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 transglycosyla