the pathogen . The protocol achieves high sensitivity and specificity with simple interpretation of genomovar status.

J Clin Microbiol, 2002 Sep, 40(9), 3300 - 7
Comparative assessment of genotyping methods for epidemiologic study of Burkholderia cepacia genomovar III; Coenye T et al.; We analyzed a collection of 97 well-characterized Burkholderia cepacia genomovar III isolates to evaluate multiple genomic typing systems, including pulsed-field gel electrophoresis (PFGE), BOX-PCR fingerprinting and random amplified polymorphic DNA (RAPD) typing . The typeability, reproducibility, and discriminatory power of these techniques were evaluated, and the results were compared to each other and to data obtained in previous studies by using multilocus restriction typing (MLRT) . All methods showed excellent typeability . PFGE with SpeI was more reproducible than RAPD and BOX-PCR fingerprinting . The discriminatory power of the methods was variable, with PFGE and RAPD typing having a higher index of discrimination than BOX-PCR fingerprinting . In general, the results obtained by PFGE, BOX-PCR fingerprinting, and MLRT were in good agreement . Our data indicate that different genomic-based methods can be used to type B . cepacia genomovar III isolates depending on the situation and the epidemiologic question being addressed . PFGE and RAPD fingerprinting are best suited to addressing small-scale studies (i.e., local epidemiology), whereas BOX-PCR fingerprinting is more appropriate for large-scale studies (i.e., global epidemiology) . In this regard, BOX-PCR fingerprinting can be considered a rapid and easy alternative to MLRT.

J Clin Microbiol, 2002 Sep, 40(9), 3198 - 203
Comparison of rapid, automated ribotyping and DNA macrorestriction analysis of Burkholderia pseudomallei; Inglis TJ et al.; An automated ribotyping device (RiboPrinter) was used to determine the ribotypes of a collection of Burkholderia pseudomallei isolates . In a preliminary evaluation with the restriction enzymes BamHI and EcoRI, the protocol with EcoRI was more discriminating . The reproducibilities of the ribotypes obtained with EcoRI (EcoRI ribotypes) were determined by testing three levels of bacterial loads . The performance of the manufacturer's software was assessed by comparing the machine-optimized ribotypes with the type determined from the original gel image analyzed with Bionumerics software . The library of B . pseudomallei EcoRI ribotypes was then compared with the ribotypes obtained by DNA macrorestriction analysis of XbaI digests by pulsed-field gel electrophoresis . The typeability of B . pseudomallei by EcoRI ribotyping was 100%, and the discrimination index was 0.94 . The slightly greater discrimination provided by DNA macrorestriction analysis (0.96) was achieved at the expense of a significantly longer processing time of 6 days, although the method was only half the cost of automated ribotyping . Typeability by macrorestriction analysis was lower (97%) unless a thiourea step was added to neutralize the action of Tris-dependent endonucleases . The digital record of B . pseudomallei isolates analyzed thus far provides a useful resource for future epidemiological studies and will help shorten the response time in the event of a further melioidosis outbreak or the deliberate release of B . pseudomallei as a biohazard.

J Bacteriol, 2002 Sep, 184(18), 5011 - 7
prbA, a gene coding for an esterase hydrolyzing parabens in enterobacter cloacae and Enterobacter gergoviae strains; Valkova N et al.; The new gene prbA encodes an esterase responsible for the hydrolysis of the ester bond of parabens in Enterobacter cloacae strain EM . This gene is located on the chromosome of strain EM and was cloned by several PCR approaches . The prbA gene codes for an immature protein of 533 amino acids, the first 31 of which represent a proposed signal peptide yielding a mature protein of a putative molecular mass of 54.6 kDa . This enzyme presents analogies with other type B carboxylesterases, mainly of eukaryotic origin . The cloning and expression of the prbA gene in a strain of Escherichia coli previously unable to hydrolyze parabens resulted in the acquisition of a hydrolytic capacity comparable to the original activity of strain EM, along with an increased resistance of the transformed strain to methyl paraben . The presence of homologues of prbA was tested in additional ubiquitous bacteria, which may be causative factors in opportunistic infections, including Enterobacter gergoviae, Enterobacter aerogenes, Pseudomonas agglomerans, E . coli, Pseudomonas aeruginosa, and Burkholderia cepacia . Among the 41 total strains tested, 2 strains of E . gergoviae and 1 strain of Burkholderia cepacia were able to degrade almost completely 800 mg of methyl paraben liter(-1) . Two strains of E . gergoviae, named G1 and G12, contained a gene that showed high homology to the prbA gene of E . cloacae and demonstrated comparable paraben esterase activities . The significant geographical distance between the locations of the isolated E . cloacae and E . gergoviae strains suggests the possibility of an efficient transfer mechanism of the prbA gene, conferring additional resistance to parabens in ubiquitous bacteria that represent a common source of opportunistic infections.

J Biochem Mol Biol Biophys, 2002 Feb, 6(1), 45 - 53
Phage display of recombinant antibodies toward Burkholderia pseudomallei exotoxin; Nathan S et al.; We have used the phagemid pComb3H to construct recombinant phages displaying the single chain variable fragment (ScFv) towards exotoxin of Burkholderia pseudomallei . Variable heavy and light chain fragments were amplified from the hybridoma 6E6A8F3B line, with a wide spectrum of primers specific to mouse antibody genes . Through overlapping extension polymerase chain reaction, the heavy and light chain fragments were linked to form the ScFv which was subsequently cloned into the phage display vector and transformed into ER2537 cells to yield a complexity of 10(8) clones . The transformants were screened by four rounds of biopanning against the exotoxin and resulted in selective enrichment of exotoxin-binding antibodies by 301 fold . The phage pool from the final round of selection displayed antibodies of high-affinity to the exotoxin as demonstrated by ELISA . Several clones were selected randomly from this pool and analysed by restriction enzyme digestion, fingerprinting and sequencing . Restriction analysis confirmed that all clones carried a 700-800 bp insert whose sequences, in general, corresponded to that of mouse IgG . Fingerprinting profiles delineated the antibodies into two families with different CDR sequences.

Infect Immun, 2002 Sep, 70(9), 5290 - 4
A mutant of Burkholderia pseudomallei, auxotrophic in the branched chain amino acid biosynthetic pathway, is attenuated and protective in a murine model of melioidosis; Atkins T et al.; Using a transposon mutagenesis approach, we have identified a mutant of Burkholderia pseudomallei that is auxotrophic for branched chain amino acids . The transposon was shown to have interrupted the ilvI gene encoding the large subunit of the acetolactate synthase enzyme . Compared to the wild type, this mutant was significantly attenuated in a murine model of disease . Mice inoculated intraperitoneally with the auxotrophic mutant, 35 days prior to challenge, were protected against a challenge dose of 6,000 median lethal doses of wild-type B . pseudomallei.

Mol Gen Mikrobiol Virusol, 2002, (2), 19 - 22
{Cloning and expression of 7.55 kb KPNI fragment of Burkholderia pseudomallei PPM1 plasmid in Escherichia coli}; Zakharova IB et al.; BamHI, SalI, PstI, and KpnI fragments of pPM1 (B . pseudomallei 12.95 kb plasmid) were cloned in E . coli . The recombinant clones carrying a 7.55 kb KpnI fragment of pPM1 were highly resistant to several aminoglycosides (streptomycin, kanamycin, and gentamycin) and fluoroguinolones (perfloxacin, ofloxacin) . Two outer membrane proteins (23 and 27 kDa) absent in E . coli and capable to form 120 kDa oligomer complex were detected by the Western blot method in the strain carrying recombinant pS19 plasmid . The integration of a cloned 7.55 kb sequence in the chromosome was observed by the dot and Southern hybridization analysis in the clones carrying recombinant plasmids pS12 and pS14.

Microbiology, 2002 Aug, 148(Pt 8), 2439 - 48
The principal determinants for the structure of the substrate-binding pocket are located within a central core of a biphenyl dioxygenase alpha subunit; Zielinski M et al.; Protein engineering by segment exchange was used to distinguish between regions of major and minor influence on the structure of the substrate-binding pocket of a biphenyl dioxygenase (BDO) . Eight chimaeric enzyme systems were generated that each consisted of a hybrid hydroxylase alpha subunit (BphA1) containing segments from Burkholderia sp . strain LB400 and Rhodococcus globerulus P6, and of a hydroxylase beta subunit (BphA2), a ferredoxin (BphA3) and a ferredoxin reductase (BphA4) from strain LB400 . All hybrid bphA1 genes were expressed at high levels . Seven of the resulting fusion subunits functionally interacted with the other polypeptides of the dioxygenase system to yield catalytically active enzymes . Changes in the regiospecificity of substrate attack, monitored by the formation of seventeen different dioxygenation products obtained from seven chlorobiphenyls, were used to monitor effects of segment exchanges on the structure of the BDO substrate-binding site . Exchanges of neither the beta subunit nor the N- and C-terminal regions of the alpha subunit exerted significant influences . All BDO regions that showed major effects on the substrate-binding pocket were located between approximately positions 165 and 395 of the alpha subunit . Within this part of the enzyme, in addition to segments identified previously, a subregion which is involved in ligation of the mononuclear iron significantly influenced the regiospecificity of substrate dioxygenation . Moreover, the results indicate that the construction of appropriate hybrid genes may be used as a general strategy to overcome problems in obtaining heterologous BDO activities in Escherichia coli or other host organisms.

Harefuah, 2002 May, 141 Spec No, 88 - 91, 119
{Glanders--a potential disease for biological warfare in humans and animals}; Lehavi O et al.; Infection with Burkholderia mallei (formerly Pseudomonas mallei) can cause a subcutaneous infection known as "farcy" or can disseminate to condition known as Glanders . It is primarily a disease affecting horses, donkeys and mules . In humans, Glanders can produce four types of disease: localized form, pulmonary form, septicemia, and chronic form . Necrosis of the tracheobronchial tree and pustular skin lesions characterize acute infection with B . mallei . Other symptoms include febrile pneumonia, if the organism was inhaled, or signs of sepsis and multiple abscesses, if the skin was the port of entry . Glanders is endemic in Africa, Asia, the Middle East, and Central and South America . Glanders has low contiguous potential, but because of the efficacy of aerosolized dissemination and the lethal nature of the disease, B . mallei was considered a candidate for biological warfare . During World War I, Glanders was believed to have been spread to infect large numbers of Russian horses and mules on the Eastern front . The Japanese infected horses, civilians and prisoners of war during World War II . The USA and the Soviet Union have shown interest in B . mallei in their biological warfare program . The treatment is empiric and includes mono or poly-therapy with Ceftazidime, Sulfadiazine, Trimethoprim + Sulfamethoxazol, Gentamicin, Imipenem etc . Aggressive control measures essentially eliminated Glanders from the west . However, with the resurgent concern about biological warfare, B . mallei is now being studied in a few laboratories worldwide . This review provides an overview of the disease and presents the only case reported in the western world since 1949.

J Antimicrob Chemother, 2002 Aug, 50(2), 265 - 9
Influence of taxonomic status on the in vitro antimicrobial susceptibility of the Burkholderia cepacia complex; Nzula S et al.; The Burkholderia cepacia complex is a diverse group of human pathogens that cause life-threatening lung infections in patients with cystic fibrosis or chronic granulomatous disease, and in patients requiring intensive care . Most previous antimicrobial susceptibility studies of these bacteria were performed before recent major revisions in the taxonomy of these bacteria . We determined the in vitro susceptibility of 65 B . cepacia complex isolates from clinical and environmental sources, representing six genomovars . Although intrinsic resistance is considered to be a common feature of the B . cepacia complex, MICs of individual antimicrobials varied widely and resistance was not exhibited by all members of the group.

Respirology, 2002 Sep, 7(3), 241 - 5
Effect of Burkholderia cepacia infection in the clinical course of patients with cystic fibrosis: a pilot study in a Sydney clinic; Soni R et al.; BACKGROUND: Colonisation with Burkholderia cepacia complex in patients with cystic fibrosis (CF) has been associated with adverse outcomes . The aim of the present study was to determine the actuarial survival of CF patients colonized with B . cepacia and to evaluate the efficacy of the Royal Prince Alfred Hospital segregation policy . A secondary aim was to characterize the specific genomovars and strains of B . cepacia isolated in an Australian clinic . METHODS: Retrospective review of spirometric and microbiological data on all patients colonized with B . cepacia . Each B . cepacia-colonized subject was matched with three case-control subjects . Phenotype and genomovar typing, random amplified polymorphic DNA strain type and B . cepacia epidemic strain marker analyses were performed . The effect of B . cepacia colonization on transplant-free survival was estimated by Cox's proportional hazards regression using the entire clinic population . RESULTS: Fifteen patients were colonized with B . cepacia, of whom six (40%) had died from CF-related disease by August 1998, compared with 30 of 173 (17.3%) of the entire clinic population . Cepacia status had a significant adverse effect on survival, with a hazard ratio of 2.16 (95% confidence interval 1.0-4.69; P = 0.05) . The outcome was variable in subgroups of B . cepacia . DISCUSSION: Colonization with B . cepacia had a significant adverse effect on survival within the study population . Genomovar and strain typing contributed usefully in accessing the effectiveness of the hospital's segregation policy in preventing cross-colonization.

Appl Environ Microbiol, 2002 Aug, 68(8), 3750 - 8
Culture-based and non-growth-dependent detection of the Burkholderia cepacia complex in soil environments; Miller SC et al.; Burkholderia cepacia complex (Bcc) bacteria reside in soil, plant rhizospheres, and water, but their prevalence and distribution in outdoor environments is not clear . We sampled a variety of soil and rhizosphere environments with which people may have contact: playgrounds, athletic fields, parks, hiking trails, residential yards, and gardens . A total of 91 sites was sampled in three large U.S . cities . In the first phase of the study, putative Bcc isolates were recovered on Burkholderia cepacia selective agar and trypan blue tetracycline medium and subsequently examined for biochemical reactivity and growth at 32 and 22 degrees C . Isolates were further examined by PCR assays targeting Bcc-specific ribosomal DNA and recA gene sequences . Among the 1,013 bacterial isolates examined, 68 were identified as Bcc; 14 (15%) of 91 sampled sites yielded Bcc isolates . In the second phase, DNA was extracted directly from soil samples and examined with PCR assays targeting Bcc 16S rRNA gene sequences . Either 82 or 93% of the soil samples were positive for at least one Bcc genomovar, depending on the PCR assay system used . Cloning and sequencing were performed to check the specificity of the PCR assays . Sequence analysis of the 463-bp 16S rRNA inserts from eight clones indicated that all were from members of the Bcc . The four soil samples from which these clones were generated did not yield isolates identified as Bcc . Based on PCR detection, Bcc appears to be prevalent in soil from urban and suburban environments . Culture-based recovery of Bcc may underestimate environmental populations.

Mol Cell Probes, 2002 Jun, 16(3), 217 - 22
A simple method to detect and differentiate Burkholderia pseudomallei and Burkholderia thailandensis using specific flagellin gene primers; Sonthayanon P et al.; We have previously shown that Burkholderia pseudomallei, the causative pathogen of melioidosis, may be discriminated from the closely related non-pathogenic species Burkholderia thailandensis by the presence of a 15 base pair deletion in the flagellin gene of B . thailandensis . Using specific flagellin gene primers flanking the distinctive region, PCR products of 191 and 176 bp in size were detected for B . pseudomallei and B . thailandensis, respectively . The sensitivity of detection is 20-80 colony forming units/reaction of B . pseudomallei and B . thailandensis cell suspension . To mimic the expected environmental situation, mixed populations of the two species were analyzed . The results showed that the PCR-based method could be use to distinguish the two species in a duplex reaction . In addition, we have developed a simplified method for direct PCR-based detection from soil samples . The result indicated that about 200 colonies of bacteria per reaction could be detected . This method can be applied to epidemiological studies, especially for investigating the ecological relationship between these two species in the environment.

Clin Exp Dermatol, 2002 Jun, 27(4), 280 - 2
Cutaneous melioidosis; Tran D et al.; Melioidosis is a rare tropical disease caused by infection with the bacterium Burkholderia pseudomallei . It occurs predominantly in south-east Asia, northern and central Australia and central and south America . Patients often present to the internal medicine physicians with a severe, potentially fatal sepsis . We report three patients who presented to our dermatology department with cutaneous melioidosisPublication Types:

Case Reports

What Is Protein?, What Is Bioremediation?, What Is Nitrification?, What Is Functional Genomics?, What Is Fermentation?, i, Bacteriology, o, Microbes, e, Microorganisms, e, Bacterium, a, Microbe, s, Bacillus, r, Multidrug resistant, i, Thermophile, n, B. anthracis, r, Prokaryotes, s, Streptococci, o, Clostridia, a, Antibiotics, r, S. cerevisiae, e, Lactobacillus, i, Microorganism, c, Yeasts, o, Escherichia coli, r, Salmonella, n, Enterobacters, s, Erythromycin, o, Escherichia coli, r, Functional genomics, o, Cryptococci, o, Streptomycin, o, Antibiotics

Scientific Publications - Work Done by Microbiology Reader Bioscreen C
Agricultural Microbiology Anaerobic Microbiology Antimicrobial Susceptibility Artificial Atmosphere Bioassay of Antibiotics Biofilm Microbiology Bioreactor Technology Biotechnology Cell Biology Clinical Microbiology Environmental Microbiology Experiments with Yeast	Fermentation Food Microbiology Functional Genomics Gene Technology Growth Media Development Growth Rate and Lag Time Industrial Microbiology Medical/Pharmaceutical Field Microbiological Assay Microbiological Research Microbiology of Cosmetics go to a specific theme...	Military Microbiology Molecular Microbiology Mutagenicity and Genotoxicity Oral Microbiology Patents Postantibiotic Studies Soil Microbiology Spore Microbiology Veterinary Microbiology Waste/Wastewater Treatment Water Microbiology Wine Microbiology

� 2005 Transgalactic Ltd (manufacturer of Bioscreen C software) | Privacy Statement | P.O. Box 1393, 00101 Helsinki, Finland, phone: +358 9 85172920, fax: +358 9 8749481, e-mail: microbiology@bionewsonline.com

Last modified: May 25, 2005