J Infect Dis, 2002 Jul 15, 186(2), 286 - 9 Epub 2002 Jun 27. Demonstration of a cell-mediated immune response in melioidosis; Ketheesan N et al.; Melioidosis is a bacterial infection caused by Burkholderia pseudomallei . The aim of this study was to determine whether a cell-mediated adaptive immune response against B . pseudomallei developed in patients who had recovered from melioidosis . Lymphocyte proliferation assays were done on peripheral blood mononuclear cells from patients (n=13) and control subjects (n=10) to determine the lymphocyte response to B . pseudomallei antigens . Production of interferon-gamma and interleukin-10 was also determined . Activation of T cell subsets was assessed by fluorescence-activated cell sorter analysis, using antibodies to CD4, CD8, and CD69 antigens . Lymphocyte proliferation and interferon-gamma production in response to B . pseudomallei antigens were significantly higher (P<.001 for both) in patients than in control subjects . There was also an increase in the percentage of activated CD4+ (P<.004) and activated CD8+ T cells (P<.035) in cell cultures from patients . The development of such a cell-mediated immune response in patients may be essential for their survival. J Med Microbiol, 2002 Jul, 51(7), 539 - 47 Characterisation of an acapsular mutant of Burkholderia pseudomallei identified by signature tagged mutagenesis; Atkins T et al.; A Burkholderia pseudomallei mutant which was attenuated in a mouse model of melioidosis was identified by a signature tagged mutagenesis approach . The transposon was shown to be inserted into a gene within the capsular biosynthetic operon . Compared with the wild-type bacteria this mutant demonstrated a 10(5)-fold increase in the median lethal dose in a mouse model and it did not react with a monoclonal antibody against high mol . wt polysaccharide of B . pseudomallei . To determine the kinetics of infection, mice were dosed intraperitoneally (i.p.) and intravenously (i.v.) with mutant and wild-type bacteria . After i.p challenge, the number of mutant bacteria in the peritoneal cavity declined, whereas wild-type bacteria proliferated . When administered by the i.v . route, the mutant was able to cause disease but the time to death was increased compared with the wild type . Mice were dosed with the mutant and subsequently challenged with wild-type B . pseudomallei, but the mutant failed to induce a protective immune response. J Med Microbiol, 2002 Jul, 51(7), 533 - 8 Burkholderia cepacia complex infection in patients with cystic fibrosis; Mahenthiralingam E et al.; The word 'complex' has several meanings and synonyms such as composite, obsession, heterogeneous, mixed and network, can all be used in its place . Our obsession with bacteria from the Burkholderia cepacia complex started in the early 1990s . In less than 10 years, we have seen the status of this bacterium move from: (i) a lesser known pseudomonad opportunist pathogen, (ii) to devastating infections transmitted between patients with cystic fibrosis (CF), (iii) through divisions into several new species, and (iv) now on towards one of the largest gram-negative genome sequencing projects . For microbiologists, hospital infection control officers, caregivers, and most of all the CF community, the changes in our understanding of the taxonomy, epidemiology and pathogenesis of the bacterium 'B . cepacia' are complex. Cornea, 2002 Aug, 21(6), 602 - 3 Burkholderia gladioli keratitis associated with consecutive recurrent endophthalmitis; Ritterband D et al.; PURPOSE: To report a case of Burkholderia gladioli keratitis with consecutive endophthalmitis . METHODS: Case report and literature review . RESULTS: A 76-year-old man with a history of diabetes mellitus developed bacterial keratitis and consecutive endophthalmitis in the corneal graft of the left eye . Corneal, aqueous, and vitreous cultures yielded Burkholderia gladioli . Emergent keratoplasty, pars plana vitrectomy, and injection of intravitreal antibiotics led to resolution of the infection and improved vision . Four months later, the patient developed recurrent Burkholderia keratitis and endophthalmitis, necessitating a total keratoplasty and repeat injection of intravitreal antibiotics . CONCLUSION: This is the first report, to our knowledge, of ocular Burkholderia gladioli infection, an uncommon aerobic, gram-negative rod, recently subclassified from the genus Pseudomonas based on DNA-rRNA homology studies. ANZ J Surg, 2002 Jun, 72(6), 408 - 10 Primary melioidotic prostatic abscess: presentation, diagnosis and management; Tan JK et al.; INTRODUCTION: In South-East Asia and Northern Australia, melioidosis (infection with Burkholderia pseudomallei) is a known cause of severe community-acquired sepsis . However, melioidosis presenting primarily as prostatic abscesses is very rare . METHODS: The presenting features, investigations and management outcome of five patients who developed melioidotic prostatic abscesses from 1997 to 2000 were reviewed in the present study . RESULTS: The mean age at presentation was 53 years (range: 29-69) . Old age and diabetes mellitus were predisposing factors . All patients had a fever of at least 38.5 degrees C and presented with obstructive urinary symptoms culminating in urinary retention . Presence of prostatic abscess was demonstrated by transrectal ultrasound in all cases . The abscesses were drained with transurethral resection of the prostate . One patient required re-resection while another patient developed severe septic shock requiring intensive care and -inotropic support . There was no mortality in our series . CONCLUSIONS: Elderly diabetic men presenting with fever and urinary tract obstruction in endemic areas may harbour an unusual but potentially life threatening melioidotic prostatic abscess . Transrectal ultrasound and bacteriological confirmation are mandatory . Prompt surgical drainage coupled with appropriate antibiotics are keys to a favourable outcome. J Bacteriol, 2002 Aug, 184(15), 4219 - 32 Origins of the 2,4-dinitrotoluene pathway; Johnson GR et al.; The degradation of synthetic compounds requires bacteria to recruit and adapt enzymes from pathways for naturally occurring compounds . Previous work defined the steps in 2,4-dinitrotoluene (2,4-DNT) metabolism through the ring fission reaction . The results presented here characterize subsequent steps in the pathway that yield the central metabolic intermediates pyruvate and propionyl coenzyme A (CoA) . The genes encoding the degradative pathway were identified within a 27-kb region of DNA cloned from Burkholderia cepacia R34, a strain that grows using 2,4-DNT as a sole carbon, energy, and nitrogen source . Genes for the lower pathway in 2,4-DNT degradation were found downstream from dntD, the gene encoding the extradiol ring fission enzyme of the pathway . The region includes genes encoding a CoA-dependent methylmalonate semialdehyde dehydrogenase (dntE), a putative NADH-dependent dehydrogenase (ORF13), and a bifunctional isomerase/hydrolase (dntG) . Results from analysis of the gene sequence, reverse transcriptase PCR, and enzyme assays indicated that dntD dntE ORF13 dntG composes an operon that encodes the lower pathway . Additional genes that were uncovered encode the 2,4-DNT dioxygenase (dntAaAbAcAd), methylnitrocatechol monooxygenase (dntB), a putative LysR-type transcriptional (ORF12) regulator, an intradiol ring cleavage enzyme (ORF3), a maleylacetate reductase (ORF10), a complete ABC transport complex (ORF5 to ORF8), a putative methyl-accepting chemoreceptor protein (ORF11), and remnants from two transposable elements . Some of the additional gene products might play as-yet-undefined roles in 2,4-DNT degradation; others appear to remain from recruitment of the neighboring genes . The presence of the transposon remnants and vestigial genes suggests that the pathway for 2,4-DNT degradation evolved relatively recently because the extraneous elements have not been eliminated from the region. Emerg Infect Dis, 2002 Jul, 8(7), 692 - 6 Infection by Ralstonia species in cystic fibrosis patients: identification of R . pickettii and R . mannitolilytica by polymerase chain reaction; Coenye T et al.; The frequency of respiratory tract infections caused by Ralstonia species in persons with cystic fibrosis (CF) and the role of these species in CF pulmonary disease are not well documented . In part, this lack of documentation may be attributed to the difficulty in accurately identifying Ralstonia species; R . mannitolilytica and R . pickettii in particular may be misidentified as other closely related species, particularly those of the Burkholderia cepacia complex . We used polyphasic analyses to identify 42 Ralstonia isolates from sputum cultures from 38 CF patients . Several isolates that could not be identified to the species level may belong to novel Ralstonia species . We demonstrated chronic colonization by using genotyping of serial isolates recovered from the same patient . To facilitate identification of R . mannitolilytica and R . pickettii, we developed 16S ribosomal DNA-based polymerase chain reaction assays that allow sensitive and specific identification of these species. Eur J Biochem, 2002 Jul, 269(13), 3321 - 8 The cold-active lipase of Pseudomonas fragi . Heterologous expression, biochemical characterization and molecular modeling; Alquati C et al.; A recombinant lipase cloned from Pseudomonas fragi strain IFO 3458 (PFL) was found to retain significant activity at low temperature . In an attempt to elucidate the structural basis of this behaviour, a model of its three-dimensional structure was built by homology and compared with homologous mesophilic lipases, i.e . the Pseudomonas aeruginosa lipase (45% sequence identity) and Burkholderia cepacia lipase (38%) . In this model, features common to all known lipases have been identified, such as the catalytic triad (S83, D238 and H260) and the oxyanion hole (L17, Q84) . Structural modifications recurrent in cold-adaptation, i.e . a large amount of charged residues exposed at the protein surface, have been detected . Noteworthy is the lack of a disulphide bridge conserved in homologous Pseudomonas lipases that may contribute to increased conformational flexibility of the cold-active enzyme. J Bacteriol, 2002 Jul, 184(14), 4003 - 17 Burkholderia thailandensis E125 harbors a temperate bacteriophage specific for Burkholderia mallei; Woods DE et al.; Burkholderia thailandensis is a nonpathogenic gram-negative bacillus that is closely related to Burkholderia mallei and Burkholderia pseudomallei . We found that B . thailandensis E125 spontaneously produced a bacteriophage, termed phiE125, which formed turbid plaques in top agar containing B . mallei ATCC 23344 . We examined the host range of phiE125 and found that it formed plaques on B . mallei but not on any other bacterial species tested, including B . thailandensis and B . pseudomallei . Examination of the bacteriophage by transmission electron microscopy revealed an isometric head and a long noncontractile tail . B . mallei NCTC 120 and B . mallei DB110795 were resistant to infection with phiE125 and did not produce lipopolysaccharide (LPS) O antigen due to IS407A insertions in wbiE and wbiG, respectively . wbiE was provided in trans on a broad-host-range plasmid to B . mallei NCTC 120, and it restored LPS O-antigen production and susceptibility to phiE125 . The 53,373-bp phiE125 genome contained 70 genes, an IS3 family insertion sequence (ISBt3), and an attachment site (attP) encompassing the 3' end of a proline tRNA (UGG) gene . While the overall genetic organization of the phiE125 genome was similar to lambda-like bacteriophages and prophages, it also possessed a novel cluster of putative replication and lysogeny genes . The phiE125 genome encoded an adenine and a cytosine methyltransferase, and purified bacteriophage DNA contained both N6-methyladenine and N4-methylcytosine . The results presented here demonstrate that phiE125 is a new member of the lambda supergroup of Siphoviridae that may be useful as a diagnostic tool for B . mallei. J Bacteriol, 2002 Jul, 184(14), 3794 - 800 Family shuffling of a targeted bphA region to engineer biphenyl dioxygenase; Barriault D et al.; In this work we used a new strategy designed to reduce the size of the library that needs to be explored in family shuffling to evolve new biphenyl dioxygenases (BPDOs) . Instead of shuffling the whole gene, we have targeted a fragment of bphA that is critical for enzyme specificity . We also describe a new protocol to screen for more potent BPDOs that is based on the detection of catechol metabolites from chlorobiphenyls . Several BphA variants with extended potency to degrade polychlorinated biphenyls (PCBs) were obtained by shuffling critical segments of bphA genes from Burkholderia sp . strain LB400, Comamonas testosteroni B-356, and Rhodococcus globerulus P6 . Unlike all parents, these variants exhibited high activity toward 2,2'-, 3,3'-, and 4,4'-dichlorobiphenyls and were able to oxygenate the very persistent 2,6-dichlorobiphenyl . The data showed that the replacement of a short segment (335TFNNIRI341) of LB400 BphA by the corresponding segment (333GINTIRT339) of B-356 BphA or P6 BphA contributes to relax the enzyme toward PCB substrates. J Environ Sci Health B, 2002 Jul, 37(4), 379 - 91 Sequence analysis and molecular characterization of a nitrocatechol dioxygenase gene from Pseudomonas putida; Walia S et al.; A Pseudomonas putida capable of degrading polychlorinated biphenyl was also found to transform 4-nitrocatechol to 3-nitro-2-hydroxy-6-oxohexa-2,4-dienoic acid (NHODA) . Crude cell extract of this bacterium exhibited an enzyme (nitrocatechol dioxygenase, Ndo) activity catalyzing this transformation . The gene encoding Ndo was cloned in E . coli . The cloned gene (ndo) expressed in E . coli had enzyme activity that degraded not only 4-nitrocatechol but also 4-chlorocatechol, 4-methylcatechol, 2,3-dihydroxybiphenyl, and 4'-chloro-2,3-dihydroxybiphenyl . Nucleotide sequence analysis of the cloned ndo exhibited an open reading frame of 939 base pairs . This sequence can encode a 313 amino acids protein of approximately molecular weight of 35 kd, which was confirmed by in vitro transcription and translation assay and SDS-PAGE analysis . A putative ribosomal binding site (GAGGAGA) was present 7 base pairs upstream from the AUG start codon and a promotor site homologous to E . coli '-10' and '-35' regulatory region was located at '-123' and '-174' area of our clone with sequences of TTGAAG and GTGACA, respectively . The deduced amino acid sequence showed 69% homology with Cdo from Burkholderia cepacia AAI . A unique insertion of 21 amino acids was found towards the N-terminal of the Ndo . Expression of ndo in strain OU83 was repressed in presence of 3-chlorobenzoic acid as judged by the decrease in the expression of ndo specific transcript. FEMS Microbiol Lett, 2002 Jun 4, 211(2), 259 - 64 Sec-dependent and Sec-independent translocation of haloacid dehalogenase Chd1 of Burkholderia cepacia MBA4 in Escherichia coli; Tsang JS et al.; 2-Haloacid dehalogenases are hydrolytic enzymes that cleave the halogen-carbon bond(s) in haloalkanoic acids . We have previously isolated a cryptic haloacid dehalogenase gene from Burkholderia cepacia MBA4 and expressed it in Escherichia coli . This recombinant protein is unusual in having a long leader sequence, a property of periplasmic enzymes . In this paper, we report the functional role of this leader sequence . Western blot analyses showed that Chd1 is translocated to the periplasm . The results on the expression of Chd1 in the presence of sodium azide suggested the cleavage of the leader to be Sec-dependent . Chimeras of Chd1 and green fluorescent protein demonstrated that the leader sequence is fully functional in translocating the fusion protein to the periplasm . The expression of the chimeras in Sec mutants supported the Sec-dependent translocation . Surprisingly, recombinant Chd1 and a chimera with no leader sequence were also found in the periplasm. Lancet, 2002 Jun 8, 359(9322), 2002 - 3 An epidemic Burkholderia cepacia complex strain identified in soil; LiPuma JJ et al.; Life threatening infection with species of the Burkholderia cepacia complex frequently occurs as a result of cross infection among individuals with cystic fibrosis . Stringent infection control measures have decreased but not eliminated such infection in this vulnerable population, implying that non-patient reservoirs contribute to ongoing acquisition . However, strains common to both the natural environment and patients with cystic fibrosis have not yet been described . By use of various genotyping methods, we have identified from agricultural soil the B cepacia genomovar III strain that is most frequently recovered from cystic fibrosis patients in the mid-Atlantic region of the USA . This finding indicates that human pathogenic strains are not necessarily distinct from environmental strains, and might help explain ongoing human acquisition despite strict infection control measures. Microb Pathog, 2002 May, 32(5), 249 - 54 Identification of a general secretory pathway in a human isolate of Burkholderia vietnamiensis (formerly B . cepacia complex genomovar V) that is required for the secretion of hemolysin and phospholipase C activities; Fehlner-Gardiner CC et al.; Members of the Burkholderia cepacia complex can secrete proteases, lipases, and hemolysins . We report in this study the identification of a general secretory pathway present in a B . vietnamiensis (formerly genomovar V) clinical isolate, which is required for the efficient secretion of phospholipase C and hemolysin activities . Southern blot hybridization experiments revealed that this general secretion pathway is highly conserved among the different genomovars of the B . cepacia complex and is homologous to a similar system described in B . pseudomallei . We also show that this pathway appears not to be necessary for intracellular survival of B . vietnamiensis within Acanthamoeba polyphaga . Jpn J Antibiot, 2002 Apr, 55(2), 154 - 80 {Yearly changes in antibacterial activities of cefozopran against various clinical isolates between 1996 and 2000--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 2000 were yearly evaluated and compared with those of other cephems, oxacephems, and carbapenems . Thirty-two species 2,697 strains of Gram-negative bacteria were isolated from the clinical materials annually collected from January to December, and consisted of Moraxella subgenus Branhamella catarrhalis (n = 125), Escherichia coli (n = 250), Citrobacter freundii (n = 153), Citrobacter koseri (n = 97), Klebsiella pneumoniae (n = 150), Klebsiella oxytoca (n = 100), Enterobacter aerogenes (n = 50), Enterobacter cloacae (n = 125), Serratia marcescens (n = 153), Proteus mirabillis (n = 103), Proteus vulgaris (n = 77), Morganella morganii (n = 141), Providencia spp . (P . alcalifaciens, P . rettgeri, P . stuartii; n = 154), Pseudomonas aeruginosa (n = 211), Pseudomonas putida (n = 49), Burkholderia cepacia (n = 102), Stenotrophomonas maltophilia (n = 101), Haemophilus influenzae (n = 210), Acinetobactor baumannii (n = 63), Acinetobactor Iwoffii (n = 30), Bacteroides fragilis group (B . fragilis, B . vulgatus, B . distasonis, B . ovatus, B . thetaiotaomicron; n = 129), and Prevotella spp . (P . melaninogenica, P . intermedia, P . bivia, P . oralis, P . denticola; n = 124) . 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 . lowffii throughout 5 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 65-time difference during 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 the antibacterial activity of CZOP against H . influenzae was suggested. Arch Microbiol, 2002 Jul, 178(1), 65 - 70 Epub 2002 Apr 30. Aciduric Proteobacteria isolated from pH 2.9 soil; Curtis P et al.; Acidic (pH 2.9) soil was used as an inoculum to culture heterotrophic bacteria at pH values of 3-4 . Four isolates were obtained; on the basis of 16S rDNA sequence, they were shown to be members of the beta- and gamma-Proteobacteria . The three isolates that were most closely related to Burkholderia spp . had simple nutritional requirements and could grow in glucose-mineral salts media; two of these used a broad array of organic substrates . The 16S rDNA sequence of the fourth isolate was most similar (96%) to Frateuria aurantia . The isolates were aciduric rather than acidophilic; their pH ranges for growth were approximately 3.5-8 . Unlike many bacteria whose acid tolerance represents the capacity to survive acid exposure, these microorganisms carried out exponential growth at pH<4 and their growth rates at pH 3.9 ranged from 60 to 98% of those found at pH 7 . The cell yields on glucose of two strains were identical at pH 4 and pH 7 . The acidic soils appeared to contain a very diverse bacterial community as assessed by denaturing gradient gel electrophoresis fingerprinting of PCR amplicons of a portion of the 16S rDNA gene . Electronic supplementary material to this paper can be obtained by using the Springer LINK server located at http://dx.doi.org/10.1007/s00203-002-0427-1. Mol Microbiol, 2002 Jun, 44(5), 1185 - 97 Characterization of Burkholderia pseudomallei infection and identification of novel virulence factors using a Caenorhabditis elegans host system; Gan YH et al.; The environmental saphrophyte Burkholderia pseudomallei is the causative agent of melioidosis, a systemic, potentially life-threatening condition endemic to many parts of south-east Asia and northern Australia . We have used the soil nematode Caenorhabditis elegans as a model host to characterize the mechanisms by which this bacterium mounts a successful infection . We find that C . elegans is susceptible to a broad range of Burkholderia species, and that the virulence mechanisms used by this pathogen to kill nematodes may be similar to those used to infect mammals . We also find that the specific dynamics of the C . elegans-B . pseudomallei host-pathogen interaction can be highly influenced by environmental factors, and that nematode killing results at least in part from the presence of a diffusible toxin . Finally, by screening for bacterial mutants attenuated in their ability to kill C . elegans, we genetically identify several new potential virulence factors in B . pseudomallei . The use of C . elegans as a model host should greatly facilitate future investigations into how B . pseudomallei can interact with host organisms. Infect Immun, 2002 Jul, 70(7), 3953 - 8 Analogous cytokine responses to Burkholderia pseudomallei strains contrasting in virulence correlate with partial cross-protection in immunized mice; Ulett GC et al.; Cytokine mRNA levels were assessed in Burkholderia pseudomallei-susceptible BALB/c mice and B . pseudomallei-resistant C57BL/6 mice following administration of a sublethal dose of less virulent (LV) B . pseudomallei, a candidate immunogen tested for protection against a highly virulent (HV) challenge . Compared on the basis of the bacterial loads, the cytokine patterns induced by HV and LV B . pseudomallei were similar, involving gamma interferon, interleukin-10, and other cytokines . Partial cross-protection between B . pseudomallei strains is shown to be associated with cytokine profiles involving both type 1 and type 2 cytokines. J Biochem Biophys Methods, 2002 Apr 18, 51(2), 115 - 20 Microwave-assisted rapid characterization of lipase selectivities; Bradoo S et al.; A rapid screening procedure for characterization of lipase selectivities using microwaves was developed . The rate of reaction of various commercial lipases (porcine pancreas, Mucor miehei, Candida rugosa, Pseudomonas cepacia) as well as lipases from laboratory isolates-Bacillus stearothermophilus and Burkholderia cepacia RGP-10 for triolein hydrolysis was 7- to 12-fold higher in a microwave oven as compared to that by pH stat . The esterification of sucrose/methanol and ascorbic acid with different fatty acids was also achieved within 30 s in a microwave using porcine pancreas, B . stearothermophilus SB-1 and B . cepacia RGP-10 lipases . The relative rates and selectivity of the lipases both for hydrolytic and synthesis reactions remains unaltered . However, the rate of reaction was dynamically enhanced when exposed to microwaves . Microwave-assisted enzyme catalysis can become an attractive procedure for rapid characterization of large number of enzyme samples and substrates, which otherwise is a cumbersome and time-consuming exercise. Mol Plant Microbe Interact, 2002 Jun, 15(6), 515 - 21 Enhanced resistance to seed-transmitted bacterial diseases in transgenic rice plants overproducing an oat cell-wall-bound thionin; Iwai T et al.; Bacterial attack is a serious agricultural problem for growth of rice seedlings in the nursery and field . The thionins purified from seed and etiolated seedlings of barley are known to have antimicrobial activity against necrotrophic pathogens; however, we found that no endogenous rice thionin genes alone are enough for resistance to two major seed-transmitted phytopathogenic bacteria, Burkholderia plantarii and B . glumae, although rice thionin genes constitutively expressed in coleoptile, the target organ of the bacteria . Thus, we isolated thionin genes from oat, one of which was overexpressed in rice . When wild-type rice seed were germinated with these bacteria, all seedlings were wilted with severe blight . In the seedling infected with B . plantarii, bacterial staining was intensively marked around stomata and intercellular spaces . However, transgenic rice seedlings accumulating a high level of oat thionin in cell walls grew almost normally with bacterial staining only on the surface of stomata . These results indicate that the oat thionin effectively works in rice plants against bacterial attack. FEMS Immunol Med Microbiol, 2002 Jun 3, 33(2), 143 - 9 Burkholderia anthina sp . nov . and Burkholderia pyrrocinia, two additional Burkholderia cepacia complex bacteria, may confound results of new molecular diagnostic tools; Vandamme P et al.; Nineteen Burkholderia cepacia-like isolates of human and environmental origin could not be assigned to one of the seven currently established genomovars using recently developed molecular diagnostic tools for B . cepacia complex bacteria . Various genotypic and phenotypic characteristics were examined . The results of this polyphasic study allowed classification of the 19 isolates as an eighth B . cepacia complex genomovar (Burkholderia anthina sp . nov.) and to design tools for its identification in the diagnostic laboratory . In addition, new and published data for Burkholderia pyrrocinia indicated that this soil bacterium is also a member of the B . cepacia complex . This highlights another potential source for diagnostic problems with B . cepacia-like bacteria. FEMS Microbiol Lett, 2002 May 7, 210(2), 181 - 5 A liquid-based method for the assessment of bacterial pathogenicity using the nematode Caenorhabditis elegans; Smith MP et al.; Caenorhabditis elegans has previously been used as an alternative to mammalian models of infection with bacterial pathogens . We have developed a liquid-based assay to measure the effect of bacteria on the feeding ability of C . elegans . Using this assay we have shown that Pseudomonas aeruginosa strain PA14, Burkholderia pseudomallei and Yersinia pestis were able to inhibit feeding of C . elegans strain N2 . An increase in sensitivity of the assay was achieved by using C . elegans mutant phm-2, in place of the wild-type strain . Using this assay,P . aeruginosa PA01 inhibited the feeding of C . elegans mutant phm-2 . Such liquid-based feeding assays are ideally suited to the high-throughput screening of mutants of bacterial pathogens. Microb Ecol, 2000 Apr, 39(3), 211 - 221 Catabolic and Genetic Diversity of Degradative Bacteria from Fuel-Hydrocarbon Contaminated Aquifers; Stapleton RD et al.; A BSTRACTSubsurface sediments were recovered from two aquifers contaminated with petroleum hydrocarbons in order to isolate and characterize indigenous microorganisms capable of biodegrading fuel-related compounds . These sediments had been previously studied using catabolic gene probes providing putative demonstration of significant biodegradation potential . Based on 16S rDNA sequence analysis, the isolates were phylogenetically similar to common soil microorganisms, including members of the genera Pseudomonas, Ralstonia, Burkholderia, Sphingomonas, Flavobacterium, and Bacillus . A total of 26 isolates were obtained using a vapor-plate enrichment technique with the volatile aromatic hydrocarbons toluene, ethylbenzene, p-xylene, naphthalene, and JP-4 jet fuel . JP-4, toluene, and ethylbenzene served as growth substrates for more than 80% of the isolates, while less than 10% of the organisms grew on the aromatic compounds benzene and o-xylene . Subsequent assays monitoring the evolution of (14)CO(2) indicated that only seven isolates were able to completely mineralize target compounds . One isolate, designated CAFB-naph-5, was able to completely mineralize the monoaromatic compounds salicylate and toluene, as well as the polyaromatic compound naphthalene . Molecular probing of the isolates showed four isolates hybridized with DNA probes targeting genes commonly associated with hydrocarbon-degrading bacteria . The isolates also demonstrated broad ability to grow in the presence of the antimicrobial agents streptomycin, tetracycline rifampicin, carbenicillin, nalidixic acid, kanamycin, and ampicillin . The results of the study demonstrate the biochemical and biodegradative capabilities of microorganisms isolated from contaminated aquifer systems and provide closure for indirect molecular monitoring of degradative potential in contaminated environments. Can J Microbiol, 2002 Apr, 48(4), 285 - 94 A N2-fixing endophytic Burkholderia sp . associated with maize plants cultivated in Mexico; Estrada P et al.; In the frame of a survey of potentially endophytic N2-fixing Burkholderia associated with maize in Mexico, its country of origin, the soil of an indigenous maize field near Oaxaca was studied . Under laboratory conditions, plant seedlings of two ancient maize varieties were used as a trap to select endophyte candidates from the soil sample . Among the N2 fixers isolated from inside plant tissues and able to grow on PCAT medium, the most abundant isolates belonged to genus Burkholderia (API 20NE, rrs sequences) . Representative isolates obtained from roots and shoots of different plants appeared identical (rrs and nifH RFLP), showing that they were closely related . In addition, their 16S rDNA sequences differed from described Burkholderia species and, phylogenetically, they constituted a separate deep-branching new lineage in genus Burkholderia . This indicated that these isolates probably constituted a new species . An inoculation experiment confirmed that these N2-fixing Burkholderia isolates could densely colonize the plant tissues of maize . More isolates of this group were subsequently obtained from field-grown maize and teosinte plants . It was hypothesized that strains of this species had developed a sort of primitive symbiosis with one of their host plants, teosinte, which persisted during the domestication of teosinte into maize. J Mol Evol, 2002 Jun, 54(6), 815 - 24 Identification and evolutionary analysis of putative cytoplasmic mcpA-like protein in a bacterial strain living in symbiosis with a mycorrhizal fungus; Minerdi D et al.; In this paper we report the identification and characterization of a DNA region containing putative mcpA-like gene coding for a Methyl Accepting Chemotaxis Protein (MCP) and belonging to a Burkholderia endosymbiont of the arbuscular mycorrhizal fungus Gigaspora margarita . A genomic library of total DNA extracted from the fungal spores, representative of the bacterial genome, was used to investigate the prokaryotic genome . PCR experiments with primers designed on the Burkholderia mcpA-like gene and Southern blot analysis demonstrate that they actually belong to the genome of G . margarita endosymbiont . The expression of the mcpA-like gene in the fungal spores was demonstrated by RT-PCR experiments . The detailed comparative analysis of the bacterial MCPs available in databases allowed to draw a possible evolutionary pathway leading to the present-day mcpA genes . Accordingly, the ancestor of the mcpA-like genes was the result of a domain shuffling event involving two ancestral mini-genes encoding a PAS-PAC and a MA domains, respectively, followed by the elongation of the PAS-PAC moiety . The following evolutionary divergence involved not only point mutations, but also larger rearrangements (insertions and deletions) at the 3' end of the gene. Pediatr Nurs, 2000 May-Jun, 26(3), 325 - 8 Burkholderia cepacia in cystic fibrosis: implications for nursing practice; Henskens JE et al.; Burkholderia Cepacia (B . cepacia) has emerged in the last 15 years as a dangerous pathogen among individuals with cystic fibrosis (CF) . Although this pathogen affects a relatively small percentage of the total CF population, it has had an enormous impact on the CF community because of the increased morbidity and mortality associated with it . Many CF centers have instituted stringent infection control practices in an effort to prevent the spread of this bacterium . Nurses who work with CF patients are faced with the challenge of continuing to care for patients who are B . cepacia positive, without putting unaffected individuals at risk . It is incumbent upon these nurses to educate themselves, coworkers, patients, and families about B . cepacia . Education should include the significance of colonization, transmission, treatment options, and infection control practices and interventions to deal with the psychosocial impact B . cepacia has on affected individuals and their families. FEMS Microbiol Lett, 2002 Apr 23, 210(1), 49 - 54 PCR cloning of polyhydroxyalkanoate biosynthesis genes from Burkholderia caryophylli and their functional expression in recombinant Escherichia coli; Hang X et al.; The PCR cloning strategy for type II polyhydroxyalkanoate (PHA) biosynthesis genes established previously for Pseudomonas was successfully applied to Burkholderia caryophylli strain AS 1.2741 . The whole pha locus containing PHA synthase genes phaC1, phaC2 and PHA depolymerase gene phaZ was cloned . The complete open reading frames of phaC1(Bc), phaC2(Bc) and phaZ(Bc) were identified . Sequence analyses of the phaC1(Bc), phaZ(Bc) and phaC2(Bc) showed more than 77.7%, 73.7% and 68.5% identities compared with the corresponding pha loci of the known Pseudomonas strains, respectively . The functional expression of the phaC1(Bc) or phaC2(Bc) in Escherichia coli strain KM32B (fadB deleted mutant) showed the abilities of PHA production by the estimated PHA synthase genes . Over 1% PHA consisting of 3-hydroxyhexanoate (3HHx), 3-hydroxyoctanoate (3HO) and 3-hydroxydecanoate (3HD) was detected from cells of recombinant E . coli KM32B (pHXM11) harboring phaC1(Bc), grown on octanoate . At the same time over 3% of PHA consisting of 3HO and 3HD was produced from cells of recombinant E . coli KM32B (pHXM21) harboring phaC2(BC), grown on decanoate . Results showed the PCR cloning strategy developed previously can be applied to non-Pseudomonas strains such as Burkholderia in this case . This result also provided evidence for the presumption that the Burkholderia strain possesses not only polyhydroxybutyrate synthase genes, but also synthase for medium-chain-length polyhydroxyalkanoates consisting of 3HHx, 3HO and 3HD. Antimicrob Agents Chemother, 2002 Jun, 46(6), 1793 - 9 Antibacterial activities and characterization of novel inhibitors of LpxC; Clements JM et al.; Lipid A is the hydrophobic anchor of lipopolysaccharide (LPS) and forms the major lipid component of the outer monolayer of the outer membrane of gram-negative bacteria . Lipid A is required for bacterial growth and virulence, and inhibition of its biosynthesis is lethal to bacteria . UDP-3-O-(R-3-hydroxymyristoyl)-N-acetylglucosamine deacetylase (LpxC) is a metalloenzyme that catalyzes the second step in the biosynthesis of lipid A . Inhibitors of LpxC have previously been shown to have antibiotic activities . We have screened a metalloenzyme inhibitor library for antibacterial activities against an Escherichia coli strain with reduced LpxC activity . From this screen, a series of sulfonamide derivatives of the alpha-(R)-amino hydroxamic acids, exemplified by BB-78484 and BB-78485, have been identified as having potent inhibitory activities against LpxC in an in vitro assay . Leads from this series showed gram-negative selective activities against members of the Enterobacteriaceae, Serratia marcescens, Morganella morganii, Haemophilus influenzae, Moraxella catarrhalis, and Burkholderia cepacia . BB-78484 was bactericidal against E . coli, achieving 3-log killing in 4 h at a concentration 4 times above the MIC, as would be predicted for an inhibitor of lipid A biosynthesis . E . coli mutants with decreased susceptibility to BB-78484 were selected . Analysis of these mutants revealed that resistance arose as a consequence of mutations in the fabZ or lpxC genes . These data confirm the antibacterial target of BB-78484 and BB-78485 and validate LpxC as a target for gram-negative selective antibacterials. Environ Microbiol, 2002 Apr, 4(4), 238 - 45 Effects of two different application methods of Burkholderia ambifaria MCI 7 on plant growth and rhizospheric bacterial diversity; Ciccillo F et al.; In order to acquire a better understanding of the effects of the different delivery modes of bacterial inoculants on plant growth and on the community structure of rhizosphere bacterial populations, Burkholderia ambifaria MCI 7 (formerly B . cepacia MCI 7) was inoculated into the rhizosphere of maize plants by either seed adhesion or incorporation into soil . Plant growth was evaluated at different inoculum concentrations . The community structure of rhizosphere bacterial populations was evaluated by analysing the restriction patterns of the DNA coding for 16S rRNA amplified by polymerase chain reaction (PCR) (ARDRA) of 745 bacterial isolates . A number of diversity indices (richness, Shannon diversity, evenness and mean genetic distance) were calculated for each bacterial population isolated from control and treated plants according to the concept of the r/K strategy . Moreover, the analysis of molecular variance (AMOVA) method was applied to estimate the genetic differences among the various bacterial populations . Our results showed that the method of application can be an essential element in determining the effects of the inoculant on plant growth . In fact, when applied as a maize seed treatment, B . ambifaria MCI 7 promoted plant growth significantly; on the contrary, when incorporated into soil, the same strain reduced plant growth markedly . As far as the bacterial community structure is concerned, B . ambifaria MCI 7 affected the indigenous microflora of treated plants according to the application method: seed treatment brought about an abrupt decrease in bacterial diversity, whereas incorporation into soil increased bacterial diversity . Moreover, changes in bacterial diversity were limited to r-strategist bacteria . In conclusion, B . ambifaria MCI 7 can act as both a plant growth-promoting rhizobacterium and a deleterious rhizobacterium depending on the inoculation method. Microbiol Immunol, 2002, 46(3), 143 - 50 Antigenic relatedness between Burkholderia pseudomallei and Burkholderia mallei; Anuntagool N et al.; Burkholderia pseudomallei and Burkholderia mallei are causative agents of distinct diseases, namely, melioidosis and glanders, respectively . The two species are very closely related, based on DNA-DNA homology, base sequence of the 16S rRNA, and phenotypic characteristics . Based on the use of polyclonal antisera, B . pseudomallei and B . mallei are also found to be antigenically closely related to one another . We previously reported the production of monoclonal antibodies (MAbs) against B . pseudomallei antigens; one group was specific for the 200-kDa exopolysaccharide present on the surface of all B . pseudomallei isolates, and the other was specific for the lipopolysaccharide (LPS) structure present on more than 95% of the B . pseudomallei tested . In the present study, we showed that the MAbs against 200-kDa antigen of B . pseudomallei cross-reacted with a component present also in some B . mallei isolates (3/6), but the positive immunoblot reaction was noted below the 200-kDa position . On the other hand, none of the six B . mallei isolates reacted with the MAb specific for B . pseudomallei LPS . It was of interest to observe that only the 3 exopolysaccharide-positive B . mallei isolates reacted with a commercial MAb against B . mallei LPS . The data presented suggest that B . mallei can be classified antigenically into two types based on their reactivities with different MAbs, i.e., the presence or absence of exopolysaccharide and the types of lipopolysaccharide . The heterogeneity of the LPS from these two closely related organisms is most likely related to the differences in its O-polysaccharide side chain. Int J Antimicrob Agents, 2002 May, 19(5), 427 - 9 A comparison of antibiotic susceptibility testing methods for cotrimoxazole with Burkholderia pseudomallei; Piliouras P et al.; Melioidosis is caused by the Gram-negative soil saprophyte, Burkholderia pseudomallei and is endemic in tropical and subtropical regions of southeast Asia and northern Australia . Cotrimoxazole has been traditionally used for the therapy of melioidosis despite results indicating resistance often produced in the disc diffusion test against B . pseudomallei . This inconsistency was addressed by comparing this method with the agar dilution, MicroScan and E-test methods . The results demonstrated that by disc diffusion, 41.3% of 80 B . pseudomallei clinical isolates tested were susceptible to cotrimoxazole, whereas the MicroScan, agar dilution and the E-test demonstrated 92.5, 90 and 97.5% of the isolates to be susceptible, respectively . These results indicate that an MIC based method is required to test the susceptibility of B . pseudomallei against cotrimoxazole. FEMS Microbiol Lett, 2002 Mar 19, 209(1), 99 - 106 Microbiological characterisation of Burkholderia cepacia isolates from cystic fibrosis patients: investigation of the exopolysaccharides produced; Lagatolla C et al.; Eleven strains of Burkholderia cepacia were isolated directly from clinical specimens: 10 from sputum of cystic fibrosis patients, and one from a vaginal swab . They were biochemically identified using API20NE and confirmed by a PCR-based assay . The genomovar characterisation obtained by specific PCR amplification revealed seven strains belonging to genomovar I, three belonging to genomovar IIIA and one belonging to genomovar IV . All isolates were also typed by ribotyping and random amplification of polymorphic DNA analysis . Some of the characterised strains were examined for the ability to produce exopolysaccharides, with the aim of correlating the genomovar with the exopolysaccharide structure . The polysaccharides were analysed by means of methylation analysis and 1H-NMR spectroscopy in order to determine structural similarities . It was shown that different strains are capable of producing chemically different polysaccharides. J Aerosol Med, 2002 Spring, 15(1), 51 - 7 A randomized double-blinded placebo-controlled crossover trial of nebulized taurolidine in adult cystic fibrosis patients infected with Burkholderia cepacia; Ledson MJ et al.; Burkholderia cepacia is an aggressive pathogen that colonizes cystic fibrosis (CF) patients, causing greatly increased morbidity and mortality . It is resistant to most antibiotics, but sensitive in vitro to a novel agent, taurolidine . This has not previously been used against B . cepacia, nor given in nebulized form . We assessed the effect of nebulized taurolidine on United Kingdom epidemic (ET12) B . cepacia infection in 20 adult CF patients attending our regional adult cystic fibrosis outpatient clinic using a prospective, randomized, double-blinded placebo-controlled crossover trial . Nebulized taurolidine (4 mL 2% solution) or saline (4 mL 0.9% solution) was given twice daily . Each arm lasted 4 weeks, with a 2-week intervening washout period . Sputum B . cepacia colony counts (primary outcome measure), spirometry, and symptoms (secondary outcome measures) were assessed . Eighteen patients completed the study . There was no change in B . cepacia colony counts or spirometry, nor symptom scores . We conclude that, although taurolidine is well tolerated in nebulized form, in this study it had no in vivo anti-B . cepacia activity. Environ Sci Technol, 2002 Apr 1, 36(7), 1579 - 83 Root turnover: an important source of microbial substrates in rhizosphere remediation of recalcitrant contaminants; Leigh MB et al.; The growth dynamics and phenolic content of mulberry (Morus sp.) fine roots (<1 mm diameter) were determined and examined in relationship to rhizosphere remediation of recalcitrant soil contaminants . Root turnover measurements of rhizotron-grown plants showed that 58% of the fine roots produced during a 6-month growing season (June-November) died at the end of the season . The concentration of phenolic compounds in fine roots increased approximately 2-fold during the later stages of the season, and the total phenolic content of dead fine roots reached a maximum value of 38 mg/g dry weight . The late-season increase in total phenolics was primarily due to accumulation of three different flavones (morusin, morusinol, and kuwanon C) . These three flavones were shown to support the growth of the bacterium Burkholderia sp . LB400, a degrader of polychlorinated biphenyls (PCBs) . Thus, it has been established that, upon death, the fine roots of mulberry can serve as a source of substrate for PCB-degrading bacteria . These results establish for the first time that the chemical content and turnover rate of fine roots should be considered an important aspect of rhizosphere remediation. Biodegradation, 2001, 12(5), 349 - 57 Soil microbial population dynamics following bioaugmentation with a 3-chlorobenzoate-degrading bacterial culture . Bioaugmentation effects on soil microorganisms; Gentry TJ et al.; Changes in microbial populations were evaluated following inoculation of contaminated soil with a 3-chlorobenzoate degrader . Madera sandy loam was amended with 0, 500, or 1,000 microg 3-chlorobenzoate g(-1) dry soil . Selected microcosms were inoculated with the degrader Comamonas testosteroni BR60 . Culturable bacterial degraders were enumerated on minimal salts media containing 3-chlorobenzoate . Culturable heterotrophic bacteria were enumerated on R2A . Isolated degraders were grouped by enterobacterial repetitive intergenic consensus sequence-polymerase chain reaction fingerprints and identified based on 16S ribosomal-DNA sequences . Bioaugmentation increased the rate of degradation at both levels of 3-chlorobenzoate . In both the 500 and 1,000 microg 3-chlorobenzoate g(-1) dry soil inoculated microcosms, degraders increased from the initial inoculum and decreased following degradation of 3-CB . Inoculation delayed the development of indigenous 3-chlorobenzoate degrading populations . It is unclear if inoculation altered the composition of indigenous degrader populations . In the uninoculated soil, degraders increased from undetectable levels to 6.6 x 10(7) colony-forming-units g(-1) dry soil in the 500 microg 3-chlorobenzoate g(-1) dry soil microcosms, but none were detected in the 1,000 microg 3-chlorobenzoate g(-1) dry soil microcosms . Degraders isolated from uninoculated soil were identified as one of two distinct Burkholderia species . In the uninoculated soil, numbers of culturable heterotrophic bacteria initially decreased following addition of 1,000 microg 3-chlorobenzoate g(-1) dry soil . Inoculation with C . testosteroni reduced this negative impact on culturable bacterial numbers . The results indicate that bioaugmentation may not only increase the rate of 3-chlorobenzoate degradation but also reduce the deleterious effects of 3-chlorbenzoate on indigenous soil microbial populations. Pest Manag Sci, 2002 May, 58(5), 451 - 8 Identification of alternatives for the management of foliar nematodes in floriculture; Jagdale GB et al.; The foliar nematodes, Aphelenchoides spp, have emerged as important pests of ornamentals in North America during the last decade . Due to the ban on the use of potentially toxic pesticides, there are currently no nematicides registered to manage foliar nematodes on ornamentals . Therefore, we have evaluated a biological {Burkholderia cepacia (syn Pseudomonas cepacia)}, two plant products {clove (Syzygium aromaticum) extract and Nimbecidine (azadirachtin)} and twelve chemical pesticides registered for the management of insects, mites, slugs or diseases of ornamentals, against Aphelenchoides fragariae on the most popular ornamental, hosta (Hosta spp), for two consecutive years . We found ZeroTol (270 g liter-1 peroxyacetic acid), currently labeled as a broad-spectrum fungicide/algicide, to be a very potent nematicide that killed 100% of the nematodes in water suspension . It also caused over 70% reduction in A fragariae population in soil and in the leaves without any phytotoxicity . B cepacia caused 67-85% reduction in A fragariae population in leaves and 50% reduction in the soil whereas insecticidal soap caused over 72% reduction in leaves and 61% reduction in the soil . Clove extract and Nimbecidine did not show any potential for the control of A fragariae on hosta . Although all twelve chemical pesticides were effective in reducing the population of A fragariae in the soil 45 days after treatment (DAT), only diazinon 475 g liter-1 EC, trichlorfon 800 g kg-1 SP, ethoprophos 100 g kg-1 GR, oxamyl 100 g kg-1 GR and ZeroTol caused over 70% reduction in nematode population compared with the control . In the leaves, only diazinon EC, trichlorfon SP, insecticidal soap, oxamyl GR and ZeroTol consistently caused over 70% nematode population reduction compared with the control at 45 DAT in both years . Thus, only diazinon EC, trichlorfon SP, oxamyl GR and ZeroTol consistently caused over 70% reduction in nematode population both in soil and leaves . Due to the recent ban by the US Environmental Protection Agency on the use of the first three of these formulations, only ZeroTol would serve as an effective tool to manage foliar nematodes in ornamentals . Although not as effective as ZeroTol in the soil, insecticidal soap is the only other alternative for foliar nematode management. Br Med Bull, 2002, 61, 81 - 96 Persistent and aggressive bacteria in the lungs of cystic fibrosis children; Hart CA et al.; There have been enormous improvements in life expectancy of patients with cystic fibrosis, especially with improved nutrition and better understanding of the basic cellular defects . However, infection in particular with Pseudomonas aeruginosa and Burkholderia cepacia, has the greatest effect in decreasing life expectancy . Although infections can be prevented by rigorous infection control procedures, early aggressive antimicrobial chemotherapy and established infection managed by antibiotics, they are not completely effective . A greater understanding of how the bacteria evade the host defences and produce infection is needed. J Infect Dis, 2002 May 15, 185(10), 1454 - 62 Epub 2002 Apr 23. Multilocus restriction typing: a novel tool for studying global epidemiology of Burkholderia cepacia complex infection in cystic fibrosis; Coenye T et al.; Burkholderia cepacia complex infections contribute significantly to mortality and morbidity in persons with cystic fibrosis (CF) . The aim of this study was to evaluate the use of a novel typing method, multilocus restriction typing (MLRT), for investigation of the global epidemiology of B . cepacia complex genomovar III, the species most commonly encountered in CF . In the MLRT method, variation at several loci is indexed by restriction analysis of polymerase chain reaction-amplified genes . Data obtained by MLRT and pulsed-field gel electrophoresis analysis of a large number of B . cepacia genomovar III isolates (including isolates belonging to epidemic lineages and environmental isolates) show a strong correlation . MLRT extends the utility of isolate genotyping by allowing comparisons of isolates collected in studies of larger scale (both temporal and spatial) . The portability of MLRT data will facilitate comparison of data obtained in different laboratories . In addition, data obtained with MLRT can be used in studies of bacterial population structure. J Med Microbiol, 2002 May, 51(5), 374 - 84 Distribution of type III secretion gene clusters in Burkholderia pseudomallei, B . thailandensis and B . mallei; Rainbow L et al.; Burkholderia pseudomallei, the causative agent of melioidosis, carries a cluster of genes closely related in organisation to the type III secretion (TTS) system gene clusters of the plant pathogens Ralstonia solanacearum and Xanthomonas spp . The TTS gene cluster (TTS1) is present only in B . pseudomallei and not in avirulent B . thailandensis . Adjacent to the gene cluster encoding putative secreton structural proteins lie a number of open reading frames (ORFs) encoding putative proteins with little or no homology to known proteins, with the exception of one predicted protein with homology to Pseudomonas syringae HrpK . In both R . solanacearum and Xanthomonas spp., genes in this location encode secreted effector proteins . RT-PCR analysis indicated that TTS genes, including two of these ORFs, are expressed in broth at 37 degrees C . Analysis of genome sequence data identified a second cluster of TTS genes (TTS2) present in both B . pseudomallei and B . mallei (99% identity) . However, B . mallei appears to lack the TTS1 gene cluster . PCR assays indicated that TTS2 was also present in B . thailandensis . TTS1 and TTS2 are similar in gene organisation, but nucleotide sequences are sufficiently divergent to suggest that the two TTS systems may have different roles. Respir Res . 2002;3(1):18. Binding of protegrin-1 to Pseudomonas aeruginosa and Burkholderia cepacia; Albrecht MT et al.; BACKGROUND: Pseudomonas aeruginosa and Burkholderia cepacia infections of cystic fibrosis patients' lungs are often resistant to conventional antibiotic therapy . Protegrins are antimicrobial peptides with potent activity against many bacteria, including P . aeruginosa . The present study evaluates the correlation between protegrin-1 (PG-1) sensitivity/resistance and protegrin binding in P . aeruginosa and B . cepacia . METHODS: The PG-1 sensitivity/resistance and PG-1 binding properties of P . aeruginosa and B . cepacia were assessed using radial diffusion assays, radioiodinated PG-1, and surface plasmon resonance (BiaCore) . RESULTS: The six P . aeruginosa strains examined were very sensitive to PG-1, exhibiting minimal active concentrations from 0.0625-0.5 microg/ml in radial diffusion assays . In contrast, all five B . cepacia strains examined were greater than 10-fold to 100-fold more resistant, with minimal active concentrations ranging from 6-10 microg/ml . When incubated with a radioiodinated variant of PG-1, a sensitive P . aeruginosa strain bound considerably more protegrin molecules per cell than a resistant B . cepacia strain . Binding/diffusion and surface plasmon resonance assays revealed that isolated lipopolysaccharide (LPS) and lipid A from the sensitive P . aeruginosa strains bound PG-1 more effectively than LPS and lipid A from resistant B . cepacia strains . CONCLUSION: These findings support the hypothesis that the relative resistance of B . cepacia to protegrin is due to a reduced number of PG-1 binding sites on the lipid A moiety of its LPS. Ned Tijdschr Geneeskd, 2002 Apr 13, 146(15), 723 - 5 {Melioidosis}; Leeuwenburgh I et al.; A 34-year-old woman presented two weeks after a visit to Burma with fever peaking up to 39 degrees C, chills, non-productive cough, headache, muscle pain, shortness of breath and a painful swelling on the left lower leg . She was treated immediately with intravenous amoxycillin-clavulanic acid . The Gram negative causative agent of melioidosis, Burkholderia (previously Pseudomonas) pseudomallei, was cultured from samples taken beforehand . The patient then received ceftazidime . She recovered . In view of the risk of relapse she was treated with amoxycillin-clavulanic acid for a further six months . Melioidosis is endemic in Southeast Asia and Northern Australia . It is rarely seen outside these areas . The clinical spectrum of the disease is wide and varies from fulminating sepsis to a subclinical disease and may affect any organ system, usually the lungs . The mortality of the septicaemic form after adequate treatment is 40% . Surviving patients have a high relapse rate (4-20%) . Melioidosis can become chronic with formation of abscesses or can remain subclinical for many years, probably because the microorganism can survive within phagocytic cells with a risk of reactivation at moments of immunosuppression . The optimal treatment consists of ceftazidime intravenously for at least two weeks followed by an eradication phase consisting of oral antibiotics for at least 3 months. J Hazard Mater, 2002 May 3, 92(1), 89 - 102 Effects of n-hexadecane and PM-100 clay on trichloroethylene degradation by Burkholderia cepacia; French WT et al.; Trichloroethylene (TCE) is a non-flammable, volatile organochlorine compound which was a widely used degreasing agent, anesthetic, and coolant prior to 1960, but has since been placed on the Environmental Protection Agency's (EPA) list of priority pollutants . The inadequate disposal practices for TCE have created numerous TCE-contaminated superfund sites . The most commonly employed practice for remediating TCE-contaminated sites is to purge the contaminant from the source and trap it onto an adsorbent which is disposed of in a landfill or by incineration . This investigation was undertaken to evaluate the effectiveness of Burkholderia cepacia strain G4 (G4) to regenerate used sorbents by degrading TCE from the sorbent directly or indirectly . The results of this investigation showed that G4 was capable of reducing TCE attached to PM-100 clay but at significantly reduced rate due to the slow desorption rate . Conversely, it was shown that G4 was capable of degrading TCE dissolved in n-hexadecane at the same rate as systems without n-hexadecane present . The reduction in TCE degradation when the TCE is attached to the PM-100 clay could be overcome by solvent rinsing the TCE from the clay with subsequent removal of the TCE from the n-hexadecane by G4. J Appl Microbiol, 2002, 92(5), 992 - 1004 Molecular epidemiology of cystic fibrosis-linked Burkholderia cepacia complex isolates from three national referral centres in Ireland; Crowley D et al.; AIMS: Burkholderia cepacia is a Gram-negative bacterium associated with increasing morbidity and mortality and is readily transmitted among infected cystic fibrosis (CF) patients . The B . cepacia complex consists of five distinct subgroups, termed genomovars . A collection of 17 presumptive B . cepacia isolates, obtained from three national CF referral centres located in different geographical regions in Ireland, was studied . The aim of this study was to investigate these isolates using molecular subtyping protocols for evidence of genetic relationships and for the presence of antibiotic resistance-encoding class 1 integron structures . METHODS AND RESULTS: Genomovar classifications were assigned to each isolate based on HaeIII enzyme profiles of their recA locus . Genetic relationships among this collection were also assessed after restriction fragment length polymorphism (RFLP)-mediated analysis of the 16S rDNA locus and DNA amplification fingerprinting (DAF) . The surface expression of the cable pilus gene (cblA) may facilitate an early step in the infection process . All isolates were tested by amplification strategies for this marker . Burkholderia cepacia is known to be resistant to several antimicrobial agents . Resistance typing showed that the majority were resistant to three or more common antimicrobial agents . Five of the 17 isolates were resistant to sulphonamide, a characteristic linked with the presence of class 1 integrons . Gene cassettes containing beta-lactamase (oxa) and aminoglycoside acetyltransferase (aac(6')-1a) encoding genes were identified by polymerase chain reaction amplification . CONCLUSIONS: Most of the isolates in this study were classified as genomovar III and were indistinguishable based on their corresponding 16S rDNA-RFLP profiles, whilst DAF further subtyped the collection . The cblA marker was identified in 47% of the isolates, many of which clustered in the genomovar III group . Class 1 integrons with recombined gene cassettes containing bla-OXA and aac(6')-1a genes were identified . SIGNIFICANCE AND IMPACT OF THE STUDY: This study demonstrates the application of molecular methods to investigate B . cepacia, a well-recognized human pathogen, cultured from Irish CF patients . Genomovar III was the most common genomic type identified . DNA fingerprinting further subtyped the latter isolates, facilitating a more detailed description of the molecular epidemiology . Drug resistance in these organisms can be explained, at least in part, by the presence of class 1 integrons . Development of targeted infection control strategies could be facilitated using these applied methods. Curr Opin Infect Dis, 2002 Apr, 15(2), 127 - 32 Melioidosis; Dance DA; Melioidosis is an important public health problem in some regions, and a potential bioweapon . Recent reports confirm that it is endemic in China, Taiwan and Laos, but the true incidence in most countries is unknown, and the ecology poorly understood . Potable water was the source of two recent outbreaks . The epidemiology and clinical manifestations of the disease in Australia are similar to those in Thailand, although prostatic abscesses and neurological manifestations are more common and parotid abscesses less so . Mycotic aneurysms are not uncommon . Patients with cystic fibrosis are at risk of pulmonary melioidosis . Comparison with the avirulent Burkholderia thailandensis has identified capsular polysaccharide as an important virulence determinant in Burkholderia pseudomallei . Diagnosis still relies on culture, and a throat swab is a worthwhile sample . Several beta-lactams, such as meropenem, reduce the mortality, and long courses of cotrimoxazole-containing regimes are needed to prevent relapse . The value of adjunctive treatments, such as granulocyte colony-stimulating factor, warrants further evaluation. Appl Microbiol Biotechnol, 2002 Apr, 58(5), 612 - 7 Epub 2002 Feb 08. A molybdenum-containing dehydrogenase catalyzing an unusual 2-hydroxylation of nicotinic acid; Schrader T et al.; An enzyme of Ralstonia/ Burkholderia strain DSM 6920 catalyzing the initial hydroxylation of 6-methylnicotinic acid at position 2 was purified to apparent homogeneity . It also catalyzed the unusual conversion of nicotinic acid to 2-hydroxynicotinic acid and was therefore designated as nicotinic acid dehydrogenase (NDH) . Native NDH had a molecular mass of 280 kDa and was composed of subunits of 75, 30 and 16 kDa . It contained molybdenum, iron, acid-labile sulfur and FAD in a ratio of 1.6:7.3:8.0:0.6 mol(-1) of native enzyme . The molybdenum cofactor was characterized as molybdopterin cytosine dinucleotide . Zinc was identified as an additional metal ion in a molar ratio of 1.8 mol mol(-1) of native enzyme . Purified NDH exhibited a maximal specific activity of 22.6 micromol nitro blue tetrazoliumchloride reduced min(-1) mg(-1) of protein, using nicotinic acid as electron donor . The apparent K(m) value for nicotinic acid was determined to be 154 microM . Pyridine-3,5-dicarboxylic acid and quinoline-3-carboxylic acid were further substrates, but exhibited significantly different activity pH optima . Several artificial electron acceptors were reduced by NDH, but no activity was detected with NAD or O(2) . NDH was inactivated upon incubation with cyanide, but no loss of activity was obtained in the presence of arsenite. Infect Immun, 2002 May, 70(5), 2715 - 20 Differential persistence among genomovars of the Burkholderia cepacia complex in a murine model of pulmonary infection; Chu KK et al.; Cystic fibrosis patients infected with strains from different genomovars of the Burkholderia cepacia complex can experience diverse clinical outcomes . To identify genomovar-specific determinants that might be responsible for these differences, we developed a pulmonary model of infection in BALB/c mice . Mice were rendered leukopenic by administration of cyclophosphamide prior to intranasal challenge with 1.6 x 10(4) bacteria . Five of six genomovar II strains persisted at stable numbers in the lungs until day 16 with minimal toxicity, whereas zero of seven genomovar III strains persisted but resulted in variable toxicity . We have developed a chronic pulmonary model of B . cepacia infection which reveals differences among genomovars in terms of clinical infection outcome. Infect Immun, 2002 May, 70(5), 2319 - 25 Nonviable Burkholderia mallei induces a mixed Th1- and Th2-like cytokine response in BALB/c mice; Amemiya K et al.; Nonviable cell preparations of Burkholderia mallei, the causative agent of glanders, were evaluated as potential vaccine candidates in a BALB/c murine model . Three different B . mallei cell preparations plus Alhydrogel were evaluated: a heat-killed preparation, an irradiation-inactivated preparation, and a preparation of a capsule-negative mutant strain which had been irradiation inactivated . BALB/c mice were vaccinated twice with the different B . mallei preparations, and spleens and sera were collected to determine their cellular and humoral immune responses . All three bacterial cell preparations had essentially the same results in two cellular immune response assays . In a splenocyte proliferation assay, the amount of cell proliferation in response to the homologous immunogen, concanavalin A, or lipopolysaccharide was similar for all the cell preparations . Also, splenocytes from the inoculated mice expressed interleukin 2 (IL-2), gamma interferon, and small amounts of IL-4 and IL-5, and more IL-10 cytokine in the presence of the homologous antigen . When the immunoglobulin subclasses from these mice were examined, they all produced higher levels of IgG1 than IgG2a subclasses . The higher ratio of IgG1 to IgG2a was not due to the amount of the immunogen or the adjuvant (Alhydrogel) used in the BALB/c mice . The cell preparations did not protect the vaccinated mice from a live challenge (>300 50% lethal doses) . Our results suggest that in BALB/c mice, a mixed T-helper-cell-like response to nonviable B . mallei is obtained, as demonstrated by a Th1- and Th2-like cytokine response and a Th2-like subclass immunoglobulin response . This may be the reason for the inability of the B . mallei cells that were examined as candidate vaccines to protect the mice from a live challenge. Intern Med J, 2002 Apr, 32(4), 143 - 8 An audit of the use of granulocyte colony-stimulating factor in septic shock; Stephens DP et al.; BACKGROUND: Granulocyte colony-stimulating factor (G-CSF) stimulates the production of neutrophils and modulates the function and activity of developing and mature neutrophils . In septic shock, the immune system can be considered one of the failing organ systems . G-CSF improves immune function and may be a useful adjunctive therapy in patients with septic shock . AIM: To evaluate the introduction of G-CSF as an adjunct to our standard treatment for community-acquired septic shock . METHODS: We performed a prospective data collection and analysis to determine whether the addition of G-CSF to our standard treatment for community-acquired septic shock was associated with improved hospital outcome, compared with an historical cohort of similar patients . We included all patients admitted to the Intensive Care Unit (ICU) with community-acquired septic shock between December 1998 and March 2000 . Patients received 300 microg G-CSF intravenously daily for 10 days in addition to our standard treatment for community-acquired septic shock . G-CSF was discontinued early if the patient was discharged from ICU before 10 days or if the absolute neutrophil count exceeded 75 x 10(6)/mL . RESULTS: A total of 36 patients with community-acquired septic shock, an average Apache 2 score of 26.7, and a predicted mortality of 0.79, were treated with G-CSF from December 1998 to March 2000 . Hospital mortality was 31% compared with an historical cohort of 11 similar patients with a hospital mortality of 73% (P = 0.018) . In the subgroup of patients with melioidosis septic shock, the hospital survival improved from 5% to 100% (P < 0.0001) . No significant adverse events occurred as a result of the administration of G-CSF . CONCLUSION: G-CSF is a safe adjunctive therapy in community-acquired septic shock and may be associated with improved outcome . The use of G-CSF in septic shock should undergo further investigation to define subgroups of patients who may benefit from G-CSF . The use of G-CSF in patients with septic shock due to Burkholderia pseudomallei is recommended. Chest, 2002 Apr, 121(4), 1117 - 22 Effects of an intensive 4-week summer camp on cystic fibrosis: pulmonary function, exercise tolerance, and nutrition; Blau H et al.; STUDY OBJECTIVES: Cystic fibrosis (CF) patients prefer exercise to most other forms of therapy, although objective improvement remains controversial . Israeli CF patients have attended a summer program in Switzerland for many years with subjective improvement . However, CF camps worldwide have been cancelled recently, due to fears of cross-infection with resistant organisms . Therefore, we evaluated the effect of attending the camp on pulmonary function, exercise tolerance, and nutritional state in CF patients . DESIGN: Weight, resting pulmonary function, incremental exercise test results, and sputum culture findings were assessed before and after a 4-week intensive summer camp . SETTING: Davos, Switzerland (altitude, 1,500 m) . PATIENTS: Thirteen Israeli CF patients (seven women and six men) with an age range of 9 to 25 years who had mild-to-moderate lung disease . No patients had Burkholderia cepacia detected in their sputum . INTERVENTIONS: The program included a high-calorie diet, chest physiotherapy, daily mountain climbing, and indoor activities . Arterial oxygen saturation (SaO(2)) was maintained at > 88% during exertion . RESULTS: Exercise tolerance improved significantly . The peak work capacity increased by 12.7%, the maximal oxygen uptake increased by 10%, and minute ventilation increased by 18.5% (p < 0.0005) . Of the calculated parameters, the anaerobic threshold improved by 17% . Ventilation was always the limiting factor during exercise, although it improved . There was no significant change in resting lung function and pulse or in SaO(2) decline at maximal exercise . The mean weight gain was 1 kg . No patient acquired B cepacia . CONCLUSIONS: An intensive summer camp improved exercise tolerance and nutrition in CF patients . This may explain improved patient well-being despite unchanged values for resting lung function . The reinstitution of summer camps, with special care to avoid cross-infection, should be considered. Wei Sheng Yan Jiu, 1999 Jul, 28(4), 232 - 5 {Sequencing and analysis of 16S rDNA sequences for P . cocovenenans subsp farinofermentans}; Jiao Z et al.; In order to fix the phylogenetic taxonomic position of P . cocovenenans subsp . farinofermentans and describe its relationships with other closed species, seven primers were designed to amplify and sequence the 16S rDNA of 4 strains of it by using a clonal sequencing or direct sequencing method . The 16S rDNA sequence of the 4 strains were aligned with the 16S rDNA sequences of other species in genus Burkholderia, and the polygenetic tree was produced by using a Clustal V and treeview software . The results showed that P . cocovenenans subsp . farinofermentans was in a high homology with Burkholderia gladioli and Burkholderia cocovenenans, and they formed an independent phylogenetic clustal of genus Burkholderia. Trans R Soc Trop Med Hyg, 2002 Jan-Feb, 96(1), 72 - 6 Cerebral melioidosis in Singapore: a review of five cases; Chadwick DR et al.; A variety of neurological manifestations of infection with Burkholderia pseudomallei have been described including cerebral abscesses, which are a well-recognized form of neurological melioidosis . The optimal antibiotic therapy for this condition has not been defined; however, combinations of intravenous antibiotics are frequently used in the early stages . Five cases of melioidosis involving brain abscesses are described which presented in Singapore over the past 3 years (1997-2000), 4 of which cases had evidence of disseminated infection . Despite profound neurological deficits and low Glasgow Coma Scale scores at presentation in 3 of these cases, all survived after surgical drainage and prolonged courses of intravenous ceftazidime or imipenem, and only 2 of whom had residual neurological impairment . One incidental finding on computed tomography (CT) or magnetic resonance imaging (MRI) scans not described before as an association with cerebral melioidosis was sinusitis in 4 out of the 5 cases. J Clin Microbiol, 2002 Apr, 40(4), 1188 - 93 Six-year molecular analysis of Burkholderia cepacia complex isolates among cystic fibrosis patients at a referral center for lung transplantation; Heath DG et al.; Over a 6-year period, Burkholderia cepacia complex species were isolated from cystic fibrosis (CF) patients receiving care at The University of North Carolina Hospitals (clinic CF patients) and from those referred from other treatment centers . Fifty-six isolates collected from 30 referred patients and 26 clinic CF patients were characterized by pulsed-field gel electrophoresis (PFGE) and were assayed by PCR to detect the cable pilin gene, cblA . PFGE results indicated that six separate clusters (clusters A to F) were present among the 56 isolates and that three clusters (clusters A, B, and E) consisted only of isolates from referred patients infected with B . cepacia complex isolates prior to referral . However, one cluster (cluster C) consisted of isolates from four CF patients, and hospital records indicate that this cluster began with an isolate that came from a referred patient and that spread to three clinic CF patients . Cluster D consisted of two isolates from clinic CF patients, and hospitalization records are consistent with nosocomial, patient-to-patient spread . cblA was present in only 4 of the 56 isolates and included isolates in cluster E from the referred patients . Our results indicate a lack of spread of a previously characterized, transmissible clone from referred patients to our clinic CF population . Only two instances of nosocomial, patient-to-patient spread could be documented over the 6-year period . An additional spread of an isolate (cluster F) from a referred patient to a clinic patient could not be documented as nosocomial and may have been the result of spread in a nonhospitalized setting . The majority (36 of 56) of our B . cepacia complex-infected CF patients harbor isolates with unique genotypes, indicating that a diversity of sources account for infection . These data suggest that CF patients infected with B . cepacia complex and referred for lung transplantation evaluation were not a major source of B . cepacia complex strains that infected our resident CF clinic population. J Clin Pathol, 2002 Apr, 55(4), 309 - 11 Misidentification of a genomovar of Burkholderia cepacia by recA restriction fragment length polymorphism; Moore JE et al.; An 8 year old girl with cystic fibrosis presented with a pulmonary exacerbation from which Burkholderia cepacia was cultured . Subsequent polymerase chain reaction restriction fragment length polymorphism analysis of the recA gene suggested the presence of B cepacia Genomovar V (Burkholderia vietnamiensis); however, on subsequent sequence typing, this isolate was confirmed as B cepacia Genomovar IIIb . This report outlines the potential difficulties in the correct characterisation of the various genomovars within the B cepacia complex of organisms, which has particularly important implications for patient segregation and infection control. Protein Eng, 2002 Feb, 15(2), 147 - 52 Modifying the chain-length selectivity of the lipase from Burkholderia cepacia KWI-56 through in vitro combinatorial mutagenesis in the substrate-binding site; Yang J et al.; The mature lipase of Burkholderia cepacia KWI-56 was synthesized in an enzymatically active form using an in vitro Escherichia coli S30 coupled transcription/translation system by expressing the mature lipase gene (rlip) in the presence of its specific activator . To investigate the substrate specificity of the lipase comprehensively, a large number of mutant lipases were constructed and analyzed in a high throughput manner by combining overlapping PCR and in vitro protein synthesis . In this paper, Phe119 and Leu167, which are located in the acyl portion of the substrate-binding pocket of the lipase of B.cepacia KWI-56, were substituted with six hydrophobic amino acid residues by the in vitro combinatorial mutagenesis . The wild-type and 35 mutant genes amplified by PCR were directly used as templates for the in vitro transcription/translation . The acyl chain-length selectivity of the in vitro expressed lipases against p-nitrophenyl butyrate, p-nitrophenyl caprylate and p-nitrophenyl palmitate, was compared by their relative hydrolysis rates . Two mutant lipases, L167V and F119A/L167M, which showed a significant shift in substrate selectivity were further expressed in vivo and refolded in vitro . It was found that L167V raised its preference for the short-chain ester, whereas F119A/L167M improved its selectivity for the long-chain ester. Appl Environ Microbiol, 2002 Apr, 68(4), 1728 - 34 Carbon isotope fractionation during aerobic biodegradation of trichloroethene by Burkholderia cepacia G4: a tool to map degradation mechanisms; Barth JA et al.; The strain Burkholderia cepacia G4 aerobically mineralized trichloroethene (TCE) to CO(2) over a time period of approximately 20 h . Three biodegradation experiments were conducted with different bacterial optical densities at 540 nm (OD(540)s) in order to test whether isotope fractionation was consistent . The resulting TCE degradation was 93, 83.8, and 57.2% (i.e., 7.0, 16.2, and 42.8% TCE remaining) at OD(540)s of 2.0, 1.1, and 0.6, respectively . ODs also correlated linearly with zero-order degradation rates (1.99, 1.11, and 0.64 micromol h(-1)) . While initial nonequilibrium mass losses of TCE produced only minor carbon isotope shifts (expressed in per mille delta(13)C(VPDB)), they were 57.2, 39.6, and 17.0 per thousand between the initial and final TCE levels for the three experiments, in decreasing order of their OD(540)s . Despite these strong isotope shifts, we found a largely uniform isotope fractionation . The latter is expressed with a Rayleigh enrichment factor, epsilon, and was -18.2 when all experiments were grouped to a common point of 42.8% TCE remaining . Although, decreases of epsilon to -20.7 were observed near complete degradation, our enrichment factors were significantly more negative than those reported for anaerobic dehalogenation of TCE . This indicates typical isotope fractionation for specific enzymatic mechanisms that can help to differentiate between degradation pathways. Appl Environ Microbiol, 2002 Apr, 68(4), 1595 - 603 Molecular method to assess the diversity of Burkholderia species in environmental samples; Salles JF et al.; In spite of the importance of many members of the genus Burkholderia in the soil microbial community, no direct method to assess the diversity of this genus has been developed so far . The aim of this work was the development of soil DNA-based PCR-denaturing gradient gel electrophoresis (DGGE), a powerful tool for studying the diversity of microbial communities, for detection and analysis of the Burkholderia diversity in soil samples . Primers specific for the genus Burkholderia were developed based on the 16S rRNA gene sequence and were evaluated in PCRs performed with genomic DNAs from Burkholderia and non-Burkholderia species as the templates . The primer system used exhibited good specificity and sensitivity for the majority of established species of the genus Burkholderia . DGGE analyses of the PCR products obtained showed that there were sufficient differences in migration behavior to distinguish the majority of the 14 Burkholderia species tested . Sequence analysis of amplicons generated with soil DNA exclusively revealed sequences affiliated with sequences of Burkholderia species, demonstrating that the PCR-DGGE method is suitable for studying the diversity of this genus in natural settings . A PCR-DGGE analysis of the Burkholderia communities in two grassland plots revealed differences in diversity mainly between bulk and rhizosphere soil samples; the communities in the latter samples produced more complex patterns. J Antimicrob Chemother, 2002 Apr, 49(4), 619 - 24 Identification of an immunodominant drug efflux pump in Burkholderia cepacia; Wigfield SM et al.; Burkholderia cepacia, a major pathogen amongst individuals with cystic fibrosis (CF), is intrinsically resistant to most clinically available antibiotics . We report the identification of an immunodominant antigen in CF patients infected with B . cepacia, a multidrug-resistance efflux pump called BcrA . The bcrA gene encodes a 46 kDa peptide with 14 potential alpha-helices that belongs to the major facilitator superfamily of drug transporters . A recombinant Escherichia coli strain was constructed containing the bcrA gene, which resulted in a four-fold increase in resistance to tetracycline and an eight-fold increase in resistance to nalidixic acid . These results demonstrate that the bcrA gene is part of a drug efflux system that is potentially a major contributor to the high-level antibiotic resistance observed in B . cepacia and thus a potential target for novel therapeutics. Arch Microbiol, 2002 Mar, 177(3), 267 - 73 Epub 2002 Jan 18. Characterization of a novel insertion sequence, IS Bp1, in Burkholderia pseudomallei; Woo PC et al.; During screening for antigenic proteins in Burkholderia pseudomallei, a novel insertion sequence, IS Bp1, was found by sequence similarity searches . IS Bp1 contains two overlapping ORFs of 261 bp ( orfA) and 852 bp ( orfB), encoding 87 and 284 amino acid residues, respectively, and an imperfect inverted repeat . The putative protein encoded by orfA (OrfA) is similar to the OrfA in insertion sequences of the IS 3 family in other bacteria, showing 49% and 76% amino acid identity and similarity, respectively, with the transposase encoded by IS D1 of Desulfovibrio vulgaris vulgaris . The putative protein encoded by orfB (OrfB) is similar to the OrfB in insertion sequences of the IS 3 family in other bacteria, showing 43% and 62% amino acid identity and similarity, respectively, with the transposase encoded by IS 1222 of Enterobacter agglomerans . Sequence analysis of OrfA showed the presence of an alpha-helix-turn-alpha-helix motif, as well as the putative leucine zipper at its 3' end, for possible DNA binding to the terminal inverted repeats . Sequence analysis of OrfB showed the presence of a DDE motif of aspartic acid, aspartic acid, and glutamic acid, a highly conserved motif present in OrfB of other members of the IS 3 family . Furthermore, several other conserved amino acid residues, including the arginine residue located seven amino acids downstream from the glutamic acid residue, were observed . PCR amplification of the IS Bp1 gene showed a specific band in 65% of the 26 B . pseudomallei strains tested . Southern blot hybridization after XhoI or SacI digestion showed nine different patterns of hybridization . The number of copies of IS Bp1 in those strains that possessed the insertion sequence ranged from three to 12 . Using several insertion sequences and a combination of insertion-sequence-based and non-insertion-sequence-based methods such as ribotyping will probably increase the discriminatory power of molecular typing in B . pseudomallei. Clin Microbiol Infect, 2002 Jan, 8(1), 47 - 9 Differential invasion of respiratory epithelial cells by members of the Burkholderia cepacia complex; Keig PM et al.; To investigate whether there are differences between members of the Burkholderia cepacia complex in their ability to invade human respiratory epithelial cells, 11 strains belonging to genomovars I-V were studied in an antibiotic protection assay using the A549 cell line . Strains belonging to genomovars II and III were more invasive than those of genomovars I, IV and V . There was also intra-genomovar variation in invasiveness . No correlation between invasiveness and other putative virulence factors of importance in B . cepacia infection in individuals with cystic fibrosis, cable pilus and B . cepacia epidemic strain marker was identified. Mol Gen Mikrobiol Virusol, 2002, (1), 7 - 11 {Burkholderia thailandensis: biological properties, identification and taxonomy}; Iliukhin VI et al.; Burkholderia pseudomallei-like microorganisms have been isolated from soil and water in regions with endemic melioidosis . These strains have biochemical and antigenic profiles identical to melioidosis agents, except that they differ by virulence and L-arabinose (vir-, ara+) . There are minor differences between these species by rRNA sequence . DNA hybridization and, more so, positive transformation of DNA auxotrophic mutants of B . pseudomallei by cell lysates of B . thailandensis and B . mallei confirmed the homology of these species' genomes . These members of the Burkholderia genus (pseudomallei, mallei, and thailandensis) can be regarded as a supraspecies taxon: pseudomallei group . B . thailandensis strains are not virulent for guinea pigs and slightly virulent for golden hamsters . Immunization with live cultures of B . thailandensis protected more than 50% guinea pigs challenged with 200 LD50 B . pseudomallei 100 . B . thailandensis is suggested as a potential melioidosis vaccine. Antimicrob Agents Chemother, 2002 Apr, 46(4), 1132 - 5 Cloning and expression of class A beta-lactamase gene blaA(BPS) in Burkholderia pseudomallei; Cheung TK et al.; The beta-lactamase gene blaA(BPS) in Burkholderia pseudomallei was cloned and expressed in Escherichia coli . BPS-1 is a cephalosporinase with an isoelectric point of 7.7 . Sequence analysis of BPS-1 revealed conserved motifs typical of class A beta-lactamases and a relationship to the PenA (in B . cepacia) and BlaI (in Yersinia enterocolitica) lineages. Emerg Infect Dis, 2002 Feb, 8(2), 181 - 7 Epidemiology of Burkholderia cepacia complex in patients with cystic fibrosis, Canada; Speert DP et al.; The Burkholderia cepacia complex is an important group of pathogens in patients with cystic fibrosis (CF) . Although evidence for patient-to-patient spread is clear, microbial factors facilitating transmission are poorly understood . To identify microbial clones with enhanced transmissibility, we evaluated B . cepacia complex isolates from patients with CF from throughout Canada . A total of 905 isolates from the B . cepacia complex were recovered from 447 patients in 8 of the 10 provinces; 369 (83%) of these patients had genomovar III and 43 (9.6%) had B . multivorans (genomovar II) . Infection prevalence differed substantially by region (22% of patients in Ontario vs . 5% in Quebec) . Results of typing by random amplified polymorphic DNA analysis or pulsed-field gel electrophoresis indicated that strains of B . cepacia complex from genomovar III are the most potentially transmissible and that the B . cepacia epidemic strain marker is a robust marker for transmissibility. Cell Microbiol, 2002 Feb, 4(2), 73 - 86 Interaction of cblA/adhesin-positive Burkholderia cepacia with squamous epithelium; Sajjan U et al.; A highly transmissible strain of Burkholderia cepacia from genomovar III carries the cable pilin gene, expresses the 22 kDa adhesin (cblA +ve/Adh +ve), binds to cytokeratin 13 (CK13) and is invasive . CK13 is expressed abundantly in the airway epithelia of cystic fibrosis (CF) patients . We have now investigated whether binding of cblA +ve/Adh +ve B . cepacia to CK13 potentiates bacterial invasion and epithelial damage using bronchial epithelial cell cultures differentiated into either squamous (CK13-enriched) or mucociliary (CK13-deficient) epithelia . Three different B . cepacia isolates (cblA +ve/Adh +ve, cblA +ve/Adh -ve and cblA -ve/Adh -ve) showed minimal binding to mucociliary cultures, and did not invade or cause cell damage . In contrast, the cblA +ve/Adh +ve isolate, but not others, bound to CK13-expressing cells in squamous cultures, caused cytotoxicity and stimulated IL-8 release within 2 h . By 24 h, this isolate invaded and migrated across the squamous culture, causing moderate to severe epithelial damage . A specific antiadhesin antibody, which blocked the initial binding of the cblA +ve/Adh +ve isolate to CK13, significantly inhibited all the pathologic effects . Transmission electron microscopy of squamous cultures incubated with the cblA +ve/Adh +ve isolate, revealed bacteria on the surface surrounded by filopodia by 2 h, and within the cells in membrane-bound vesicles by 24 h . Bacteria were also observed free in the cytoplasm, surrounded by intermediate filaments containing CK13 . These findings suggest that binding of B . cepacia to CK13 is an important initial event and that it promotes bacterial invasion and epithelial damage. Infect Immun, 2002 Apr, 70(4), 1799 - 806 Role of flagella in host cell invasion by Burkholderia cepacia; Tomich M et al.; Burkholderia cepacia is an important opportunistic human pathogen that affects immunocompromised individuals, particularly cystic fibrosis (CF) patients . Colonization of the lungs of a CF patient by B . cepacia can lead not only to a decline in respiratory function but also to an acute systemic infection, such as bacteremia . We have previously demonstrated that a CF clinical isolate of B . cepacia, strain J2315, can invade and survive within cultured respiratory epithelial cells . In order to further characterize the mechanisms of invasion of B . cepacia, we screened a transposon-generated mutant library of strain J2315 for mutants defective in invasion of A549 respiratory epithelial cells . Here we describe isolation and characterization of a nonmotile mutant of B . cepacia with reduced invasiveness due to disruption of fliG, which encodes a component of the motor-switch complex of the flagellar basal body . We also found that a defined null mutation in fliI, a gene encoding a highly conserved ATPase required for protein translocation via the flagellar type III secretion system, also resulted in loss of motility and a significant reduction in invasion . Both mutants lacked detectable intracellular flagellin and failed to export detectable amounts of flagellin into culture supernatants, suggesting that disruption of fliG and fliI impaired flagellar biogenesis . The reduction in invasion did not appear to be due to defective adherence of the flagellar mutants to A549 cells, suggesting that functional flagella and motility are required for full invasiveness of B . cepacia . Our findings indicate that flagellum-mediated motility may facilitate penetration of host epithelial barriers by B . cepacia, contributing to establishment of infection and systemic spread of the organism. Infect Control Hosp Epidemiol, 2002 Feb, 23(2), 103 - 6 Outbreak of burkholderia cepacia in the adult intensive care unit traced to contaminated indigo-carmine dye; Gravel D et al.; We report an unusual cluster of Burkholderia cepacia in patients . Environmental cultures identified indigo-carmine dye used in enteral feeding as the reservoir . Compared with the controls, the cases were significantly more likely to have received tube feedings tinted with this dye . This outbreak was terminated with the removal of the dye from hospital inventory. Carbohydr Res, 2002 Mar 15, 337(6), 557 - 9 Quantitative production of 2-acetamido-2-deoxy-D-glucose from crystalline chitin by bacterial chitinase; Pichyangkura R et al.; Finely powdered alpha- and beta-chitin can be completely hydrolyzed with chitinase (EC 3.2.1.14) and beta-N-acetylhexosaminidase (EC 3.2.1.52) for the production of 2-acetamido-2-deoxy-D-glucose (GlcNAc) . Crude chitinase from Burkholderia cepacia TU09 and Bacillus licheniformis SK-1 were used to digest alpha- and beta-chitin powder . Chitinase from B . cepacia TU09 produced GlcNAc in greater than 85% yield from beta- and alpha-chitin within 1 and 7 days, respectively . B . licheniformis SK-1 chitinase completely hydrolyzed beta-chitin within 6 days, giving a final GlcNAc yield of 75%, along with 20% of chitobiose . However, only a 41% yield of GlcNAc was achieved from digesting alpha-chitin with B . licheniformis SK-1 chitinase. FEMS Microbiol Lett, 2002 Jan 22, 207(1), 1 - 7 Direct detection of N-acylhomoserine lactones in cystic fibrosis sputum; Middleton B et al.; Pseudomonas aeruginosa and Burkholderia cepacia cause destructive lung disease in cystic fibrosis (CF) patients . Both pathogens employ 'quorum sensing', i.e . cell-to-cell communication, via diffusible N-acyl-L-homoserine lactone (AHL) signal molecules, to regulate the production of a number of virulence determinants in vitro . However, to date, evidence that quorum sensing systems are functional and play a role in vivo is lacking . This study presents the first direct evidence for the presence of AHLs in CF sputum . A total of 42 samples from 25 CF patients were analysed using lux-based Escherichia coli AHL biosensors . AHLs were detected in sputum from patients colonised by P . aeruginosa or B . cepacia but not Staphylococcus aureus . Furthermore, using liquid chromatography-mass spectrometry and thin layer chromatography, we confirmed the presence of N-hexanoylhomoserine lactone and N-(3-oxododecanoyl)homoserine lactone respectively in sputum samples from patients colonised by P . aeruginosa. J Hosp Infect, 2002 Mar, 50(3), 188 - 95 Burkholderia cepacia complex in cystic fibrosis and non-cystic fibrosis patients: identification of a cluster of epidemic lineages; Agodi A et al.; This study was performed in order to compare Burkholderia cepacia complex strains from cystic fibrosis (CF) and non-CF patients at the genomovar, genetic and epidemiological levels . A total of 92 B . cepacia respiratory tract isolates were obtained from patients attending the following CF centres: Catania and Palermo, Sicily; Gualdo Tadino, Central Italy, and Milan, Northern Italy . A total of 23 B . cepacia isolates were obtained from blood, surgical wound, and intravenous catheter sources of patients without CF, hospitalized in Catania and Varese, Northern Italy . Genomovar status identification, clonality and genetic relatedness determination, antibiotic susceptibility pattern determination and electron microscopy were performed . Transmission of infection was shown in both CF and non-CF patients by identifying clonality of responsible strains . In total 13 clones were involved in cross-transmission episodes . No outbreak was described involving both CF and non-CF patients . The present study indicates the existence of a distinct cluster of strains responsible for epidemics in CF and non-CF patients, based on their genetic relatedness, distinct from strains associated with no or negligible transmissibility . This result suggests that transmissibility is not only associated with a specific genomovar in CF patients, but also with a group of genetically related lineages in CF and non-CF patients . A key role is shown for both segregation measures and careful surveillance of infection, based on selective culture, molecular identification and epidemiological characterization of individual isolates . J Clin Microbiol, 2002 Mar, 40(3), 846 - 51 Burkholderia cepacia complex bacteria from clinical and environmental sources in Italy: genomovar status and distribution of traits related to virulence and transmissibility; Bevivino A et al.; Sixty-eight Burkholderia cepacia complex isolates recovered from the sputum of 53 cystic fibrosis patients and 75 isolates collected from the maize rhizosphere were compared to each other to assess their genomovar status as well as some traits related to virulence such as antibiotic susceptibility, proteolytic and hemolytic activities, and transmissibility, in which transmissibility is determined by detection of the esmR and cblA genes . Among the clinical isolates, B . cepacia genomovar III comprised the majority of isolates examined and only a very few isolates were assigned to B . cepacia genomovar I, B . stabilis, and B . pyrrocinia; among the environmental isolates a prevalence of B . cepacia genomovar III and B . ambifaria was observed, whereas few environmental isolates belonging to B . cepacia genomovar I and B . pyrrocinia were found . Antibiotic resistance analysis revealed a certain degree of differentiation between clinical and environmental isolates . Proteolytic activity and onion tissue maceration ability were found to be spread equally among both clinical and environmental isolates, whereas larger percentages of environmental isolates than clinical isolates had hemolytic activity . The esmR gene was found exclusively among isolates belonging to B . cepacia genomovar III, with a marked prevalence in clinical isolates, whereas only one clinical isolate belonging to B . cepacia genomovar III was found to bear the cblA gene . In conclusion, the results of the present study show that the species compositions of the clinical and environmental B . cepacia complex populations examined are quite different and that some of the candidate determinants related to virulence and transmissibility are not confined solely to clinical isolates but are also spread among environmental isolates belonging to different species of the B . cepacia complex. Appl Microbiol Biotechnol, 2002 Feb, 58(2), 202 - 9 Molecular detection and diversity of polycyclic aromatic hydrocarbon-degrading bacteria isolated from geographically diverse sites; Widada J et al.; Nineteen polycyclic aromatic hydrocarbon (PAH)-degrading bacteria were isolated from environmental samples in Kuwait, Indonesia, Thailand, and Japan by enrichment with either naphthalene or phenanthrene as a sole carbon source . Sequence analyses of the 16-S rRNA gene indicated that at least seven genera (Ralstonia, Sphingomonas, Burkholderia, Pseudomonas, Comamonas, Flavobacterium, and Bacillus) were present in this collection . Determination of the ability of the isolates to use PAH and its presumed catabolic intermediates suggests that the isolates showed multiple phenotypes in terms of utilization and degradation pathways . The large subunit of the terminal oxygenase gene (phnAc) from Burkholderia sp . strain RP007 hybridized to 32% (6/19) of the isolates, whilst gene probing using the large subunit of terminal oxygenase gene (pahAc) from Pseudomonas putida strain OUS82 revealed no pahAc-like genes amongst the isolates . Using three degenerated primer sets (pPAH-F/NR700, AJ025/26, and RieskeF/R), targeting a conserved region with the genes encoding the large subunit of terminal oxygenase successfully amplified material from 6 additional PAH-degrading isolates . Sequence analyses showed that the large subunit of terminal oxygenase in 4 isolates was highly homologous to the large subunit of naphthalene dioxygenase gene from Ralstonia sp . strain U2 . However, we could not obtain any information on the oxygenase system involved in the naphthalene and/or phenathrene degradation by 7 other strains . These results suggest that PAH-degrading bacteria are diverse, and that there are still many unidentified PAH-degrading bacteria. Appl Environ Microbiol, 2002 Mar, 68(3), 1047 - 54 Characterization of a chitinase gene from Stenotrophomonas maltophilia strain 34S1 and its involvement in biological control; Kobayashi DY et al.; A chitinase gene was cloned on a 2.8-kb DNA fragment from Stenotrophomonas maltophilia strain 34S1 by heterologous expression in Burkholderia cepacia . Sequence analysis of this fragment identified an open reading frame encoding a deduced protein of 700 amino acids . Removal of the signal peptide sequence resulted in a predicted protein that was 68 kDa in size . Analysis of the sequence indicated that the chitinase contained a catalytic domain belonging to family 18 of glycosyl hydrolases . Three putative binding domains, a chitin binding domain, a novel polycystic kidney disease (PKD) domain, and a fibronectin type III domain, were also identified within the sequence . Pairwise comparisons of each domain to the most closely related sequences found in database searches clearly demonstrated variation in gene sources and the species from which related sequences originated . A 51-kDa protein with chitinolytic activity was purified from culture filtrates of S . maltophilia strain 34S1 by hydrophobic interaction chromatography . Although the protein was significantly smaller than the size predicted from the sequence, the N-terminal sequence verified that the first 15 amino acids were identical to the deduced sequence of the mature protein encoded by chiA . Marker exchange mutagenesis of chiA resulted in mutant strain C5, which was devoid of chitinolytic activity and lacked the 51-kDa protein in culture filtrates . Strain C5 was also reduced in the ability to suppress summer patch disease on Kentucky bluegrass, supporting a role for the enzyme in the biocontrol activity of S . maltophilia. Clin Microbiol Infect, 1996, 2(2), 91 - 98 Meropenem in cystic fibrosis patients infected with resistant Pseudomonas aeruginosa or Burkholderia cepacia and with hypersensitivity to beta-lactam antibiotics; Ciofu O et al.; OBJECTIVE: To assess the efficacy and safety of meropenem, administered on a compassionate basis to 62 cystic fibrosis (CF) patients (age: 24plus minus6 years) with hypersensitivity reactions to beta-lactam antibiotics and/or infection by bacteria resistant to other antibiotics . METHODS: Fifty-seven patients were chronically infected with Pseudomonas aeruginosa and 5 with Burkholderia cepacia . In total, 124 courses (1 to 6/patient) of meropenem, 2 g three times a day by intravenous infusion (10 to 15 min) for 14 days, were administered . RESULTS: During treatment for P . aeruginosa the mean increase in pulmonary function (as a percentage of the predictive values) was 5.6% for FEV1 (forced expiratory volume in the first second) and 8.6% for FVC (forced vital capacity) . C-reactive protein and erythrocyte sedimentation rate (ESR) and leukocyte count decreased significantly . In courses administered for chronic infection with B . cepacia the post treatment FEV1 and FVC values were higher than the pre-treatment values, and all the inflammatory parameters decreased . The geometric means of minimal inhibitory concentration (MICs) (microg/mL) for P . aeruginosa (B . cepacia) were: tobramycin 6 (59), ciprofloxacin 1.2 (9.7), piperacillin 49 (16.3), ceftazidime 26 (23), aztreonam 26 (35), imipenem 6.4 (not determined) and meropenem 5.1 (4.8) . No statistically significant increase in the MICs of meropenem for either pathogen occurred during therapy . Of the 124 courses, 115 were tolerated without any clinical complaint . The following side effects were observed: nausea (0.8%), itching (4%), rash (3.2%), drug fever (1.6%) . CONCLUSIONS: Meropenem proved to be a valuable drug in the treatment of CF patients with chronic pulmonary infection with multiresistant P . aeruginosa and B . cepacia and with hypersensitivity reactions to other beta-lactam drugs. Clin Microbiol Infect, 1996 Aug, 2(1), 59 - 62 Enterobacterial intergenic consensus sequence polymerase chain reaction as a typing method for Burkholderia (Pseudomonas) cepacia; Cimolai N et al.; OBJECTIVE: We developed a rapid polymerase chain reaction (PCR) method, which utilizes primers from an enterobacterial repetitive consensus sequence, to differentiate among strains of Burkholderia cepacia . We then applied this method to isolates from a device-associated pseudoinfection and also compared the discriminative typing potential with that of another PCR-based method . METHODS: A simplified lysis procedure was used to generate DNA substrate for amplification by a PCR method which utilized primer ERIC2 and a reverse complement of ERIC1R . The alternative PCR-based method employed primers which are directed to Bordetella pertussis repetitive sequences . Amplification products were visually assessed after conventional agarose gel electrophoresis and staining . RESULTS: The ERIC-based method appropriately clustered strains for the device-associated pseudo-infection . In addition, there was consistency in the definition of similar or dissimilar strains between the two PCR---based systems . CONCLUSIONS: The ERIC-based PCR typing method offers sufficient discriminatory power for it to be used for epidemiologic purposes. Infect Immun, 2002 Mar, 70(3), 1081 - 6 Correlation between an in vitro invasion assay and a murine model of Burkholderia cepacia lung infection; Cieri MV et al.; Our understanding of the virulence of Burkholderia cepacia complex lung infections in cystic fibrosis patients is incomplete . There is a great deal of variability in the clinical course, from simple colonization to severe and often fatal necrotizing pneumonia, termed cepacia syndrome . Multiple subspecies (called genomovars) have been identified, and these genomovars may hold the key to understanding the variable pathogenicity . Thirty-one B . cepacia complex isolates belonging to five of the seven genomovars were examined by using a gentamicin protection assay of invasion with A549 cells . The level of epithelial cell invasion by B . cepacia in the A549 model was relatively low compared with the data obtained for other pathogens and was often variable from assay to assay . Thus, a statistical approach was used to determine invasiveness . When this model was used, one of four genomovar I strains (25%), three of eight genomovar II strains (37.5%), seven of nine genomovar III strains (77.8%), one of four genomovar IV strains (25%), and none of the four genomovar V strains examined were defined as invasive . All other strains were categorized as either noninvasive or indeterminate . Invasive, noninvasive, and indeterminate isolates belonging to genomovars II and III were subsequently tested for splenic invasion with the mouse agar bead model . Correlation between the models for six strains was demonstrated . Our results indicate that a statistical model used to determine invasiveness in an in vitro invasion assay can be used to predict in vivo invasiveness. Protein Sci, 2002 Mar, 11(3), 467 - 78 EstB from Burkholderia gladioli: a novel esterase with a beta-lactamase fold reveals steric factors to discriminate between esterolytic and beta-lactam cleaving activity; Wagner UG et al.; Esterases form a diverse class of enzymes of largely unknown physiological role . Because many drugs and pesticides carry ester functions, the hydrolysis of such compounds forms at least one potential biological function . Carboxylesterases catalyze the hydrolysis of short chain aliphatic and aromatic carboxylic ester compounds . Esterases, D-alanyl-D-alanine-peptidases (DD-peptidases) and beta-lactamases can be grouped into two distinct classes of hydrolases with different folds and topologically unrelated catalytic residues, the one class comprising of esterases, the other one of beta-lactamases and DD-peptidases . The chemical reactivities of esters and beta-lactams towards hydrolysis are quite similar, which raises the question of which factors prevent esterases from displaying beta-lactamase activity and vice versa . Here we describe the crystal structure of EstB, an esterase isolated from Burkholderia gladioli . It shows the protein to belong to a novel class of esterases with homology to Penicillin binding proteins, notably DD-peptidase and class C beta-lactamases . Site-directed mutagenesis and the crystal structure of the complex with diisopropyl-fluorophosphate suggest Ser75 within the "beta-lactamase" Ser-x-x-Lys motif to act as catalytic nucleophile . Despite its structural homology to beta-lactamases, EstB shows no beta-lactamase activity . Although the nature and arrangement of active-site residues is very similar between EstB and homologous beta-lactamases, there are considerable differences in the shape of the active site tunnel . Modeling studies suggest steric factors to account for the enzyme's selectivity for ester hydrolysis versus beta-lactam cleavage. Environ Microbiol, 2001 Dec, 3(12), 766 - 73 Detection and activity of insertion sequences in environmental strains of Burkholderia; Miche L et al.; The presence of two insertion sequences, IS406 and IS407, was tested by polymerase chain reaction (PCR) amplification in 25 strains representing 15 Burkholderia species and the close relative Ralstonia pickettii . A total of 50% of the 25 strains contained at least one of the two insertion sequences (ISs) and a statistically significant correlation was found between the occurrences of IS406 and IS407 . Moreover, PCR-RFLP studies of the amplified fragments showed that IS406 is largely conserved among all the strains tested, whereas IS407 is rather polymorphic . Transposition activity was studied in Burkholderia vietnamiensis TVV75, using the pGBG1 target plasmid . This entrapp