Burkholderia

Mol Cell Probes, 2005 Feb, 19(1), 9 - 20
Rapid presumptive identification of Burkholderia pseudomallei with real-time PCR assays using fluorescent hybridization probes; Tomaso H et al.; Burkholderia pseudomallei (the etiologic agent of melioidosis) can cause pyogenic or granulomatous lesions in almost any organ . Septicemia has a case fatality rate of >40% . Early diagnosis and appropriate antibiotic therapy are crucial for survival, but cultivation, biochemical identification, and conventional PCR of B . pseudomallei are time consuming . We established real-time PCR assays using fluorescent hybridization probes targeting the 16S rDNA, the flagellin C (fliC) and the ribosomal protein subunit S21 (rpsU) genes . The test sensitivity and specificity were assessed with a representative panel of 39 B . pseudomallei, 9 B . mallei, 126 other Burkholderia strains of 29 species, and 45 clinically relevant non-Burkholderia organisms . The detection limit for the 16S rDNA, fliC, and rpsU assay was 40, 40, and 400 genome equivalents per reaction, however, in spiked blood samples it was 300, 300, and 3000, respectively . Specificity, positive and negative predictive value of the assays was 100% . In conclusion, we recommend the use of the 16S rDNA and/or fliC real-time PCR assays for the rapid identification of B . mallei and B . pseudomallei in positive blood cultures or from suspicious bacterial colonies.

Microb Drug Resist, 2004 Winter, 10(4), 269 - 79
Three-Dimensional Model and Molecular Mechanism of Mycobacterium tuberculosis Catalase-Peroxidase (KatG) and Isoniazid-Resistant KatG Mutants; Mo L et al.; Mycobacterium tuberculosis KatG enzyme functions both as catalase for removing hydrogen peroxide (H(2)O(2)) and as peroxidase for oxidating isoniazid (INH) to active form of anti-tuberculosis drug . Although mutations in M . tuberculosis KatG confer INH resistance in tuberculous patients, structural bases for INH-resistant mutations in the KatG gene remains poorly understood . Here, three M . tuberculosis KatG mutants bearing Arg418--> Gln, Ser315 --> Thr, or Trp321 --> Gly replacement were assessed for changes in catalase-peroxidase activities and possible structure bases relevant to such changes . These three M . tuberculosis KatG mutants exhibited a marked impairment or loss of catalase-peroxidase activities . The possible structural bases for the mutant-induced loss of enzyme activities were then analyzed using a three-dimensional model of M . tuberculosis KatG protein constructed on the basis of the crystal structure of the catalase-peroxidase from Burkholderia pseudomallei . The model suggests that three M . tuberculosis KatG mutants bearing Arg418 --> Gln, Ser315 -->Thr, or Trp321--> Gly replacement affect enzyme activities by different mechanisms, although each of them impacts consequently on a heme-associated structure, the putative oxidative site . Moreover, in addition to the widely accepted substrate-binding site, M . tuberculosis KatG may bear another H(2)O(2) binding site . This H(2)O(2) binding site appears to interact with the catalytic site by a possible electron-transfer chain, a Met255-Tyr229-Trp107 triad conserved in many catalase-peroxidases . The Ser315 --> Thr mutant may have direct effect on the catalytic site by interfering with electron transfer in addition to the previously proposed mechanism of steric constraint.

Mol Cells, 2004 Dec 31, 18(3), 390 - 5
Enhanced Expression of a Gene Encoding a Nucleoside Diphosphate Kinase 1 (OsNDPK1) in Rice Plants upon Infection with Bacterial Pathogens; Cho SM et al.; A cDNA library was constructed using mRNA extracted from rice leaves infected with Xanthomonas oryzae pv . oryzae (Xoo), a bacterial leaf blight pathogen, to isolate rice genes induced by Xoo infection . Subtractive hybridization and differential screening of the cDNA library led to the isolation of many induced genes including a nucleotide diphosphate kinase 1 (OsNDPK1) and a pathogenesis-related protein 1 (OsPR1) cDNA . Nucleoside diphosphate kinases (NDPKs) are key metabolic enzymes that maintain the balance between cellular ATP and other nucleoside triphosphates (NTPs) . Three other OsNDPK genes (NP922751, OsNDPK2 and OsNDPK3) found in databases were obtained by RT-PCR . Three different programs for predicting subcellular targeting indicated that OsNDPK1 and NP922751 were non-organellar, OsNDPK2 plastidic, and OsNDPK3 mitochondrial . Only transcripts of OsNDPK1 accumulated strongly after infection with Xoo . When rice plants were infected with Burkholderia glumae, a bacterial grain/seedling rot pathogen, the pattern of expression of the rice NDPK genes was similar to that following infection with Xoo . OsNDPK1 gene expression was also strongly induced in response to exposure to salicylic acid, jasmonic acid, and abscisic acid, although the level of transcripts and their pattern of expression depended on the inducer.

J Microbiol Methods, 2005 Mar, 60(3), 417 - 22
Insight into pollutant bioavailability and toxicity using Raman confocal microscopy; Singer AC et al.; Raman confocal microscopy was used to discriminate between cultures of Burkholderia xenovorans LB400 exposed to four different common environmental pollutants: phenanthrene, dodecane, 3-chlorobiphenyl and pentachlorophenol . Evidence is presented for the application of Raman spectroscopy as a bioassay for pollutant bioavailability and toxicity.

Proteins . 2005 Jan 11; {Epub ahead of print}
Crystal structure of the ferredoxin component of carbazole 1,9a-dioxygenase of Pseudomonas resinovorans strain CA10, a novel Rieske non-heme iron oxygenase system; Nam JW et al.; The carbazole 1,9a-dioxygenase (CARDO) system of Pseudomonas resinovorans strain CA10 catalyzes the dioxygenation of carbazole; the 9aC carbon bonds to a nitrogen atom and its adjacent 1C carbon as the initial reaction in the mineralization pathway . The CARDO system is composed of ferredoxin reductase (CarAd), ferredoxin (CarAc), and terminal oxygenase (CarAa) . CarAc acts as a mediator in the electron transfer from CarAd to CarAa . To understand the structural basis of the protein-protein interactions during electron transport in the CARDO system, the crystal structure of CarAc was determined at 1.9 A resolution by molecular replacement using the structure of BphF, the biphenyl 2,3-dioxygenase ferredoxin from Burkholderia cepacia strain LB400 as a search model . CarAc is composed of three beta-sheets, and the structure can be divided into two domains, a cluster-binding domain and a basal domain . The Rieske {2Fe-2S} cluster is located at the tip of the cluster-binding domain, where it is exposed to solvent . While the overall folding of CarAc and BphF is strongly conserved, the properties of their surfaces are very different from each other . The structure of the cluster-binding domain of CarAc is more compact and protruding than that of BphF, and the distribution of electric charge on its molecular surface is very different . Such differences are thought to explain why these ferredoxins can act as electron mediators in respective electron transport chains composed of different-featured components . Proteins 2005 . (c) 2005 Wiley-Liss, Inc.

Pediatr Surg Int . 2005 Jan 12; {Epub ahead of print}
Intramural bladder-wall abscess: a late complication arising after a urethrocystoscopy?
Loertzer H, Hohne SO, Finke R, Fornara P.
Intramural bladder-wall abscesses are serious but rather rare . In the few reported cases, their aetiology has not been explicitly explained . In our case, we found a traumatic outcome induced by a urethrocystoscopy that had taken place 4 years prior to the diagnosis of abscess . To date, there has not been much published on these bladder-wall abscesses or urinary tract infections from urethrocystoscopies and Burkholderia cepacia bacteria . As a result, their pathogenesis and aetiology have not been fully explained . In this paper we report on the clinical as well as the subjective well-being of a female child who was diagnosed with a massive full-blown intramural bladder-wall abscess that developed 4 years after she had undergone a urethrocystoscopy.

Nat Rev Microbiol . 2005 Jan 10; {Epub ahead of print}
The multifarious, multireplicon Burkholderia cepacia complex; Mahenthiralingam E et al.; The Burkholderia cepacia complex (Bcc) is a collection of genetically distinct but phenotypically similar bacteria that are divided into at least nine species . Bcc bacteria are found throughout the environment, where they can have both beneficial and detrimental effects on plants and some members can also degrade natural and man-made pollutants . Bcc bacteria are now recognized as important opportunistic pathogens that can cause variable lung infections in cystic fibrosis patients, which result in asymptomatic carriage, chronic infection or 'cepacia syndrome', which is characterized by a rapid decline in lung function that can include invasive disease . Here we highlight the unique characteristics of the Bcc, focusing on the factors that determine virulence.

J Clin Microbiol, 2005 Jan, 43(1), 479 - 83
Preliminary evaluation of the API 20NE and RapID NF plus systems for rapid identification of Burkholderia pseudomallei and B . mallei; Glass MB et al.; We evaluated the API 20NE and the RapID NF Plus systems with 58 Burkholderia pseudomallei and 23 B . mallei strains for identification of these agents, but neither was reliable for confirmatory identification, with only 0 to 60% strains identified accurately . A greater diversity of strains in the system databases would be beneficial.

J Bacteriol, 2005 Jan, 187(2), 785 - 90
The BpsIR Quorum-Sensing System of Burkholderia pseudomallei; Song Y et al.; BpsIR, a LuxIR quorum-sensing homolog, is required for optimal expression of virulence and secretion of exoproducts in Burkholderia pseudomallei . Cell density-dependent expression of bpsI and bpsR, the positive regulation of bpsIR expression by BpsR, and the synthesis of N-octanoyl-homoserine lactone (C8HSL) by BpsI are described in this report.

J Bacteriol, 2005 Jan, 187(2), 415 - 21
Evolutionarily divergent extradiol dioxygenases possess higher specificities for polychlorinated biphenyl metabolites; Fortin PD et al.; The reactivities of four evolutionarily divergent extradiol dioxygenases towards mono-, di-, and trichlorinated (triCl) 2,3-dihydroxybiphenyls (DHBs) were investigated: 2,3-dihydroxybiphenyl dioxygenase (EC 1.13.11.39) from Burkholderia sp . strain LB400 (DHBD(LB400)), DHBD(P6)-I and DHBD(P6)-III from Rhodococcus globerulus P6, and 2,2',3-trihydroxybiphenyl dioxygenase from Sphingomonas sp . strain RW1 (THBD(RW1)) . The specificity of each isozyme for particular DHBs differed by up to 3 orders of magnitude . Interestingly, the K(m)(app) values of each isozyme for the tested polychlorinated DHBs were invariably lower than those of monochlorinated DHBs . Moreover, each enzyme cleaved at least one of the tested chlorinated (Cl) DHBs better than it cleaved DHB (e.g., apparent specificity constants for 3',5'-dichlorinated {diCl} DHB were 2 to 13.4 times higher than for DHB) . These results are consistent with structural data and modeling studies which indicate that the substrate-binding pocket of the DHBDs is hydrophobic and can accommodate the Cl DHBs, particularly in the distal portion of the pocket . Although the activity of DHBD(P6)-III was generally lower than that of the other three enzymes, six of eight tested Cl DHBs were better substrates for DHBD(P6)-III than was DHB . Indeed, DHBD(P6)-III had the highest apparent specificity for 4,3',5'-triCl DHB and cleaved this compound better than two of the other enzymes . Of the four enzymes, THBD(RW1) had the highest specificity for 2'-Cl DHB and was at least five times more resistant to inactivation by 2'-Cl DHB, consistent with the similarity between the latter and 2,2',3-trihydroxybiphenyl . Nonetheless, THBD(RW1) had the lowest specificity for 2',6'-diCl DHB and, like the other enzymes, was unable to cleave this critical PCB metabolite (k(cat)(app) < 0.001 s(-1)).

Ann Agric Environ Med, 2004, 11(2), 319 - 22
Studies on the occurrence of Gram-negative bacteria in ticks: Ixodes ricinus as a potential vector of Pasteurella; Stojek NM et al.; A total of 372 Ixodes ricinus ticks (101 females, 122 males, and 149 nymphs) collected by flagging in 6 mixed woodlands of eastern Poland were examined by culture for the presence of internal Gram-negative bacteria other than Borrelia burgdorferi . Adult ticks were examined in pools of 2 specimens each and nymphs were examined in pools of 3-5 specimens each . Ticks were disinfected in 70 % ethanol and homogenized in 0.85% NaCl . The diluted homogenate was inoculated onto 3 kinds of agar media: buffered charcoal yeast extract (BCYE-alpha) for isolation of fastidious Gram-negative bacteria, eosin methylene blue agar (EMB) for isolation of enterobacteria, and tryptic soya agar for isolation of all other non-fastidious Gram-negative bacteria . The Gram-negative isolates were identified with the API Systems 20E and NE microtests . A total of 9 species of Gram-negative bacteria were identified, of which the commonest were strains determined as Pasteurella pneumotropica/haemolytica, which were isolated on BCYE-alpha agar from ticks collected in all 6 examined woodlands . The total number of these strains (49) exceeded the total number of all other strains of Gram-negative bacteria recovered from ticks (30) . Of the total number of examined ticks, the minimum infection rate with Pasteurella pneumotropica/haemolytica was highest in females (18.8%), and slightly lower in males (12.3%) and nymphs (10%) . Besides Pasteurella pneumotropica/haemolytica, the following species of Gram-negative bacteria were isolated from examined ticks: Pantoea agglomerans, Serratia marcescens, Serratia plymuthica on EMB agar and Aeromonas hydrophila, Burkholderia cepacia, Chromobacterium violaceum, Pseudomonas aeruginosa, and Stenotrophomonas maltophilia on tryptic soya agar . Minimal infection rates with these bacteria were low, ranging from 0.7-5.9% . Of the isolated bacteria, Chromobacterium violaceum, Pasteurella pneumotropica/haemolytica, Pseudomonas aeruginosa, and Serratia marcescens are potentially pathogenic for man and/or animals . In particular, the common occurrence of Pasteurella pneumotropica/haemolytica in Ixodes ricinus ticks poses a potential risk of pasteurellosis for humans and animals exposed to tick bites.

Pediatr Infect Dis J, 2004 Dec, 23(12), 1169 - 71
Transmission of Burkholderia pseudomallei via breast milk in northern Australia; Ralph A et al.; Two cases of maternal to child transmission of melioidosis are reported from Australia's tropical north . One infant died of overwhelming sepsis . Both lactating mothers had mastitis . In 1 case, Burkholderia pseudomallei isolated from breast milk was identical on pulsed-field gel electrophoresis with that in blood and cerebrospinal fluid isolates from the infant.

FEMS Microbiol Lett, 2005 Jan 1, 242(1), 101 - 8
Type III secretion system cluster 3 is required for maximal virulence of Burkholderia pseudomallei in a hamster infection model; Warawa J et al.; Burkholderia pseudomallei, the etiological agent of melioidosis, is an animal pathogen capable of inducing a highly fatal septicemia . B . pseudomallei possesses three type III secretion system (TTSS) clusters, two of which (TTSS1 and TTSS2) are homologous to the TTSS of the plant pathogen Ralstonia solanacearum, and one (TTSS3) is homologous to the Salmonella SPI-1 mammalian pathogenicity island . We have demonstrated that TTSS3 is required for the full virulence of B . pseudomallei in a hamster model of infection . We have also examined the virulence of B . pseudomallei mutants deficient in several putative TTSS3 effector molecules, and found no significant attenuation of B . pseudomallei virulence in the hamster model.

Clin Infect Dis, 2005 Jan 1, 40(1), 193 - 8 Epub 2004 Dec 08.
Mycotic aneurysm due to Burkholderia pseudomallei infection: case reports and literature review; Low JG et al.; Melioidosis caused by Burkholderia pseudomallei infection is endemic in Southeast Asia and Northern Australia . Cardiovascular complications resulting in mycotic aneurysms are very rare . To our knowledge, there have only been 6 isolated case reports published in the literature to date . We report 6 cases of melioidosis in Singapore that presented as aortic aneurysms.

Chem Commun (Camb), 2005 Jan 7, (1), 130 - 2 Epub 2004 Nov 26.
Stereochemistry of the reaction catalysed by 2-hydroxy-6-keto-6-phenyl-hexa-2,4-dienoic acid 5,6-hydrolase (BphD); Li JJ et al.; The stereochemical course of the reaction catalysed by C-C hydrolase BphD from Burkholderia xenovorans LB400 occurs with replacement of a benzoyl group by hydrogen with overall retention of stereochemistry.

Syst Appl Microbiol, 2004 Nov, 27(6), 623 - 7
Burkholderia phenoliruptrix sp . nov., to accommodate the 2,4,5-trichlorophenoxyacetic acid and halophenol-degrading strain AC1100; Coenye T et al.; Strain AC1100 is well-known for its ability to degrade a variety of recalcitrant xenobiotics, including 2,4,5-trichlorophenoxyacetic acid . We performed a polyphasic-taxonomic study to determine its taxonomic position . The G+C content of strain AC1100 was 62.6 mol% . On the basis of 16S rRNA gene sequence similarity, strain AC1100 belonged to the beta-Proteobacteria and was most closely related to Burkholderia fungorum (98.3% similarity) . DNA-DNA hybridisations, comparison of protein profiles, cellular fatty acid analysis and biochemical tests allowed genotypic and phenotypic differentiation of strain AC1100 from other Burkholderia species . Our data show that strain AC1100 represents a novel species for which the name Burkholderia phenoliruptrix sp . nov . is proposed . The type strain is AC1100T (= LMG 22037T = CCUG 48558T).

Glycobiology . 2004 Dec 15; {Epub ahead of print}
Complete structural characterization of the lipid A fraction of a clinical strain of B . cepacia genomovar I lipopolysaccharide; Silipo A et al.; Burkholderia cepacia, a Gram negative bacterium ubiquitous in the environment, is a plant pathogen causing soft rot of onions . This microorganism has recently emerged as a life-threatening multiresistant pathogen in cystic fibrosis patients . An important virulence factor of B . cepacia is the lipopolysaccharide fraction . Clinical isolates and environmental strains possess LPS of high inflammatory nature which induces a high level production of cytokines . For the first time, the complete structure of the lipid A components isolated from the lipopolysaccharide fraction of a clinical strain of Burkholderia cepacia is herein described . The structural studies carried out by selective chemical degradations, mass spectrometry and NMR spectroscopy revealed multiple species differing in the acylation and in the phosphorylation patterns . The highest mass species was identified as a penta-acylated tetrasaccharide backbone containing two phosphoryl-arabinosamine residues in addition to the archetypal glucosamine disaccharide {Arap4N-L-beta-1-P>4-beta-D-GlcpN-(1>6)-alpha-D-GlcpN-1>P-1-beta-L-Arap4N} . Lipid A fatty acids substitution was also described, with two 3-hydroxytetradecanoic acids 14:0 (3-OH) in ester linkage, and two 3-hydroxyhexadecanoic acids 16:0 (3-OH) in amide-linkage, one of which was substituted by a secondary a 14:0 residue at its C-3 . Other lipid A species present in the mixture and exhibiting lower molecular weight lacked one or both beta-L-Arap4N residues.

J Heart Lung Transplant, 2004 Dec, 23(12), 1382 - 91
A novel model to study bacterial adherence to the transplanted airway: inhibition of Burkholderia cepacia adherence to human airway by dextran and xylitol; Sajjan U et al.; BACKGROUND: Lung infection with Burkholderia cepacia complex before lung transplantation in patients with cystic fibrosis is a major risk factor for decreased post-operative survival rates compared with those of patients colonized with the more common opportunistic pathogen Pseudomonas aeruginosa . Because adherence to mucosal surfaces is an important initial step in infection, we investigated the use of non-toxic neutral polysaccharides and a sugar alcohol to prevent adherence of B cepacia complex to allograft airway epithelium . METHODS: We used human airway explants prepared from donor tracheobronchial tissue to test the effect of dextrans and xylitol in inhibiting the binding of Burkholderia cepacia complex . We used immunofluorescence and electron microscopy to determine the distribution of bacteria in the explants . RESULTS: Burkholderia cepacia complex bound to the explants and was found only in the surface mucus layer . Dextran 40 kd applied before adding the bacteria decreased the number of bound organisms by 80% to 99% . Smaller molecular mass dextrans (4 and 20 kd) were ineffective . Xylitol inhibited bacterial binding by 67% to 85% . Both agents seemed to decrease the thickness of the surface mucus, suggesting that they may indirectly inhibit bacterial binding by removing adherent surface mucus . CONCLUSIONS: Treating donor lungs with dextran 40 kd or xylitol before (and possibly after) surgery may inhibit the adherence of Burkholderia cepacia complex to airways and may prevent or decrease subsequent infection of the allografts.

FEMS Immunol Med Microbiol, 2005 Jan 1, 43(1), 37 - 44
Epitope mapping of Burkholderia pseudomallei serine metalloprotease: identification of serine protease epitope mimics; Chan SW et al.; Filamentous phage random peptide libraries were used to identify the epitopes of Burkholderia pseudomallei protease by panning against IgG polyclonal sera that exhibited protease neutralizing properties . The isolated fusion peptides presented a consensus peptide sequence, TKSMALSG, which closely resembles part of the active site sequence, 435GTSMATPHVAG445, of B . pseudomallei serine metalloprotease . By comparing the consensus sequence, TKSMALSG, with the predicted three-dimensional molecular model of B . pseudomallei serine metalloprotease, it appears that the potential antibody binding epitope was buried within the molecule . This active site was conformational whereby one continuous sub-region (SMA) was located between two discontinuous sub-regions, supplied by the flanking residues in the same polypeptide . All phages selected from the biopanning with IgG polyclonal sera showed good binding towards the polyclonal antibodies when compared to the negative control . In addition, these peptide-bearing phages showed competitive inhibition of B . pseudomallei serine metalloprotease binding to the polyclonal IgG.

J Biotechnol, 2005 Jan 26, 115(2), 145 - 56
Protein engineering of toluene-o-xylene monooxygenase from Pseudomonas stutzeri OX1 for oxidizing nitrobenzene to 3-nitrocatechol, 4-nitrocatechol, and nitrohydroquinone; Vardar G et al.; Toluene-o-xylene monooxygenase (ToMO) from Pseudomonas stutzeri OX1 was found to oxidize nitrobenzene (NB) to form m-nitrophenol (m-NP, 72%) and p-NP (28%) with an initial rate of 0.098 and 0.031nmol/(minmg protein), respectively . It was also discovered that wild-type ToMO forms 4-nitrocatechol (4-NC) from m-NP and p-NP with an initial rate of 0.15 and 0.0082nmol/(minmg protein), respectively, and 3-NC (12%) and nitrohydroquinone (NHQ, 88%) from o-NP with an initial rate of 0.11 and 0.8nmol/(minmg protein), respectively . To increase the oxidation rate and alter the oxidation regiospecificity of nitro aromatics as well as to study the role of the active site residues I100, Q141, T201, and F205 of the alpha hydroxylase fragment of ToMO (TouA), DNA shuffling and saturation mutagenesis were used to generate random mutants . The mutants were initially identified by screening via a rapid agar plate assay and then were further examined by high-performance liquid chromatography (HPLC) and gas chromatography (GC) . Several mutants with higher rates of activities and with different regiospecificities were identified; for example, Escherichia coli TG1 cells expressing either TouA mutant M180T/E284G or E214G/D312N/M399V produce 4-NC 4.5- and 20-fold faster than wild-type ToMO (0.037 and 0.16nmol/minmg protein from p-NP, respectively) . TouA mutant A107T/E214A had the regiospecificity of NB changed significantly from 28% to 79% p-NP . From 200muM NB, TouA variants A101T/M114T, A110T/E392D, M180T/E284G, and E214G/D312N/M399V produce 4-NC whereas wild-type ToMO does not . From m-NP, TouA mutant I100Q produces 4-NC (37%) and NHQ (63%), whereas wild-type ToMO produces only 4-NC (100%) . Variant A107T/E214A acts like a para enzyme and forms p-cresol as the major product (93%) from toluene with enhanced activity (2.3-fold), whereas wild-type ToMO forms 32%, 21%, and 47% of o-, m-, and p-cresol, respectively . Hence, the non-specific ToMO was converted into a regiospecific enzyme, which rivals toluene 4-monooxygenase of P . mendocina KR1 and toluene o-monooxygenase of Burkholderia cepacia G4 in its specificity.

Biotechnol Lett, 2004 Nov, 26(22), 1757 - 61
Essential role of the small subunit of thermostable glucose dehydrogenase from Burkholderia cepacia; Yamaoka H et al.; The co-expression in Escherichia coli of the gamma-subunit and the catalytic alpha-subunit of the thermostable glucose dehydrogenase (GDH) from Burkholderia cepacia sp . SM4 produced 12.7 U GDH activity mg(-1) protein . A 47-amino acid, twin-arginine translocase signal peptide was identified at the amino terminus of the gamma-subunit . The expression of the alpha-subunit in the absence of the gamma-subunit or the gamma-subunit signal peptide failed to produce any detectable GDH protein or activity . The gamma-subunit may be a chaperone-like component that assists folding of the alpha-subunit polypeptide to the active form and its translocation to the periplasm.

Int J Infect Dis, 2005 Jan, 9(1), 15 - 20
Community-acquired pneumonia in northern Australia: low mortality in a tropical region using locally-developed treatment guidelines; Elliott JH et al.; OBJECTIVE:: To investigate the epidemiology and outcome of adult community-acquired pneumonia (CAP) in tropical Australia . METHODS:: A prospective study was performed of all adult patients with CAP admitted to the Royal Darwin Hospital, a major hospital in tropical northern Australia . A standard definition of CAP was used and data collected on demographics, risk factors, history, examination, investigations, treatment and outcome . Locally-developed treatment guidelines were used . RESULTS:: One hundred and sixty-seven adults were included in the analysis . Aboriginal people were over-represented, younger and were more likely to have risk factors for CAP . The most frequent pathogens isolated were Streptococcus pneumoniae and Burkholderia pseudomallei . 'Atypical pneumonia' organisms were uncommon . Treatment guidelines included penicillin for mild pneumonia but emphasised coverage of Burkholderia pseudomallei in those with risk factors, especially during the monsoon season . The mortality rate from pneumonia was low with three deaths in 167 cases (1.8%) . CONCLUSIONS:: International guidelines for the management of CAP have been based on populations and organisms from temperate regions and may not necessarily be applicable to tropical regions . Guidelines based upon local epidemiology must therefore be developed . This study illustrates how mortality can be minimised using a process of determining local CAP etiology, developing treatment guidelines and auditing patient management.

Ann Clin Microbiol Antimicrob . 2004 Dec 15;3(1):26.
Organisms isolated from adults with Cystic Fibrosis; McManus TE et al.; BACKGROUND: Patients with cystic fibrosis {CF} have frequent pulmonary exacerbations associated with the isolation of bacterial organisms from sputum samples . It is not clear however, if there are differences in the types of additional organisms isolated from patients who are infected with Burkholderia cepacia complex {BCC} or Pseudomonas aerugionsa {PA} in comparison to those who are not infected with either of these organisms {NI} . METHODS: Adult patients attending the regional CF unit were followed over a two year period and patients were assigned to three groups depending on whether they were known to be chronically infected with BCC, PA or NI . We compared the numbers and types of organisms which were isolated in each of these groups . RESULTS: Information was available on a total of 79 patients; BCC 23, PA 30 and NI 26 . Total numbers of organisms isolated, expressed as median and IQR for each group, {P = 0.045} and numbers of co-infecting organisms {P = 0.003} were significantly higher in the BCC group compared to PA, and in the PA group {P < 0.001, p = 0.007 respectively} compared to NI patients . The pattern of co-infecting organisms was similar in all three groups . CONCLUSIONS: Total numbers of organisms isolated and numbers of co-infecting organisms were significantly higher in the BCC group compared to PA, and in the PA group compared to NI patients . Types of co-infecting organisms are similar in all groups of patients.

J Med Microbiol, 2004 Dec, 53(Pt 12), 1177 - 82
Evaluation of lipopolysaccharide and capsular polysaccharide as subunit vaccines against experimental melioidosis; Nelson M et al.; Burkholderia pseudomallei is the causative agent of melioidosis, which is a major cause of morbidity and mortality in endemic regions . Currently there is no human vaccine against melioidosis . In this study, LPS or capsular polysaccharide was used to immunize BALB/c mice . The different polysaccharide antigens induced antibody responses . Mice vaccinated with LPS developed predominantly IgM and IgG3 responses . Contrastingly, mice vaccinated with capsular polysaccharide developed a predominantly IgG2b response . After immunization, mice were challenged by the intra-peritoneal route and an increased mean time to death was observed compared with unvaccinated controls . Immunization with LPS provided an optimal protective response . Mice challenged by the aerosol route showed a small increase in the mean time to death compared with the unvaccinated controls . The passive transfer of antigen from immunized into naive mice provided protection against a subsequent challenge . This study is the first time antigens protective by active immunization have been identified and suggests that polysaccharides have potential as vaccine candidates against melioidosis.

J Clin Microbiol, 2004 Dec, 42(12), 5871 - 4
Identification and discrimination of Burkholderia pseudomallei, B . mallei, and B . thailandensis by real-time PCR targeting type III secretion system genes; Thibault FM et al.; Burkholderia pseudomallei and B . mallei are two highly pathogenic bacteria, responsible for melioidosis and glanders, respectively . The two are closely related and can also be mistaken for B . thailandensis, a nonpathogenic species . To improve their differential identification, we describe a hydrolysis probe-based real-time PCR method using the uneven distribution of type III secretion system genes among these three species.

J Clin Microbiol, 2004 Dec, 42(12), 5537 - 41
Burkholderia cepacia is associated with pulmonary hypertension and increased mortality among cystic fibrosis patients; Fauroux B et al.; The aim of the study was to evaluate the impact of Burkholderia cepacia on cardiovascular status and mortality in cystic fibrosis . Seven patients infected with B . cepacia were matched with 31 patients not infected with this organism for gender, age, height, weight, genotype, and percent predicted forced expiratory volume in one second, partial arterial oxygen pressure, and pancreatic sufficiency status . The pulmonary artery systolic pressure, as assessed by transthoracic echocardiography, was significantly higher in patients infected with B . cepacia (61.3 +/- 17.2 mm Hg) than in controls (37.3 +/- 13.9 mm Hg; P = 0.02), and the mean acceleration time was significantly lower (77 +/- 33 ms versus 108 +/- 25 ms; P = 0.02) . The 6-month mortality was significantly higher in patients infected with B . cepacia (57% versus 16%; P = 0.02) . Six of the seven patients infected with B . cepacia harbored the same ribotype (genomovar II, B . multivorans) . Pulmonary hypertension was significantly more frequent in patients infected by B . cepacia and could contribute to the increased mortality rate.

J Clin Microbiol, 2004 Dec, 42(12), 5477 - 83
Isolates of Burkholderia pseudomallei from Northern Australia are distinct by multilocus sequence typing, but strain types do not correlate with clinical presentation; Cheng AC et al.; Melioidosis is the disease caused by the saprophytic organism Burkholderia pseudomallei . Previous studies have suggested some strain tropism and differential virulence . In this study, we defined strains by multilocus sequence typing (MLST) of isolates taken from the Top End of Australia's Northern Territory and compared the results with those of other strains typed worldwide . We specifically sought clinical and geographical correlates of strain types . Among 87 Australian isolates, 48 sequence types were defined . None of the sequence types in this study has been found elsewhere in the world . Strains were distributed widely throughout the region, and the different presentations of disease, including neurological and prostatic infection, were associated with many different strains . There was excellent congruence between pulsed-field gel electrophoresis and MLST, and the two typing methods had a similar level of strain discrimination . The work suggests that host and environmental factors may be more important in determining disease presentation than infecting strain type . It is possible that the distinct but diverse strain types found in this study reflect Australia's geographical isolation over many millions of years.

Clin Neuropathol, 2004 Sep-Oct, 23(5), 195 - 203
The neuropathology of melioidosis: two cases and a review of the literature; Koszyca B et al.; Melioidosis is an infectious disease caused by Burkholderia pseudomallei and is hyperendemic in the Top End of the Northern Territory of Australia, as well as being widespread throughout tropical south east Asia . The infection is primarily acquired via the inoculation of compromised surface tissues by contaminated soils and water and it can cause an acute, rapidly fatal illness . Although pneumonia is the commonest manifestation, neurological presentations have been described, most notably encephalomyelitis . This paper presents the neuropathology of 2 fatal cases of neurological melioidosis and reviews the relevant literature.

Biotechnol Prog, 2004 Nov-Dec, 20(6), 1697 - 704
Production and characterization of poly-beta-hydroxyalkanoate copolymers from Burkholderia cepacia utilizing xylose and levulinic acid; Keenan TM et al.; Poly(beta-hydroxybutyrate-co-beta-hydroxyvalerate) (P(3HB-co-3HV)) copolymers were prepared via shake-flask fermentations of Burkholderia cepacia (formerly Pseudomonas cepacia) containing 2.2% (w/v) xylose and concentrations of levulinic acid ranging from 0.07% to 0.67% (w/v) . Periodic harvest of shake-flask cultures from 48 to 92 h post-inoculation yielded 4.4-5.3 g/L of dry cell biomass, containing 42-56% (w/w) P(3HB-co-3HV), with optimal product yield occurring between 66 and 74 h . Growth and PHA accumulation enhancement were observed with concentrations of levulinic acid from 0.07 to 0.52% (w/v), producing dry cell biomass and P(3HB-co-3HV) yields of 9.5 and 4.2 g/L, respectively, at the 0.52% (w/v) concentration of levulinic acid . Representative samples were subjected to compositional analysis by 300 MHz 1H and 150 MHz 13C NMR, indicating that these random copolymers contained between 0.8 and 61 mol % 3-hydroxyvalerate (3HV) . Solvent-cast film samples were characterized by differential scanning calorimetry, which demonstrated melting temperatures (Tm) to decrease in a pseudoeutectic fashion from 174.3 degrees C (0.8 mol % 3HV) to a minimum of 154.2 degrees C (25 mol % 3HV) and the glass transition temperatures (Tg) to decrease linearly from 2.1 to -11.9 degrees C as a function of increasing mol % 3HV . Thermogravimetric analysis of the copolymer series showed the temperature for onset of thermal decomposition (T(decomp)) to vary as a function of mol % 3HV from 273.4 to 225.5 degrees C . Intrinsic viscosities (eta) varied from 3.2 to 5.4 dL/g, as determined by dilute solution viscometry . Viscosity average molecular weights (Mv) of the copolymers were determined to range from 469 to 919 kDa, indicating that these P(3HB-co-3HV) copolymers are of sufficient molecular mass for commercial application.

Peptides, 2004 Dec, 25(12), 2055 - 61
Antimicrobial activity of Bac7 fragments against drug-resistant clinical isolates; Benincasa M et al.; Ten peptides from 13 to 35 residues in length and covering the whole sequence of the Pro-rich peptide Bac7 were synthesized to identify the domain responsible for its antimicrobial activity . At least 16 residues of the highly cationic N-terminal sequence were required to maintain the activity against Gram-negative bacteria . The fragments Bac7(1-35) and, to a lesser extent, Bac7(1-16) proved active against a panel of antibiotic-resistant clinical isolates of Gram-negative bacteria, with the notable exception of Burkholderia cepacia . In addition, when tested against fungi, the longer fragment was also active against collection strains and clinical isolates of Cryptococcus neoformans, but not towards clinical isolates of Candida albicans.

J Hosp Infect, 2005 Jan, 59(1), 46 - 52
Nosocomial bloodstream infections with Burkholderia stabilis; Otag F et al.; Burkholderia stabilis was grown from blood cultures of seven patients presenting with signs and symptoms of septicaemia in the intensive care unit at Mersin University Hospital, Mersin, Turkey between July and October 2002 . Four patients had one B . stabilis-positive blood culture, two patients had two, and one patient had four . Isolates from six of seven patients had the same resistotype and random amplified polymorphic DNA analysis type . Despite treatment with ciprofloxacin and imipenem, to which the strains were susceptible, all patients died one to eight days after isolation of B . stabilis from their blood . B . stabilis should be regarded as an opportunistic pathogen that may cause nosocomial bloodstream infections.

J Mol Biol, 2005 Jan 7, 345(1), 21 - 8
Structural characterization of the Ser324Thr variant of the catalase-peroxidase (KatG) from Burkholderia pseudomallei; Deemagarn T et al.; The Ser315Thr variant of the catalase-peroxidase KatG from Mycobacterium tuberculosis imparts resistance to the pro-drug isonicotinic acid hydrazide (isoniazid) through a failure to convert it to the active drug, isonicotinoyl-NAD . The equivalent variant in KatG from Burkholderia pseudomallei, Ser324Thr, has been constructed, revealing catalase and peroxidase activities that are similar to those of the native enzyme . The other activities of the variant protein, including the NADH oxidase, the isoniazid hydrazinolysis and isonicotinoyl-NAD synthase activities are reduced by 60-70% . The crystal structure of the variant differs from that of the native enzyme in having the methyl group of Thr324 situated in the entrance channel to the heme cavity, in a modified water matrix in the entrance channel and heme cavity, in lacking the putative perhydroxy modification on the heme, in the multiple locations of a few side-chains, and in the presence of an apparent perhydroxy modification on the indole nitrogen atom of the active-site Trp111 . The position of the methyl group of Thr324 creates a constriction or narrowing of the channel leading to the heme cavity, providing an explanation for the lower reactivity towards isoniazid and the slower rate of isonicotinoyl-NAD synthesis.

Cell Mol Biol (Noisy-le-grand), 2004 Jul, 50(5), 525 - 36
Bacterial diversity of a soil sample from Schirmacher Oasis, Antarctica; Shivaji S et al.; The bacterial diversity of a soil sample collected in the vicinity of Lake Zub, Schirmacher Oasis, Antarctica, was determined both by establishing pure colonies of culturable bacteria and by cloning the total 16S rDNA of the soil and establishing the phylogeny of the clones . Analysis of the 16S rRNA gene clones indicated that the bacteria belonged to the classes alpha-proteobacteria, beta-proteobacteria, gamma-proteobacteria, Gemmatimonas, Bacteriodetes, Actinobacteria, Chloroflexi and Chlamydiae . In addition, seven clones were categorized as unidentified and unculturable in the classes of beta-Proteobacteria, Actinobacteria, Chloroflexi and Chlamydiae . Further, the culturable bacteria from the same site were identified as belonging to the genera Pseudomonas, Sphingobacterium, Arthrobacter, Micrococcus, Brevondimonas, Rhodococcus and Microbacterium . These results identify for the first time the presence of bacteria belonging to the genera Brevundimonas, Microbacterium, Rhodococcus, Serratia, Enterobacter, Rhodopseudomonas, Sphingomonas, Acidovorax, Burkholderia, Nevskia, Gemmatimonas, Xanthomonas and Flexibacter in Antarctica . Further, comparison of the Antarctic soil bacterial diversity with other cold habitats of Antarctica like from sediments, ice and cyanobacterial mat samples indicated that the bacterial diversity in soil was similar to the diversity observed in the continental shelf sediment sample . The Antarctic soil clones also resembled the bacterial diversity of soils from other geographical regions, but were unique in that none of the clones from the soil belonged to the uncultured Y, O, G, A and B groups common to all soil samples.

Zh Mikrobiol Epidemiol Immunobiol, 2004 Sep-Oct, (5), 109 - 13
{Identification and differential diagnostics of pathogenic Burkholderia}
{Immune response in experimental animals immunized with Burkholderia pseudomallei surface antigens}
{No authors listed}

The influence of the chromatographic fractions of B . pseudomallei surface antigenic complex (C, C1, D, H) on immune response in white rats and white mice was under study . These antigenic complexes were noted to produce perceptible stimulating effect on the immune system of white rats, in contrast to that of white mice . The immunization of the mice the above-mentioned fractions suppressed the phagocytic activity of peritoneal macrophages (PM) and slightly enhanced cell-mediated immunity . In experiments on white rats, fraction C induced the growth of specific antibody titers and stimulated the phagocytic activity of PM, as well as the indices of delayed hypersensitivity (DH) . Fraction D showed a lower level of the induction of the phagocytic activity of PM and was inactive in the manifestation of cell-mediated immunity, but induced a high level of humoral immunity . Antigenic complexes C1 and H increased the phagocytic activity of PM and DH characteristics with a low level of antibody production . The studied fractions of the causative agent of melioidosis decreased the content of bactericidal cationic proteins (BCP) in rat blood neutrophils, and in mice a decreased content of BCP in phagocytes was registered . The fractions increased the activity of myeloperoxidase in blood neutrophils in mice and rats . As revealed with the use of immunoelectrophoresis, SDS PAAG electrophoresis and immunoblotting, the surface antigenic complex contained proteins of 18, 22, 39 kD and glycoproteins 42, 55, 90 kD . The latter glycoprotein was found in all the fractions under study, having protective properties.

Emerg Infect Dis, 2004 Nov, 10(11), 1957 - 9
Burkholderia cenocepacia Vaginal Infection in Patient with Smoldering Myeloma and Chronic Hepatitis C; Petrucca A; We report a case of a vaginal infection caused by a strain of Burkholderia cenocepacia . The strain was isolated from vaginal swab specimens from a 68-year-old woman with smoldering myeloma and chronic hepatitis C virus infection who was hospitalized for abdominal abscess . Treatment with piperacillin/tazobactam eliminated B . cenocepacia infection and vaginal symptoms.

Int J Syst Evol Microbiol, 2004 Nov, 54(Pt 6), 2237 - 9
'Candidatus Burkholderia calva' and 'Candidatus Burkholderia nigropunctata' as leaf gall endosymbionts of African Psychotria; Van Oevelen S et al.; Phylogenetic 16S rRNA gene analysis was used to assign the bacterial leaf-nodulating endosymbionts of two tropical African Psychotria species to the genus Burkholderia . The microsymbionts of the different Psychotria hosts were recognized as distinct and novel species of Burkholderia on the basis of relatively low intersequence similarities and sufficiently large evolutionary distances when compared with each other and their closest validly named neighbours . The obligate endosymbiotic nature of the bacteria prevented their in vitro cultivation and the deposition of type strains to culture collections . Therefore, the provisional status Candidatus is assigned to the bacterial partners of Psychotria calva and Psychotria nigropunctata, with the proposal of the names 'Candidatus Burkholderia calva' and 'Candidatus Burkholderia nigropunctata', respectively.

Int J Syst Evol Microbiol, 2004 Nov, 54(Pt 6), 2155 - 62
Burkholderia tropica sp . nov., a novel nitrogen-fixing, plant-associated bacterium; Reis VM et al.; In an ecological survey of nitrogen-fixing bacteria isolated from the rhizosphere and as endophytes of sugarcane, maize and teosinte plants in Brazil, Mexico and South Africa, a new phylogenetically homogeneous group of N(2)-fixing bacteria was identified within the genus Burkholderia . This polyphasic taxonomic study included microscopic and colony morphology, API 20NE tests and growth on different culture media at different pH and temperatures, as well as carbon source assimilation tests and whole-cell protein pattern analysis . Analysis of 16S rRNA gene sequences showed 99.2-99.9 % similarity within the novel species and 97.2 % similarity to the closest related species, Burkholderia sacchari . The novel species was composed of four distinct amplified 16S rDNA restriction analysis groups . The DNA-DNA reassociation values within the novel species were greater than 70 % and less than 42 % for the closest related species, B . sacchari . Based on these results and on many phenotypic characteristics, a novel N(2)-fixing species is proposed for the genus Burkholderia, Burkholderia tropica sp . nov., with the type strain Ppe8(T) (=ATCC BAA-831(T)=LMG 22274(T)=DSM 15359(T)) . B . tropica was isolated from plants grown in geographical regions with climates ranging from temperate subhumid to hot humid.

Eur J Cardiothorac Surg, 2004 Dec, 26(6), 1180 - 6
Long-term outcome of lung transplantation for cystic fibrosis--Danish results; Bech B et al.; OBJECTIVE: Over the last decades improvements in medical therapies have delayed the progression of lung disease in cystic fibrosis (CF) . However, lung disease is still the most common cause of premature death, and lung transplantation today is the only treatment for end-stage lung disease in patients with CF . We present a retrospective review of the outcome of CF patients transplanted in Denmark since start of the national lung transplantation programme in 1992 . METHODS: In a 10-year period, 47 patients with CF were listed for lung transplantation; 29 patients underwent transplantation and 18 patients died while waiting for donor organs . Eleven patients received en block double lung transplantation with direct bronchial artery revascularization and 18 patients received bilateral sequential lung transplantation . Median age at transplantation was 29 years (range 11-50) . RESULTS: The perioperative mortality (< or =30 days) was 3.5% (1/29 patients) . Actuarial survival of transplanted patients at 1, 3, 5 and 8 years was 89, 80, 80 and 70%, respectively . Actuarial survival of non-transplanted patients on the waiting list at 1 and 2 years was 28 and 11% (P<0.0001) . Causes of death of transplanted patients were: respiratory failure on day 7 (n=1), bronchiolitis obliterans syndrome (n=2), infection (Cytomegalovirus, Aspergillus fumigatus) (n=2), bronchial anastomosis dehiscence (n=1) . Pulmonary function (FEV1% predicted) improved from median 20% (range 13-31) pre-transplant to 71% (range 19-118) after 5 years (P<0.0001) . Renal function (51Cr-EDTA clearance) decreased from median 97 ml/min (range 45-190) pre-transplant to 32 ml/min (range 8-84) 6 months after transplantation (P<0.001) . Three patients (11%) received dialysis post-transplant of whom two underwent kidney transplantation . Immunosuppressive induction therapy with rabbit-antithymocyte-globulin compared to daclizumab resulted in fewer treatments for acute rejection within the first 3 months post-transplant (P=0.05 at 5-8 weeks) . Burkholderia multivorans was present in three patients pre-transplant with satisfying long-term outcome in one patient . CONCLUSIONS: Lung transplantation is a well-established life-extending treatment for patients with CF and end-stage lung disease . The operative mortality is low and CF patients have a significant early survival benefit after lung transplantation . Satisfying long-term results can be achieved in this young and severely ill group of patients.

Diagn Mol Pathol, 2004 Dec, 13(4), 247 - 53
Development of 5' nuclease real-time PCR assays for the rapid identification of the burkholderia mallei//burkholderia pseudomallei complex; Tomaso H et al.; Burkholderia pseudomallei is the causative agent of melioidosis and was classified as a biologic agent by the Centers for Disease Control and Prevention (Atlanta, GA) . Acute melioidosis has a case fatality rate of >40%, and septicemia is fatal in up to 90% . The aim of the study was to design 5'-nuclease real-time PCR assays for the rapid and reliable identification of the B . mallei/B . pseudomallei complex . Real-time PCR assays using TaqMan probes targeting the 16S rDNA and fliC were developed on an ABI Prism 7000 sequence detection system (Applied Biosystems, Foster City, CA) . Specificity was assessed with 64 B . pseudomallei, nine B . mallei, 126 other Burkholderia strains of 29 species, and 45 clinically relevant non-Burkholderia organisms . Sensitivity, specificity, and positive and negative predictive value of the assays were 100% . Discrimination between B . pseudomallei and B . mallei, an organism which can be regarded as a clone of B . pseudomallei, could not be achieved . A probit analysis revealed that 7.5 and 52 genome equivalents (GE) of B . pseudomallei could be detected using the fliC and the 16S rDNA assays (P = .05), respectively . In spiked blood samples, the detection limit was approximately 300 and 3.000 GE for fliC and the 16S rDNA, respectively . In conclusion, we recommend the simultaneous use of the 16S rDNA and fliC real-time PCR assays for the rapid and specific identification of the B . mallei/B . pseudomallei complex in positive blood cultures or from suspicious bacterial colonies allowing the early onset of appropriate antibiotic therapy.

Pharmacotherapy, 2004 Nov, 24(11), 1641 - 5
Empiric treatment of multidrug-resistant Burkholderia cepacia lung exacerbation in a patient with cystic fibrosis: application of pharmacodynamic concepts to meropenem therapy; Kuti JL et al.; A 31-year-old man with cystic fibrosis was diagnosed with multidrug-resistant Burkholderia cepacia pneumonia . Meropenem 2000 mg every 8 hours was administered as a 3-hour infusion to maximize pharmacodynamic exposure; oral minocycline 100 mg twice/day was also given . Blood samples were collected to confirm meropenem concentrations . Concentrations above the mimimum inhibitory concentration (MIC) of 8 microg/ml were achieved for 52% of the dosing interval, which is greater than what is required for a bactericidal effect . The patient's condition improved, he was discharged, and completed a 3-week course of the antibiotic regimen . After 6 months, he had remained at his baseline level of health . This case demonstrates that pharmacodynamic principles can be used to design an antibiotic dosing regimen that can achieve optimal exposures when the MIC is above that considered susceptible to conventional dosing strategies.

Biochem Biophys Res Commun, 2004 Dec 10, 325(2), 414 - 20
Homology modeling and S(N)2 displacement reaction of fluoroacetate dehalogenase from Burkholderia sp . FA1; Zhang Y et al.; Fluoroacetate dehalogenase (EC 3.8.1.3) catalyzes the dehalogenation of fluoroacetate and other haloacetates . In order to investigate the relation between the structure and the function, and understand the reaction mechanism of the enzyme, a 3D model of fluoroacetate dehalogenase FAc-DEX FA1 was built by homology-based modeling . The 3D model was optimized by unconstrained molecular dynamics simulation . Furthermore, the optimized 3D model was assessed by comparison of specific properties with two known protein structures . From the final 3D model, we find that the main residues involved in the active site in FAc-DEX FA1 were Phe34, Trp148, Tyr147, Tyr212, Asp104, and His271; especially Asp104 was the key nucleophilic residue in substrate binding . A reaction model including Asp104 and the substrate fluoroacetate was then constructed and used to characterize explicit enzymatic reactions . In order to further illustrate catalytic properties, the equilibrium geometries, energies, and frequencies of stationary points (reactants, products, and transition states) of the reaction model were calculated at the B3LYP/6-31G level of theory in both gas phase and solution . The results showed that the reaction in gas was dynamically more favorable than in solution.

Appl Environ Microbiol, 2004 Nov, 70(11), 6789 - 99
Engineering of chimeric class II polyhydroxyalkanoate synthases; Niamsiri N et al.; PHA synthase is a key enzyme involved in the biosynthesis of polyhydroxyalkanoates (PHAs) . Using a combinatorial genetic strategy to create unique chimeric class II PHA synthases, we have obtained a number of novel chimeras which display improved catalytic properties . To engineer the chimeric PHA synthases, we constructed a synthetic phaC gene from Pseudomonas oleovorans (phaC1Po) that was devoid of an internal 540-bp fragment . Randomly amplified PCR products (created with primers based on conserved phaC sequences flanking the deleted internal fragment) were generated using genomic DNA isolated from soil and were substituted for the 540-bp internal region . The chimeric genes were expressed in a PHA-negative strain of Ralstonia eutropha, PHB(-)4 (DSM 541) . Out of 1,478 recombinant clones screened for PHA production, we obtained five different chimeric phaC1Po genes that produced more PHA than the native phaC1Po . Chimeras S1-71, S4-8, S5-58, S3-69, and S3-44 exhibited 1.3-, 1.4-, 2.0-, 2.1-, and 3.0-fold-increased levels of in vivo activity, respectively . All of the mutants mediated the synthesis of PHAs with a slightly increased molar fraction of 3-hydroxyoctanoate; however, the weight-average molecular weights (Mw) of the PHAs in all cases remained almost the same . Based upon DNA sequence analyses, the various phaC fragments appear to have originated from Pseudomonas fluorescens and Pseudomonas aureofaciens . The amino acid sequence analyses showed that the chimeric proteins had 17 to 20 amino acid differences from the wild-type phaC1Po, and these differences were clustered in the same positions in the five chimeric clones . A threading model of PhaC1Po, developed based on homology of the enzyme to the Burkholderia glumae lipase, suggested that the amino acid substitutions found in the active chimeras were located mostly on the protein model surface . Thus, our combinatorial genetic engineering strategy proved to be broadly useful for improving the catalytic activities of PHA synthase enzymes.

Cell Microbiol, 2004 Dec, 6(12), 1127 - 38
Burkholderia cepacia complex isolates survive intracellularly without replication within acidic vacuoles of Acanthamoeba polyphaga; Lamothe J et al.; We have previously demonstrated that isolates of the Burkholderia cepacia complex can survive intracellularly in murine macrophages and in free-living Acanthamoeba . In this work, we show that the clinical isolates B . vietnamiensis strain CEP040 and B . cenocepacia H111 survived but did not replicate within vacuoles of A . polyphaga . B . cepacia-containing vacuoles accumulated the fluid phase marker Lysosensor Blue and displayed strong blue fluorescence, indicating that they had low pH . In contrast, the majority of intracellular bacteria within amoebae treated with the V-ATPse inhibitor bafilomycin A1 localized in vacuoles that did not fluoresce with Lysosensor Blue . Experiments using bacteria fluorescently labelled with chloromethylfluorescein diacetate demonstrated that intracellular bacteria remained viable for at least 24 h . In contrast, Escherichia coli did not survive within amoebae after 2 h post infection . Furthermore, intracellular B . vietnamiensis CEP040 retained green fluorescent protein within the bacterial cytoplasm, while this protein rapidly escaped from the cytosol of phagocytized heat-killed bacteria into the vacuolar lumen . Transmission electron microscopy analysis confirmed that intracellular Burkholderia cells were structurally intact . In addition, both Legionella pneumophila- and B . vietnamiensis-containing vacuoles did not accumulate cationized ferritin, a compound that localizes within the lysosome . Thus, our observations support the notion that B . cepacia complex isolates can use amoebae as a reservoir in the environment by surviving without intracellular replication within an acidic vacuole that is distinct from the lysosomal compartment.

J Vet Med B Infect Dis Vet Public Health, 2004 Sep, 51(7), 305 - 20
Melioidosis in animals: a review on epizootiology, diagnosis and clinical presentation; Sprague LD et al.; Melioidosis, an infectious disease caused by Burkholderia pseudomallei is an emerging disease with high impact on animals and man . In different animal species, the clinical course varies and delayed diagnosis poses risks for the dissemination of the agent in non-endemic areas . Not only migration and transport of animals around the world but also tourism increases the risk that melioidosis can leave its endemic boundaries and establish itself elsewhere . Detection of the agent is a major challenge, as the agent has to be handled in laboratories of biosafety level 3 and test kits are not yet commercially available . Veterinarians and doctors should be aware of melioidosis not only as an agent of public interest but also in terms of a bioterrorist attack . The aim of this review is to describe the agent, its aetiology, the manifestation in a variety of animal species as well as to describe diagnostic procedures, typing techniques and countermeasures.

Mol Microbiol, 2004 Nov, 54(4), 921 - 34
Quorum sensing and the LysR-type transcriptional activator ToxR regulate toxoflavin biosynthesis and transport in Burkholderia glumae; Kim J et al.; Burkholderia glumae BGR1 produces a broad-host range phytotoxin, called toxoflavin, which is a key pathogenicity factor in rice grain rot and wilt in many field crops . Our molecular and genetic analyses of toxoflavin-deficient mutants demonstrated that gene clusters for toxoflavin production consist of four transcriptional units . The toxoflavin biosynthesis genes were composed of five genes, toxA to toxE, as Suzuki et al . (2004) reported previously . Genes toxF to toxI, which are responsible for toxoflavin transport, were polycistronic and similar to the genes for resistance-nodulation-division (RND) efflux systems . Using Tn3-gusA reporter fusions, we found that ToxR, a LysR-type regulator, regulates both the toxABCDE and toxFGHI operons in the presence of toxoflavin as a coinducer . In addition, the expression of both operons required a transcriptional activator, ToxJ, whose expression is regulated by quorum sensing . TofI, a LuxI homologue, was responsible for the biosynthesis of both N-hexanoyl homoserine lactone and N-octanoyl homoserine lactone (C8-HSL) . C8-HSL and its cognate receptor TofR, a LuxR homologue, activated toxJ expression . This is the first report that quorum sensing is involved in pathogenicity by the regulation of phytotoxin biosynthesis and its transport in plant pathogenic bacteria.

Genome Res, 2004 Nov, 14(11), 2295 - 307
Patterns of large-scale genomic variation in virulent and avirulent Burkholderia species; Ong C et al.; The human diseases melioidosis and glanders are caused by the bacteria Burkholderia pseudomallei and B . mallei respectively, and both species are regarded as potential biowarfare agents . We used B . pseudomallei DNA microarrays to compare the genomes of several clinical and environmental isolates of B . pseudomallei, B . mallei, and B . thailandensis, a closely related but avirulent species . Open reading frames (ORFs) deleted between the three species were associated with diverse cellular functions, including nitrogen and iron metabolism, quorum sensing, and polysaccharide production . Deleted ORFs in B . mallei exhibited significant genomic clustering, whereas deletions in B . thailandensis were more uniformly dispersed, suggesting that B . mallei and B . thailandensis may have diverged from B . pseudomallei and each other via distinct mechanisms . The genomes of independent B . pseudomallei isolates were highly conserved with a large-scale variance of less than 3% between isolates, and at least three distinct molecular subtypes could be defined . An analysis of subtype-specific genomic regions suggests that DNA loss has played an important role in the evolutionary radiation of B . pseudomallei in the natural environment . Our results raise several hypotheses concerning the possible mechanisms underlying the diverse biological properties exhibited by members of the Burkholderia family.

Eur Respir J, 2004 Nov, 24(5), 798 - 804
Impaired pulmonary status in cystic fibrosis adults with two mutated MBL-2 alleles; Davies JC et al.; Mannose-binding lectin has recently been identified as a modifier of severity in cystic fibrosis, although studies have produced differing results and the mechanism of action remains unclear . The current authors have studied large cohorts of adults (n=298) and children (n=260) to explore this apparent relationship further . Adults with two structural mutations, but not heterozygotes, had significantly reduced lung function and oxygen saturations, more frequent hospital admissions and raised systemic inflammatory markers . This was not related to increased rates of infection with Pseudomonas aeruginosa, and there was no increased susceptibility to Burkholderia cepacia . None of these findings was mirrored in the paediatric cohort . In conclusion, severe mannose-binding lectin deficiency appears to be detrimental to cystic fibrosis adults, although heterozygotes are not affected . It is suggested that this is not related to impaired complement-mediated bacterial killing, and a link with the host inflammatory response is hypothesised . If mannose-binding lectin replacement is developed as a new approach to treatment for this disease, the present study would suggest that the small group of severely deficient patients with two structural mutations may be the group to benefit.

Thorax, 2004 Nov, 59(11), 955 - 9
Association between Stenotrophomonas maltophilia and lung function in cystic fibrosis; Goss CH et al.; BACKGROUND: Stenotrophomonas maltophilia (SM) is a Gram-negative non-fermenting bacteria cultured from the sputum of patients with cystic fibrosis (CF) . To date, no information is available regarding the effect of this organism on lung function in CF . METHODS: A cohort study was conducted to assess the effect of SM on lung function among CF patients aged > or =6 years in the CF Foundation National Patient Registry from 1994 to 1999 . Repeated measures regression was used to assess the association between SM and lung function . RESULTS: The cohort consisted of 20 755 patients with median age at entry of 13.8 years and median follow up time of 3.8 years; 2739 patients (13%) were positive at least once for SM and 18 016 (87%) were never positive . After adjusting for sex, height and age, patients with SM had a mean forced expiratory volume in 1 second which was 0.09 l less (95% CI 0.05 to 0.14) than those without SM . The mean rate of decline associated with SM positivity was 0.025 l/year (95% CI 0.012 to 0.037) but, after adjusting for confounders (sex, height, weight, intravenous antibiotic courses, hospital admissions, pancreatic insufficiency, and Pseudomonas aeruginosa and Burkholderia cepacia status), the mean rate of decline decreased to 0.008 l/year (-0.008, 95% CI -0.019 to 0.003) . CONCLUSIONS: Although CF patients with SM have worse lung function at the time of positivity, no association was found between SM and increased rate of decline after controlling for confounders.

Thorax, 2004 Nov, 59(11), 952 - 4
Recovery of Burkholderia cenocepacia strain PHDC from cystic fibrosis patients in Europe; Coenye T et al.; BACKGROUND: Burkholderia cenocepacia can cause life threatening respiratory tract infections in patients with cystic fibrosis (CF) and has a significant impact on survival . There is extensive evidence for patient to patient spread and nosocomial transmission of this organism, and several widespread B cenocepacia strains have been described including the transatlantic ET12 clone . A study was performed to compare B cenocepacia isolates recovered from CF patients receiving care in several European countries and strains isolated from other clinical samples and the environment, with reference isolates from the epidemic B cenocepacia strain PHDC which has so far only been recovered from CF patients and soil in the USA . METHODS: A large collection of B cenocepacia isolates, including a large number recovered from CF patients receiving care in several European countries, Canada and the USA, were genotyped by means of randomly amplified polymorphic DNA typing (RAPD) and rep-PCR using the BOX-A1R primer (BOX-PCR) . RESULTS: Nineteen Burkholderia cenocepacia isolates cultured from clinical samples in Europe (18 recently recovered from CF patients in France and Italy and one recovered in 1964 from urine in the UK) showed RAPD fingerprinting patterns that were similar to patterns obtained from isolates of B cenocepacia strain PHDC . Subsequent analysis of these isolates using BOX-PCR confirmed that the European isolates and strain PHDC represent the same clone . CONCLUSION: Strain PHDC represents a second transatlantic B cenocepacia clone capable of colonising patients with CF.

Thorax, 2004 Nov, 59(11), 948 - 51
Burkholderia cenocepacia and Burkholderia multivorans: influence on survival in cystic fibrosis; Jones AM et al.; INTRODUCTION: Burkholderia cepacia infection has been associated with a poor prognosis for patients with cystic fibrosis (CF) . It is now recognised that organisms classified as B cepacia comprise a number of distinct genomic species each known as a genomovar of the B cepacia complex (BCC) . The outcome of infection for CF patients with individual genomovars is unknown . The clinical outcome of infection with the two most commonly isolated genomovars (B cenocepacia and B multivorans) was studied at a specialist CF centre between 1982 and 2003 . METHODS: The numbers of patients who progressed from initial to chronic infection were assessed . Control groups were created by matching patients with chronic BCC infection by percentage forced expiratory volume in 1 second with patients with Pseudomonas aeruginosa infection . Outcome measures were survival time, deaths from "cepacia syndrome", rate of decline in spirometry and body mass index (BMI), and treatment requirements . RESULTS: Forty nine patients had an initial infection with either B multivorans (n = 16) or B cenocepacia (n = 33); 8/16 and 31/33, respectively, developed chronic infection (p<0.001) . Deaths from "cepacia syndrome" occurred in both BCC groups . Patients with B cenocepacia infection had a shorter survival than patients with P aeruginosa infection (p = 0.01) . There was no difference in survival between CF patients infected with B multivorans and P aeruginosa . There were no observed differences in changes in spirometry and BMI or treatment requirements between the BCC groups and respective controls . CONCLUSION: In CF, the genomovar status of BCC may influence both the likelihood of progression from initial to chronic infection and the overall survival of the patients.

MMWR Morb Mortal Wkly Rep, 2004 Oct 29, 53(42), 988 - 90
Laboratory exposure to Burkholderia pseudomallei--Los Angeles, California, 2003; Centers for Disease Control and Prevention (CDC); On July 26, 2003, the Los Angeles County Department of Health Services (LACDHS) received a report that a local clinical laboratory had isolated from specimens Burkholderia pseudomallei, a category B biologic terrorism agent and the causative organism for melioidosis, which is endemic to certain tropical areas . Because laboratory workers had manipulated cultures of the organism, CDC was asked to assist in the subsequent investigation . This report summarizes the results of that investigation, which included assessment of laboratory exposures, postexposure chemoprophylaxis, and serologic testing of exposed laboratory workers . The findings underscore the need to reinforce proper laboratory practices and the potential benefits of chemoprophylaxis after laboratory exposures.

Curr Opin Pulm Med, 2004 Nov, 10(6), 515 - 23
The use of macrolide antibiotics in patients with cystic fibrosis; Saiman L; PURPOSE OF REVIEW: There has been much recent interest in the use of macrolide antibiotics as chronic suppressive therapy in patients with cystic fibrosis . Three recent randomized, placebo-controlled trials have been conducted . RECENT FINDINGS: All three trials used similar regimens of azithromycin, and lung function improved after 3 to 6 months of treatment . The relative change in forced expiratory volume in 1 second predicted improved between 3.6% and 6.2% . Furthermore, the azithromycin treatment groups had improvement in a variety of secondary outcomes related to pulmonary exacerbations, including a reduction in antibiotic use (both intravenous and oral) and hospitalization rate . Furthermore, azithromycin was well tolerated: Only nausea, diarrhea, and wheezing (described as mild to moderate) occurred more frequently in the azithromycin group compared with the placebo group . The evidence for the clinical benefit of azithromycin in cystic fibrosis has been summarized in a Cochrane review in which a meta-analysis confirmed a significant improvement in forced expiratory volume in 1 second among the 286 pooled participants . SUMMARY: Azithromycin has entered the therapeutic armamentarium for patients with cystic fibrosis who are chronically infected with Pseudomonas aeruginosa . Improved lung function, a reduction in pulmonary exacerbations and antibiotic use, and weight gain are potential benefits of this drug . Future studies should address the use of azithromycin in other cystic fibrosis patient populations, including those patients without chronic infection with P . aeruginosa, children younger than 6 years of age, and those infected with Burkholderia cepacia complex . The mechanism of action of macrolide antibiotics in cystic fibrosis remains unknown.

Glycobiology . 2004 Oct 27; {Epub ahead of print}
Nitrogen-fixing bacterium Burkholderia brasiliensis produces a novel yersiniose A-containing O-polysaccharide; Mattos KA et al.; Burkholderia brasiliensis, a Gram-negative diazotrophic endophytic bacterium, was first isolated from roots, stems and leaves of rice plant in Brazil . The polysaccharide moiety was released by ammonolysis from the B . brasiliensis lipopolysaccharide (LPS), allowing the unambiguous characterization of a 3,6-dideoxy-4-C-(1-hydroxyethyl)-D-xylo-hexose (Yersiniose A), an uncommon feature for Burkholderia LPS . The complete structure of the yersiniose A-containing O-antigen was identified by sugar and methylation analyses and nuclear magnetic resonance spectroscopy . Our results show that the repeating oligosaccharide motif of LPS O-chain consists of a branched tetrasaccharide with the following structure: -->2-alpha-L-Rhap-(1-->3)-{alpha-YerAp-(1-->2)}-alpha-L-Rhap-(1-->3)-alpha-L-Rhap-(1-->

J Antimicrob Chemother, 2004 Dec, 54(6), 1134 - 8 Epub 2004 Oct 27.
Antibiotic susceptibility of 65 isolates of Burkholderia pseudomallei and Burkholderia mallei to 35 antimicrobial agents; Thibault FM et al.; OBJECTIVES: Fifty isolates of Burkholderia pseudomallei and 15 isolates of Burkholderia mallei were tested for their susceptibilities to 35 antimicrobial agents, including agents not previously tested against these bacteria . METHODS: MICs were determined by agar dilution in Mueller-Hinton medium . RESULTS: Among the antibiotics tested, lower MICs were obtained with imipenem, ceftazidime, piperacillin, piperacillin/tazobactam, doxycycline and minocycline . Fluoroquinolones and aminoglycosides had poor activities . A single clinical isolate of B . pseudomallei was resistant to ceftazidime, co-amoxiclav and doxycycline but remained susceptible to imipenem . CONCLUSIONS: Although B . mallei MICs are often lower, the overall results underline the importance of resistance in both species . The susceptibilities measured are consistent with the current recommendations for the treatment of B . pseudomallei and B . mallei infections.

Phytochemistry, 2004 Nov, 65(22), 2957 - 66
Protein phosphorylation in Nicotiana tabacum cells in response to perception of lipopolysaccharides from Burkholderia cepacia; Gerber IB et al.; Bacterial LPS have the ability to act as modulators of the innate immune response in plants . Complex and largely unresolved perception systems exist for LPS on the plant cell surfaces that lead to the activation of multiple intracellular defense signaling pathways . The aim of the present study was to investigate the perception mechanism of cultured Nicotiana tabacum cells towards LPS from Burkholderia cepacia (LPS(B.cep.)), with regard to the role of protein phosphorylation during signal perception-related responses to gain a better understanding of the chemosensory perception of LPS elicitor signals in plant cells . In vivo labeling of protein phosphorylation events during signal transduction indicated the rapid phosphorylation of several proteins with the hyperphosphorylation of two proteins of 28 and 2 kDa, respectively . Significant differences and de novo LPS-induced phosphorylation were also observed with two-dimensional analysis . The protein kinase inhibitor, staurosporine, totally inhibited the extracellular alkalinization response induced by LPS(B.cep.), while the oxidative burst was only partially inhibited by staurosporine . Inhibition of protein phosphatase activity by calyculin A intensified the LPS(B.cep.) responses . The results indicate that perception- and signal transduction responses during LPS(B.cep.) elicitation of tobacco cells require a balance between the actions of certain protein kinases and protein phosphatases.

Am J Respir Crit Care Med, 2005 Jan 15, 171(2), 158 - 64 Epub 2004 Oct 22.
One-year Outcome after Severe Pulmonary Exacerbation in Adults with Cystic Fibrosis; Ellaffi M et al.; We retrospectively studied the outcomes of adult patients with cystic fibrosis (CF) hospitalized for severe pulmonary exacerbations (69 cases) between January 1997 and June 2001 . Cases were treated either in the Pulmonary Department (n = 46) or in the intensive care unit (ICU) (n = 23) depending on severity . Noninvasive mechanical ventilation was used in 61% (14 of 23) and 33% (15 of 46) of cases treated in the ICU and the Pulmonary Department groups, respectively . Invasive ventilation was necessary in 4 of 23 cases treated in the ICU . The 1-year survival rate was 52% (12 of 23) and 91% (42 of 46) in the ICU and the Pulmonary Department groups, respectively . Lung transplantation was performed in two patients from the ICU group and in five patients from the Pulmonary Department group after hospital discharge . Factors predictive of death were prior colonization with Burkholderia cepacia and rapid decline in FEV(1) before admission and severity of exacerbations (severity of hypoxemia and hypercapnia, simplified acute physiology score II and logistic organ dysfunction (LOD) scores, requirement of noninvasive mechanical ventilation, and hospitalization in the ICU) in the univariate analysis and were prior colonization with B . cepacia, the severity of hypoxemia at admission, and hospitalization in the ICU in the multivariate analysis . In 1-year survivors, pulmonary exacerbation did not affect the progression of the disease.

Infect Immun, 2004 Nov, 72(11), 6589 - 96
Quorum sensing: a transcriptional regulatory system involved in the pathogenicity of Burkholderia mallei; Ulrich RL et al.; Numerous gram-negative bacterial pathogens regulate virulence factor expression by using a cell density mechanism termed quorum sensing (QS) . An in silico analysis of the Burkholderia mallei ATCC 23344 genome revealed that it encodes at least two luxI and four luxR homologues . Using mass spectrometry, we showed that wild-type B . mallei produces the signaling molecules N-octanoyl-homoserine lactone and N-decanoyl-homoserine lactone . To determine if QS is involved in the virulence of B . mallei, we generated mutations in each putative luxIR homologue and tested the pathogenicities of the derivative strains in aerosol BALB/c mouse and intraperitoneal hamster models . Disruption of the B . mallei QS alleles, especially in RJ16 (bmaII) and RJ17 (bmaI3), which are luxI mutants, significantly reduced virulence, as indicated by the survival of mice who were aerosolized with 10(4) CFU (10 50% lethal doses {LD50s}) . For the B . mallei transcriptional regulator mutants (luxR homologues), mutation of the bmaR5 allele resulted in the most pronounced decrease in virulence, with 100% of the challenged animals surviving a dose of 10 LD50s . Using a Syrian hamster intraperitoneal model of infection, we determined the LD50s for wild-type B . mallei and each QS mutant . An increase in the relative LD50 was found for RJ16 (bmaI1) (>967 CFU), RJ17 (bmaI3) (115 CFU), and RJ20 (bmaR5) (151 CFU) compared to wild-type B . mallei (<13 CFU) . These findings demonstrate that B . mallei carries multiple luxIR homologues that either directly or indirectly regulate the biosynthesis of an essential virulence factor(s) that contributes to the pathogenicity of B . mallei in vivo.

Res Microbiol, 2004 Nov, 155(9), 781 - 93
Genome-wide comparison reveals great inter- and intraspecies variability in B . pseudomallei and B . mallei pathogens; Monastyrskaya G et al.; Burkholderia mallei and B . pseudomallei, closely related Gram-negative bacteria, are causative agents of serious infectious diseases of humans and animals: glanders and melioidosis, respectively . Despite numerous studies of these pathogens, the detailed mechanism of their pathogenesis is still unknown . The problem is even more complicated due to natural variability of B . pseudomallei and B . mallei strains, the understanding of which is a prerequisite for rational design of tools for diagnostics, prophylaxis and therapy of the diseases . Using a subtractive hybridization technique, we compared the genomes of B . pseudomallei C-141 and B . mallei C-5 strains . A subtracted library of DNA fragments specific for B . pseudomallei C-141 and absent from B . mallei C-5 was obtained and analyzed . A variety of differences have been detected and mapped on the recently sequenced genome of B . pseudomallei K96243 . A comparative sequence analysis also revealed considerable genomic differences between B . pseudomallei C-141 and B . mallei ATCC 23344 strains sequenced at The Institute for Genomic Research (TIGR) . We also observed significant genomic differences between B . pseudomallei C-141 and B . pseudomallei K96243 . Some of the differential DNA fragments displayed similarity to different mobile elements which have not yet been described for B . pseudomallei, whereas the others matched various prophage components, components of active transport systems, different enzymes and transcription regulators . A substantial proportion of the differential clones had no database matches either at the nucleotide or protein level . The results provide evidence for great genome-wide variability of B . pseudomallei, further confirmed by Southern blot analysis of various B . pseudomallei strains . The data obtained can be useful for future development of efficient diagnostic tools allowing rapid identification of species, strains and isolates of B . mallei and B . pseudomallei.

Proc Natl Acad Sci U S A, 2004 Nov 2, 101(44), 15811 - 6 Epub 2004 Oct 21.
Innate immunity in Arabidopsis thaliana: lipopolysaccharides activate nitric oxide synthase (NOS) and induce defense genes; Zeidler D et al.; Lipopolysaccharides (LPS) are cell-surface components of Gram-negative bacteria and are microbe-/pathogen-associated molecular patterns in animal pathosystems . As for plants, the molecular mechanisms of signal transduction in response to LPS are not known . Here, we show that Arabidopsis thaliana reacts to LPS with a rapid burst of NO, a hallmark of innate immunity in animals . Fifteen LPS preparations (among them Burkholderia cepacia, Pseudomonas aeruginosa, and Erwinia carotovora) as well as lipoteichoic acid from Gram-positive Staphylococcus aureus were found to trigger NO production in suspension-cultured Arabidopsis cells as well as in leaves . NO was detected by confocal laser-scanning microscopy in conjunction with the fluorophore 4-amino-5-methylamino-2',7'-difluorofluorescein diacetate, by electron paramagnetic resonance, and by a NO synthase (NOS) assay . The source of NO was addressed by using T-DNA insertion lines . Interestingly, LPS did not activate the pathogen-inducible varP NOS, but AtNOS1, a distinct NOS previously associated with hormonal signaling in plants . A prominent feature of LPS treatment was activation of defense genes, which proved to be mediated by NO . Northern analyses and transcription profiling by using DNA microarrays revealed induction of defense-associated genes both locally and systemically . Finally, AtNOS1 mutants showed dramatic susceptibility to the pathogen Pseudomonas syringae pv . tomato DC3000 . In sum, perception of LPS and induction of NOS contribute toward the activation of plant defense responses.

J Med Microbiol, 2004 Nov, 53(Pt 11), 1053 - 64
Role of quorum sensing in the pathogenicity of Burkholderia pseudomallei; Ulrich RL et al.; Burkholderia pseudomallei is the causative agent of human and animal melioidosis . The role of quorum sensing (QS) in the in vivo pathogenicity of B . pseudomallei via inhalational exposure of BALB/c mice and intraperitoneal challenge of Syrian hamsters has not been reported . This investigation demonstrates that B . pseudomallei encodes a minimum of three luxI and five luxR homologues that are involved in animal pathogenicity . Mass spectrometry analysis of culture supernatants revealed that wild-type B . pseudomallei and the luxI mutants synthesized numerous signalling molecules, including N-octanoyl-homoserine lactone, N-decanoyl-homoserine lactone, N-(3-hydroxyoctanoyl)-L-homoserine lactone, N-(3-hydroxydecanoyl)-L-homoserine lactone and N-(3-oxotetradecanoyl)-L-homoserine lactone, which was further confirmed by heterologous expression of the B . pseudomallei luxI alleles in Escherichia coli . Mutagenesis of the B . pseudomallei QS system increased the time to death and reduced organ colonization of aerosolized BALB/c mice . Further, intraperitoneal challenge of Syrian hamsters with the B . pseudomallei QS mutants resulted in a significant increase in the LD50 . Using semi-quantitative plate assays, preliminary analysis suggests that QS does not affect lipase, protease and phospholipase C biosynthesis/secretion in B . pseudomallei . The findings of the investigation demonstrate that B . pseudomallei encodes multiple luxIR genes, and disruption of the QS alleles reduces animal pathogenicity, but does not affect exoproduct secretion.

Syst Appl Microbiol, 2004 Sep, 27(5), 517 - 26
Polyphasic characterisation of Burkholderia cepacia-like isolates leading to the emended description of Burkholderia pyrrocinia; Storms V et al.; Twenty-five Burkholderia cepacia-like isolates of human and environmental origin, comprising five different recA RFLP types, were examined by using a polyphasic taxonomic approach, including recA gene sequence analysis, 16S rRNA gene sequence analysis, DNA:DNA hybridisation studies, tDNA-PCR, fatty acid analysis and biochemical analysis . The results of the present study demonstrated that twenty-three of these strains belong to Burkholderia pyrrocinia, a B . cepacia complex species thus far comprising one single soil isolate only . An emended description of Burkholderia pyrrocinia is proposed . The taxonomic status of the remaining two isolates requires further analysis.

Lett Appl Microbiol, 2004, 39(5), 413 - 9
PCR-based identification and characterization of Burkholderia cepacia complex bacteria from clinical and environmental sources; Seo ST et al.; AIMS: To study the genotypic identification and characterization of the 119 Burkholderia cepacia complex (Bcc) strains recovered from clinical and environmental sources in Japan and Thailand . METHODS AND RESULTS: Based on the results of analysis by 16S rDNA RFLP generated after digestion with DdeI, the Bcc strains were differentiated into two patterns: pattern 1 (including Burkholderia vietnamiensis) and pattern 2 (including B . cepacia genomovar I, Burkholderia cenocepacia and Burkholderia stabilis) . All strains belonged to pattern 2 except for one strain . In the RFLP analysis of the recA gene using HaeIII, strains were separated into eight patterns designated as A, D, E, G, H, I, J and K, of which pattern K was new . Burkholderia cepacia epidemic strain marker (BCESM) encoded by emsR and the pyrrolnitrin biosynthetic locus encoded by prnC were present in 22 strains (18%) and 88 strains (74%) from all sources, respectively . All emsR-positive strains belonged to B . cenocepacia, whereas most prnC-positive strains belonged to B . cepacia genomovar I . CONCLUSIONS: Strains derived from clinical sources were assigned to B . cepacia genomovar I, B . cenocepacia, B . stabilis and B . vietnamiensis . The majority of Bcc strains from environmental sources (77 of a total 95 strains) belonged to B . cepacia genomovar I, whereas the rest belonged to B . cenocepacia . On the basis of genomovar-specific PCR and prnC RFLP analysis, strains belonging to recA pattern K were identified as B . cepacia genomovar I . SIGNIFICANCE AND IMPACT OF THE STUDY: This work provides the genotypic identification of a collection of the Bcc strains from Japan and Thailand . RFLP analysis of the prnC gene promises to be a useful method for differentiating Burkholderia pyrrocinia from B . cepacia genomovar I strains.

J Biochem Mol Biol, 2004 Sep 30, 37(5), 556 - 64
Neutralizing chimeric mouse-human antibodies against Burkholderia pseudomallei protease: expression, purification and characterization; Chan SW et al.; A recombinant Fab monoclonal antibody (Fab) C37, previously obtained by phage display and biopanning of a random antibody fragment library against Burkholderia pseudomallei protease, was expressed in different strains of Escherichia coli . E . coli strain HB2151 was deemed a more suitable host for Fab expression than other E . coli strains when grown in media supplemented with 0.2 % glycerol . The expressed Fab fragment was purified by affinity chromatography on a Protein G-Sepharose column, and the specificity of the recombinant Fab C37 towards B . pseudomallei protease was proven by Western blotting, enzyme-linked immunosorbent assay (ELISA) and by proteolytic activity neutralization . In addition, polyclonal antibodies against B . pseudomallei protease were produced in rabbits immunized with the protease . These were isolated from high titer serum by affinity chromatography on recombinant-Protein A-Sepharose . Purified polyclonal antibody specificity towards B . pseudomallei protease was proven by Western blotting and ELISA.

FEMS Microbiol Lett, 2004 Oct 15, 239(2), 319 - 23
Ocurrence of the antibiotic producing bacterium Burkholderia sp . in colonies of the leaf-cutting ant Atta sexdens rubropilosa; Santos AV et al.; Fungus garden material from recently established Atta sexdens rubropilosa colonies (6-12 months old) was sampled to detect antibiotic producing microorganisms that inhibited the growth of pathogens of insects and of the fungus gardens but did not affect their mutualistic fungus . A bacterium with activity against the entomopathogenic fungus Beauveria bassiana was isolated from 56% of the gardens tested (n=57) and identified from its biochemical profile and from 16S and 23S ribosomal DNA sequences as a member of the genus Burkholderia . The ant-associated Burkholderia isolates secreted a potent, anti-fungal agent that inhibited germination of conidia of the entomopathogenic fungi B . bassiana, Metarhizium anisopliae, of the saprophytic Verticillium lecanii, and also of a specialist fungus garden Escovopsis weberi . Growth of the ant's mutualist fungus was unaffected.

Phytother Res, 2004 Aug, 18(8), 647 - 51
The antibacterial principle of Caesalpina sappan; Xu HX et al.; Using a bioassay-directed purification scheme, the active antibacterial principle from Caesalpina sappan was isolated and identified to be brasilin . This compound showed potent activity against antibiotic-resistant bacteria, notably methicillin-resistant Staphylococcus aureus (MRSA) and vancomycin-resistant enterococci (VRE), multi-drug resistant Burkholderia cepacia as well as a number of other bacteria . The minimal inhibitory concentrations ranged from 4 to 32 microg/mL . The results from time-kill studies showed that brasilin is bactericidal against MRSA . The addition of brasilin to growing MRSA cells resulted in a rapid inhibition of incorporation of {(3)H} thymidine or {(3)H} serine into DNA and proteins, respectively . Exposure of MRSA to a sub-MIC level of brasilin for ten consecutive subcultures did not induce resistance to the compound . The Trypan blue dye exclusion test showed that brasilin lacked cytotoxicity against Vero cells . In conclusion, brasilin is an antibacterial principle from C . sappan and it has the potential to be developed into an antibiotic.

Epidemiol Infect, 2004 Oct, 132(5), 813 - 20
Preliminary report on the northern Australian melioidosis environmental surveillance project; Inglis TJ et al.; An environmental surveillance programme was developed to determine whether water supplies could be a source of Burkholderia pseudomallei as noted during previous melioidosis outbreak investigations . Water supplies to communities in the three northern Australian jurisdictions (Western Australia, Northern Territory and Queensland) were sampled periodically during 2001 and 2002 . Water and soil samples were collected from communities known to have had recent culture-positive melioidosis cases and nearby communities where no cases had been diagnosed . Clinical isolates of B . pseudomallei obtained from northern Australian patients during 2001 and 2002 were compared with the environmental B . pseudomallei isolates by ribotyping and pulsed-field gel electrophoresis . B . pseudomallei was isolated from 11 distinct locations, all in the Northern Territory, seven of which were associated with culture-positive melioidosis cases (>1 case at three locations) . Water was implicated as a possible environmental source of melioidosis in six locations . A variety of free-living amoebae including Acanthamoeba and Hartmannella spp . that are potential hosts to B . pseudomallei were recovered from environmental specimens . Culturable B . pseudomallei was not found to be widely dispersed in the environments sampled.

J Clin Microbiol, 2004 Oct, 42(10), 4824 - 7
Species distribution and ribotype diversity of Burkholderia cepacia complex isolates from French patients with cystic fibrosis; Brisse S et al.; A total of 153 Burkholderia cepacia strains obtained from 153 French patients with cystic fibrosis were identified as Burkholderia multivorans (51.6%) or Burkholderia cenocepacia (45.1%) . Eighty-two genotypes were identified using PvuII and EcoRI ribotyping . B . multivorans genotype A (found in 32 French patients) and two other genotypes were also identified among isolates from Austrian, German, Italian, and Canadian patients.

J Cyst Fibros, 2004 Aug, 3(3), 165 - 72
Exopolysaccharides produced by Burkholderia cenocepacia recA lineages IIIA and IIIB; Chiarini L et al.; Clinical and environmental strains of Burkholderia cenocepacia belonging to the recA lineages IIIA and IIIB were examined for exopolysaccharide (EPS) production . The exopolysaccharides structure was determined using mainly gas chromatography coupled to mass spectrometry and NMR spectroscopy . All the strains produced Cepacian, a highly branched polysaccharide constituted of a heptasaccharide repeating unit, composed of one rhamnose, one glucose, one glucuronic acid, one mannose and three galactose residues . This polymer is the most common exopolysaccharide produced by strains of the Burkholderia cepacia (Bcc) complex . One clinical strain produced also another polysaccharide constituted of three galactose units and one 3-deoxy-d-manno-2-octulosonic acid residues, a polymer that was previously isolated from two strains of B . cepacia genomovar I and B . cenocepacia IIIA.

J Cyst Fibros, 2004 Aug, 3(3), 159 - 63
Molecular epidemiology of Pseudomonas aeruginosa, Burkholderia cepacia complex and methicillin-resistant Staphylococcus aureus in a cystic fibrosis center; Campana S et al.; Chronic pulmonary infections caused by Pseudomonas aeruginosa, Burkholderia cepacia complex and Staphylococcus aureus are responsible for most of the morbidity and mortality of patients with cystic fibrosis (CF) . Little is known about the routes of transmission of these pathogens from environmental or hospital sources to the patients . We hypothesised that strains of P . aeruginosa, B . cepacia complex and methicillin-resistant S . aureus (MRSA) are nosocomially acquired by CF patients . Bacterial isolates were obtained from 164 patients attending the CF Centre of Florence and from the hospital environment and the strains typed using restriction enzymes and pulsed-field gel electrophoresis (PFGE) . Seventy (43%) of patients were colonised by P . aeruginosa, 6 (3.6%) by B . cepacia complex, and 11 (7%) by MRSA . Three P . aeruginosa strains were isolated from the sinks of the ward . All the MRSA isolates differed from each other . The analysis of 83 P . aeruginosa strains showed identical genotypes in five pairs of patients, whereas from the six patients infected with B . cepacia complex strains, two patients harboured identical genotypes . These pairs of patients had no contact with each other outside the CF centre and P . aeruginosa genotypes from the hospital environment differed from these clinical isolates, suggesting a possible common source of infection within or outside the centre . The study showed that, despite isolation precautions, a minimal risk of cross-infection still existed in the CF centre and that hygienic standards should be increased to further reduce this risk.

J Cyst Fibros, 2004 Jun, 3(2), 133 - 4
'Cepacia syndrome' with Burkholderia multivorans, 9 years after initial colonization; Blackburn L et al.; A 16-year-old boy with cystic fibrosis developed 'cepacia syndrome' 9 years after the first isolation of Burkholderia multivorans . It is important to recognise that 'cepacia syndrome' is not restricted to those infected with genomovar type III strains and that rapid, irreversible clinical decline can occur many years after the 1st isolation of Burkholderia cepacia complex (Bcc).

J Cyst Fibros, 2004 Jun, 3(2), 93 - 8
Clinical outcome of Burkholderia cepacia complex infection in cystic fibrosis adults; Courtney JM et al.; BACKGROUND: The Burkholderia cepacia complex (BCC) is one of the most important groups of organisms infecting cystic fibrosis (CF) patients . The aim of the study was to examine how infection with BCC affects clinical outcome . METHODS: Nineteen CF adults infected with BCC and 19 controls infected with Pseudomonas aeruginosa were studied over a 4-year period . The best forced expiratory volume in 1 s (FEV(1)) and body mass index (BMI) for each year were recorded and annual rate of decline calculated . RESULTS: The BCC infected group displayed a significantly greater reduction of FEV(1) and BMI compared to the P . aeruginosa infected group (p=0.001 and p=0.009, respectively) . Sixteen patients infected with a single Burkholderia cenocepacia strain had a significantly greater rate of FEV(1) decline compared to those infected with Burkholderia multivorans (n=3) or P . aeruginosa (p=0.01 and p<0.0001, respectively) . The rate of BMI decline was significantly greater in patients infected with B . cenocepacia compared to those with P . aeruginosa (p=0.007), but not significantly different in those with B . multivorans (p=0.29) . CONCLUSION: BCC infection is associated with an accelerated decline in pulmonary function and BMI . Infection with a single B . cenocepacia strain was associated with a more rapid decline in lung function than those infected with either B . multivorans or P . aeruginosa.

J Cyst Fibros, 2002 Dec, 1(4), 292 - 4
Burkholderia cepacia genomovar III and Burkholderia vietnamiensis double infection in a cystic fibrosis child; Magalhaes M et al.; Herein we report a case of a cystic fibrosis child who was simultaneously infected with Burkholderia cepacia genomovar III and Burkholderia vietnamiensis . After antimicrobial therapy only B . cepacia genomovar III persisted.

Bol Asoc Med P R, 2003 Nov-Dec, 95(6), 17 - 20
Severe community-acquired pneumonia and sepsis caused by Burkholderia pseudomallei associated with flooding in Puerto Rico; Christenson B et al.; Burkholderia pseudomallei (melioidosis) is usually found in endemic areas of Southeast Asia and Northern Australia . However, a few cases of confirmed melioidosis indigenous to Puerto Rico and the Americas have been reported previously . We describe the occurrence of a B . pseudomallei infection in a female with insulin-dependent diabetes mellitus exposed to flood waters in Puerto Rico . We conclude that B . pseudomallei should be considered a potential pathogen in high-risk patients with severe community-acquired pneumonia and sepsis in Puerto Rico especially in individuals exposed to flood waters during rainy seasons . A more thorough epidemiologic and microbiologic surveillance with environmental sampling may be warranted in the island.

Acta Crystallogr D Biol Crystallogr, 2004 Oct, 60(Pt 10), 1824 - 32 Epub 2004 Sep 23.
Structure of the C-terminal domain of the catalase-peroxidase KatG from Escherichia coli; Carpena X et al.; Catalase-peroxidases or KatGs, the apparent in vivo activators of the anti-tubercular pro-drug isoniazid, are active as homodimers, each subunit having two distinct but sequence- and structure-related domains . The N-terminal domain contains the haem group and is catalytically active, while the C-terminal domain lacks the cofactor . The C-terminal domain of KatG from Escherichia coli is expressed as a soluble protein which has been crystallized in triclinic, orthorhombic and tetragonal crystal forms . Packing in the orthorhombic crystals, with eight molecules in the asymmetric unit, follows the pattern of commensurate modulated structures, which explains the diversity of pseudo-origin peaks observed in the native Patterson map . The different crystal forms arise from variations in the length and sequence of the N-terminal extensions in the different constructs . Despite the variability in the N-terminal region, the overall domain conformations beginning with Pro437 are very similar both to each other and to the C-terminal domains within the native structures of the KatGs from Haloarcula marismortui and Burkholderia pseudomallei . Some structural reorganization in the C-terminal domain relative to the N-terminal domain has evolved to compensate for the absence of the haem group . A high percentage of the residues in the C-terminal domains of KatG proteins from different sources are highly conserved and these residues are spread uniformly throughout the domain . The easily folded nature and retention of structure in the C-terminal domain suggests that it may serve as a platform for the folding of the N-terminal domain and for stabilization of the molecular dimer.

Int J Syst Evol Microbiol, 2004 Sep, 54(Pt 5), 1677 - 81
Classification of the biphenyl- and polychlorinated biphenyl-degrading strain LB400T and relatives as Burkholderia xenovorans sp . nov; Goris J et al.; Strain LB400T is the best-studied polychlorinated biphenyl (PCB) degrader . This organism has previously been allocated in the genus Burkholderia, since its 16S rRNA gene sequence shows 98.6 % sequence similarity to the type strains of Burkholderia graminis and Burkholderia terricola . A polyphasic study was undertaken to clarify the actual taxonomic position of this biotechnologically important organism and of two strains, one recovered from a blood culture vial and one from a coffee plant rhizosphere, both of which resembled strain LB400T in their whole-cell protein patterns . DNA-DNA hybridization experiments revealed that the three strains represented a single novel species, for which the name Burkholderia xenovorans sp . nov . is proposed . Strains of this novel species can be differentiated phenotypically from nearly all other Burkholderia species by their inability to assimilate L-arabinose . The whole-cell fatty acid profile of B . xenovorans strains is consistent with their classification in the genus Burkholderia, with 18 : 1omega7c, 16 : 1omega7c, 16 : 0, 14 : 0 3OH, 16 : 0 3OH, 17 : 0 cyclo and 14 : 0 being the most abundant fatty acids . The G + C content of the species varies between 62.4 and 62.9 mol% . The type strain of B . xenovorans is LB400T (= LMG 21463T = CCUG 46959T = NRRL B-18064T).

Biomed Environ Sci, 2004 Jun, 17(2), 187 - 95
Bioremediation of quinoline-contaminated soil using bioaugmentation in slurry-phase reactor; Wang JL et al.; OBJECTIVE: To investigate the possibility of using bioaugmentation as a strategy for remediating quinoline-contaminated soil . METHODS: Microorganisms were introduced to the soil to assess the feasibility of enhancing the removal of quinoline from quinoline-contaminated soil . Slurry-phase reactor was used to investigate the bioremediation of quinoline-contaminated soil . HPLC (Hewlett-Packard model 5050 with an UV detector) was used for analysis of quinoline concentration . RESULTS: The biodegradation rate of quinoline was increased through the introduction of Burkholderia pickettii . Quinoline, at a concentration of 1 mg/g soil, could be removed completely within 6 and 8 hours with and without combined effect of indigenous microbes, respectively . Although the indigenous microbes alone had no quinoline-degrading ability, they cooperated with the introduced quinoline-degrader to remove quinoline more quickly than the introduced microbes alone . Bioaugmentaion process was accelerated by the increase of inoculum size and bio-stimulation . The ratio of water to soil in slurry had no significant impact on bioremediation results . CONCLUSION: Bioaugmetation is an effective way for bioremediation of quinoline-contaminated soil.

Infect Immun, 2004 Oct, 72(10), 6142 - 7
Persistence of Burkholderia multivorans within the pulmonary macrophage in the murine lung; Chu KK et al.; Differences in infection kinetics and host response between Burkholderia multivorans and Burkholderia cenocepacia were demonstrated in a pulmonary infection model in BALB/c mice . B . multivorans persisted in the lung, while B . cenocepacia was cleared . Indirect immunofluorescence and electron microscopy of B . multivorans-infected lungs localized bacteria to macrophages . Clearance of B . cenocepacia was associated with greater interleukin-1beta and neutrophil responses than the responses induced by B . multivorans.

Am J Trop Med Hyg, 2004 Sep, 71(3), 360 - 2
Contamination of hand wash detergent linked to occupationally acquired melioidosis; Gal D et al.; Two mechanics working at a garage in tropical northern Australia simultaneously developed upper limb melioidosis ulcers . Both patients had Burkholderia pseudomallei of identical pulsed-field gel electrophoresis (PFGE) type (Spe I) . Environmental sampling identified B . pseudomallei in a container of commercial hand wash detergent as the likely source of infection, although there were multiple isolates of different PFGE types to the clinical isolates.

Clin Immunol, 2004 Oct, 113(1), 22 - 8
Adaptive immunity in melioidosis: a possible role for T cells in determining outcome of infection with Burkholderia pseudomallei; Barnes JL et al.; Melioidosis is a potentially fatal disease caused by the bacterium Burkholderia pseudomallei . Individuals with subclinical melioidosis have no apparent clinical signs or symptoms, and are identified only by positive serology . The present study is the first to investigate cell-mediated immune (CMI) responses following in vitro stimulation with B . pseudomallei antigens in peripheral blood mononuclear cells (PBMC), collected under field conditions in Papua New Guinea (PNG) from individuals with exposure to B . pseudomallei (n = 13) . While five had a clinical history of melioidosis (C(+)), the remaining individuals (n = 8) were seropositive, yet healthy with no clinical history of melioidosis (S(+)/C(-)) . Proliferation and IFN-gamma production were significantly greater in lymphocyte cultures from S(+)/C(-) individuals compared to C(+) individuals (P < 0.001 and P < 0.05, respectively) . These findings demonstrate that compared to C(+) patients, individuals with subclinical melioidosis have a stronger CMI response to B . pseudomallei antigens in vitro . Such a response may be essential for protection against disease progression.

Proc Natl Acad Sci U S A, 2004 Sep 28, 101(39), 14240 - 5 Epub 2004 Sep 17.
Genomic plasticity of the causative agent of melioidosis, Burkholderia pseudomallei; Holden MT et al.; Burkholderia pseudomallei is a recognized biothreat agent and the causative agent of melioidosis . This Gram-negative bacterium exists as a soil saprophyte in melioidosis-endemic areas of the world and accounts for 20% of community-acquired septicaemias in northeastern Thailand where half of those affected die . Here we report the complete genome of B . pseudomallei, which is composed of two chromosomes of 4.07 megabase pairs and 3.17 megabase pairs, showing significant functional partitioning of genes between them . The large chromosome encodes many of the core functions associated with central metabolism and cell growth, whereas the small chromosome carries more accessory functions associated with adaptation and survival in different niches . Genomic comparisons with closely and more distantly related bacteria revealed a greater level of gene order conservation and a greater number of orthologous genes on the large chromosome, suggesting that the two replicons have distinct evolutionary origins . A striking feature of the genome was the presence of 16 genomic islands (GIs) that together made up 6.1% of the genome . Further analysis revealed these islands to be variably present in a collection of invasive and soil isolates but entirely absent from the clonally related organism B . mallei . We propose that variable horizontal gene acquisition by B . pseudomallei is an important feature of recent genetic evolution and that this has resulted in a genetically diverse pathogenic species.

Proc Natl Acad Sci U S A, 2004 Sep 28, 101(39), 14246 - 51 Epub 2004 Sep 17.
Structural flexibility in the Burkholderia mallei genome; Nierman WC et al.; The complete genome sequence of Burkholderia mallei ATCC 23344 provides insight into this highly infectious bacterium's pathogenicity and evolutionary history . B . mallei, the etiologic agent of glanders, has come under renewed scientific investigation as a result of recent concerns about its past and potential future use as a biological weapon . Genome analysis identified a number of putative virulence factors whose function was supported by comparative genome hybridization and expression profiling of the bacterium in hamster liver in vivo . The genome contains numerous insertion sequence elements that have mediated extensive deletions and rearrangements of the genome relative to Burkholderia pseudomallei . The genome also contains a vast number (>12,000) of simple sequence repeats . Variation in simple sequence repeats in key genes can provide a mechanism for generating antigenic variation that may account for the mammalian host's inability to mount a durable adaptive immune response to a B . mallei infection.

Clin Exp Immunol, 2004 Oct, 138(1), 61 - 5
Burkholderia pseudomallei stimulates low interleukin-8 production in the human lung epithelial cell line A549; Utaisincharoen P et al.; Melioidosis is a life-threatening disease caused by Burkholderia pseudomallei . The lung is the most commonly affected organ, resulting in abscess formation in patients with chronic melioidosis . Previous study has shown that B . pseudomallei was able to invade and multiply in epithelial cells . In the present study, we have demonstrated that B . pseudomallei is able to stimulate interleukin 8 (IL-8) production from the human alveolar lung epithelium cell line A549 . However, the level of IL-8 production was significantly lower than when the cells were infected with other Gram-negative bacteria such as Salmonella enterica serovar Typhi (S . typhi) which were used for comparison . The degree of IkappaBalpha degradation in the B . pseudomallei-infected cells was lower than that of the S . typhi-infected cells, suggesting that B . pseudomallei is also a poorer cell activator . Inhibition of B . pseudomallei invasion by cytochalasin D did not interfere with either IL-8 production or IkappaBalpha degradation, indicating that bacterial uptake is not required for the production of this chemokine . Thus, it appears that the signalling initiated by the interaction of B . pseudomallei with the epithelial cell surface is sufficient for epithelial cell activation.

Pathology, 2004 Aug, 36(4), 352 - 7
Isolation and identification of Burkholderia cepacia by participants in an external Quality Assurance Program (QAP) between 1994 and 1999; Taylor PC et al.; AIM: External quality assurance programs (QAPs) provide an opportunity to benchmark laboratory performance according to the profile of specimens received . Participant confidentiality is maintained within each group of laboratories whose performance is measured using similar, repetitive exercises . Isolation and identification of Burkholderia cepacia from simulated cystic fibrosis (CF) sputa was a clinically relevant exercise that provided a model for this analytical approach . METHODS: Between 1994 and 1999, six Royal College of Pathologists of Australasia (RCPA) Microbiology QAPs included four simulated CF sputa and two panels of oxidative Gram-negative bacilli . Laboratories were grouped according to experience with CF sputa disclosed by two questionnaires . Data were analysed by laboratory group for ability to isolate and identify B . cepacia . RESULTS: Three laboratory groups annually received >100 CF sputa (CF>100), 100 CF sputa or fewer, or did not regularly receive CF sputa . CF>100 laboratories inoculated more isolation media, were more likely to use selective media and were less likely to misidentify B . cepacia than the other groups . Improved performance by CF>100 laboratories was marked after the first exercise and remained at a high level compared with the other two groups . This trend in performance was also apparent for Pseudomonas aeruginosa although the numbers of errors were less than for B . cepacia . CONCLUSIONS: These exercises demonstrated consistently improved performance only among CF>100 laboratories . The future criteria for laboratory accreditation may include performance as well as participation in QAPs, placing additional burdens on organisers and participants.

J Clin Microbiol, 2004 Sep, 42(9), 4121 - 6
Correlation of wbiI genotype, serotype, and isolate source within species of the Burkholderia cepacia complex; Vinion-Dubiel AD et al.; Gram-negative bacteria of the Burkholderia cepacia complex (Bcc) are opportunistic pathogens that can infect the lungs of cystic fibrosis (CF) patients and can be transmitted among these patients, causing epidemics in the CF community . Lipopolysaccharide (LPS) is an important virulence factor of many gram-negative bacteria, with the O antigen component of LPS being responsible for serotype specificity . The goal of this work was to develop a genetic method of determining the serotype of Bcc isolates based on the conserved gene wbiI . Homologues of wbiI are found in polysaccharide biosynthesis gene clusters in other bacteria . Primers to a conserved region of the Bcc wbiI gene were able to amplify by PCR a single product in 67 of 80 Bcc isolates tested . Sequencing and restriction enzyme digestion of this wbiI PCR product revealed sufficient DNA polymorphisms to distinguish and group various isolates . In five of nine instances, Bcc isolates of a single serotype had a single wbiI restriction fragment length polymorphism (RFLP) pattern, while isolates of the other four serotypes could have multiple wbiI RFLP types . Species determination of the Bcc isolates revealed no obvious correlation between wbiI RFLP type and species . There was also no apparent correlation between wbiI RFLP type and the ability of a single Bcc isolate to infect an individual with CF . However three of five Bcc outbreaks involved isolates with the same wbiI RFLP type, indicating that wbiI RFLP typing may be a useful tool to help track Bcc outbreaks.

FEMS Immunol Med Microbiol, 2004 Oct 1, 42(2), 249 - 53
Detection of bacterial DNA in blood samples from febrile patients: underestimated infection or emerging contamination?
Peters RP, Mohammadi T, Vandenbroucke-Grauls CM, Danner SA, van Agtmael MA, Savelkoul PH.
We applied real-time broad-range polymerase chain reaction (PCR) to detect bacteraemia in blood from febrile patients . Interpretation of amplification results in relation to clinical data and blood culture outcome was complex, although the reproducibility of the PCR results was good . Sequencing analysis of the PCR products revealed the presence of Burkholderia species DNA while no Burkholderia species grew in culture . The source of this contamination was shown to be the commercial DNA isolation kit used in the automated MagNA Pure Isolation Robot . A high degree of suspicion is required when uncommon or unexpected pathogens are diagnosed by molecular methods as clinical consequences can be serious.

Trans R Soc Trop Med Hyg, 2004 Nov, 98(11), 678 - 86
The changing pattern of bloodstream infections associated with the rise in HIV prevalence in northeastern Thailand; Chierakul W et al.; A survey of bloodstream infections was conducted in the large regional hospital in Ubon Ratchatani, northeastern Thailand between 1989 and 1998, during the onset of the HIV epidemic . The incidence of Staphylococcus aureus, Escherichia coli, Klebsiella/Enterobacter and Pseudomonas aeruginosa bacteraemias remained constant whereas infections caused by Burkholderia pseudomallei, non-typhoid Salmonellae, Cryptococcus neoformans, Penicillum marneffei and to a lesser extent Streptococcus pneumoniae all rose . Burkholderia pseudomallei infections were unrelated to HIV, whereas the other infections were associated directly with HIV . Group D non-typhoid Salmonellae bloodstream infections (mainly Salmonella enteritidis) rose coincident with the increase in HIV seroprevalence, and preceded the increase in the other HIV-associated infections . Other non-typhoid Salmonella bacteraemias increased two years after the rise in group D infections, and invasive yeast infections increased four years later, coincident with the increase in AIDS . Increasing Group D non-typhoid Salmonella bloodstream infections are an early warning signal of an impending rise in AIDS.

Pediatr Infect Dis J, 2004 Sep, 23(9), 882 - 4
Hemophagocytic syndrome and Burkholderia cepacia splenic microabscesses in a child with chronic granulomatous disease; Sirinavin S et al.; Hemophagocytic syndrome, splenic microabscesses and pulmonary cavitary lesions were presented in a 17-month-old boy with prolonged fever, hepatosplenomegaly and a history of tuberculous lymphadenitis . Clinical course mimicked tuberculosis . Blood cultures were negative . Ultrasound-guided, percutaneous aspiration from splenic microabscesses grew Burkholderia cepacia . He was treated successfully with trimethoprim-sulfamethoxazole . This child with chronic granulomatous disease had an unusual clinical manifestation of B . cepacia infection.

J Med Microbiol, 2004 Oct, 53(Pt 10), 999 - 1005
Characterizing uncommon Burkholderia cepacia complex isolates from an outbreak in a haemodialysis unit; Souza AV et al.; An outbreak of bacteraemia in a haemodialysis unit where 65 episodes of infection involved 35 outpatients is reported . Burkholderia cepacia complex was the agent most frequently recovered from blood . Thirty-three environmental and clinical isolates of B . cepacia complex were characterized by whole-cell protein electrophoresis and recA-RFLP profile . Fourteen isolates were genomovar I and 16 isolates were not classifiable by their recA-RFLP pattern . Ribotyping, random amplification of polymorphic DNA (RAPD) and integron profile were used to explore the clonality of the isolates, and revealed multiple strain genotypes . Four ribotypes and RAPD types and three integron patterns were identified . The water supply was identified as the source of the outbreak, and inappropriate cleaning and a leak in the reverse osmosis tubing connection were the probable causes of contamination . B . cepacia complex was still recovered from blood of patients even after apparently adequate measures were taken and water quality standards were met, suggesting that higher standards for water quality should be adopted in haemodialysis units . The genomovars recovered here were distinct from those commonly reported for cystic fibrosis isolates.

J Microbiol, 2004 Jun, 42(2), 126 - 32
Expression and purification of a recombinant scFv towards the exotoxin of the pathogen, Burkholderia pseudomallei; Lim KP et al.; A single chain variable fragment (scFv) specific towards B . pseudomallei exotoxin had previously been generated from an existing hybridoma cell line (6E6AF83B) and cloned into the phage display vector pComb3H . In this study, the scFv was subcloned into the pComb3X vector to facilitate the detection and purification of expressed antibodies . Detection was facilitated by the presence of a hemagglutinin (HA) tag, and purification was facilitated by the presence of a histidine tag . The culture was grown at 30 degrees C until log phase was achieved and then induced with 1 mM IPTG in the absence of any additional carbon source . Induction was continued at 30 degrees C for five h . The scFv was discerned by dual processes-direct enzyme-linked immunosorbent assays (ELISA), and Western blotting . When compared to E . coli strains ER2537 and HB2151, scFv expression was observed to be highest in the E . coli strain Top10F' . The expressed scFv protein was purified via nickel-mediated affinity chromatography and results indicated that two proteins a 52 kDa protein, and a 30 kDa protein were co-purified . These antibodies, when blotted against immobilized exotoxin, exhibited significant specificity towards the exotoxin, compared to other B . pseudomallei antigens . Thus, these antibodies should serve as suitable reagents for future affinity purification of the exotoxin.

Water Res, 2004 Oct, 38(17), 3705 - 12
Amplification and attenuation of tetracycline resistance in soil bacteria: aquifer column experiments; Rysz M et al.; A growing inefficacy of antimicrobial agents to treat infectious diseases has stimulated research on the development of antibiotic resistance in bacteria in the environment . Sustained exposure of soil microorganisms to tetracycline (TC) in flow-through columns (50mg/L influent) significantly decreased the effluent concentration of total heterotrophs and selected for TC-resistant (Tet(r)) soil bacteria . This suggests that TC released to the environment from animal farms may contribute to the development and amplification of TC resistance, with soil bacteria serving as reservoirs for antibiotic resistance continuance . Burkholderia cepacia, with genetic determinants for efflux pumps that facilitate TC excretion, was the only bacterium that grew on TC-amended R2A plates . Following 300 days of exposure, TC was removed from the influent to study the recovery pattern of the microbial community . The percentage of Tet(r) hererotrophs decreased from 25% to close to the control level of 1% within 1 month of discontinuing TC exposure . This was due both to a significant rebound in the total heterotrophic population and to a significant decrease in the concentration of Tet(r) bacteria . Thus, discontinuing TC exposure or curtailing its use should enhance natural attenuation mechanisms that mitigate the spread of resistance vectors.

Appl Environ Microbiol, 2004 Sep, 70(9), 5613 - 20
Implications of amino acid substitutions in GyrA at position 83 in terms of oxolinic acid resistance in field isolates of Burkholderia glumae, a causal agent of bacterial seedling rot and grain rot of rice; Maeda Y et al.; Oxolinic acid (OA), a quinolone, inhibits the activity of DNA gyrase composed of GyrA and GyrB and shows antibacterial activity against Burkholderia glumae . Since B . glumae causes bacterial seedling rot and grain rot of rice, both of which are devastating diseases, the emergence of OA-resistant bacteria has important implications on rice cultivation in Japan . Based on the MIC of OA, 35 B . glumae field isolates isolated from rice seedlings grown from OA-treated seeds in Japan were divided into sensitive isolates (OSs; 0.5 microg/ml), moderately resistant isolates (MRs; 50 microg/ml), and highly resistant isolates (HRs; > or =100 microg/ml) . Recombination with gyrA of an OS, Pg-10, led MRs and HRs to become OA susceptible, suggesting that gyrA mutations are involved in the OA resistance of field isolates . The amino acid at position 83 in the GyrA of all OSs was Ser, but in all MRs and HRs it was Arg and Ile, respectively . Ser83Arg and Ser83Ile substitutions in the GyrA of an OS, Pg-10, resulted in moderate and high OA resistance, respectively . Moreover, Arg83Ser and Ile83Ser substitutions in the GyrA of MRs and HRs, respectively, resulted in susceptibility to OA . These results suggest that Ser83Arg and Ser83Ile substitutions in GyrA are commonly responsible for resistance to OA in B . glumae field isolates.

Appl Environ Microbiol, 2004 Sep, 70(9), 5214 - 21
Requirement of a relatively high threshold level of Mg(2+) for cell growth of a rhizoplane bacterium, Sphingomonas yanoikuyae EC-S001; Hoo H et al.; Mg(2+) is one of the essential elements for bacterial cell growth . The presence of the magnesium cation (Mg(2+)) in various concentrations often affects cell growth restoration in plant-associating bacteria . This study attempted to determine whether Mg(2+) levels in Sphingomonas yanoikuyae EC-S001 affected cell growth restoration in the host plant and what the threshold level is . S . yanoikuyae EC-S001, isolated from the rhizoplane of spinach seedlings grown from surface-sterilized seeds under aseptic conditions, displayed uniform dispersion and attachment throughout the rhizoplane and phylloplane of the host seedlings . S . yanoikuyae EC-S001 did not grow in potato-dextrose broth medium but grew well in an aqueous extract of spinach leaves . Chemical investigation of the growth factor in the spinach leaf extract led to identification of the active principle as the magnesium cation . A concentration of ca . 0.10 mM Mg(2+) or more allowed S . yanoikuyae EC-S001 to grow in potato-dextrose broth medium . Some saprophytic and/or diazotrophic bacteria used in our experiment were found to have diverse threshold levels for their Mg(2+) requirements . For example, Burkholderia cepacia EC-K014, originally isolated from the rhizoplane of a Melastoma sp., could grow even in Mg(2+)-free Hoagland's no . 2 medium with saccharose and glutamine (HSG medium) and requires a trace level of Mg(2+) for its growth . In contrast, S . yanoikuyae EC-S001, together with Bacillus subtilis IFO12113, showed the most drastic restoring responses to subsequent addition of 0.98 mM Mg(2+) to Mg(2+)-free HSG medium . Our studies concluded that Mg(2+) is more than just the essential trace element needed for cell growth restoration in S . yanoikuyae EC-S001 and that certain nonculturable bacteria may require a higher concentration of Mg(2+) or another specific essential element for their growth.

J Biol Chem, 2004 Nov 12, 279(46), 47489 - 96 Epub 2004 Aug 31.
Revisiting the regiospecificity of Burkholderia xenovorans LB400 biphenyl dioxygenase toward 2,2'-dichlorobiphenyl and 2,3,2',3'-tetrachlorobiphenyl; Barriault D et al.; 2,2'-Dichlorobiphenyl (CB) is transformed by the biphenyl dioxygenase of Burkholderia xenovorans LB400 (LB400 BPDO) into two metabolites (1 and 2) . The most abundant metabolite, 1, was previously identified as 2,3-dihydroxy-2'-chlorobiphenyl and was presumed to originate from the initial attack by the oxygenase on the chlorine-bearing ortho carbon and on its adjacent meta carbon of one phenyl ring . 2,3,2',3'-Tetrachlorobiphenyl is transformed by LB400 BPDO into two metabolites that had never been fully characterized structurally . We determined the precise identity of the metabolites produced by LB400 BPDO from 2,2'-CB and 2,3,2',3'-CB, thus providing new insights on the mechanism by which 2,2'-CB is dehalogenated to generate 2,3-dihydroxy-2'-chlorobiphenyl . We reacted 2,2'-CB with the BPDO variant p4, which produces a larger proportion of metabolite 2 . The structure of this compound was determined as cis-3,4-dihydro-3,4-dihydroxy-2,2'-dichlorobiphenyl by NMR . Metabolite 1 obtained from 2,2'-CB-d(8) was determined to be a dihydroxychlorobiphenyl-d(7) by gas chromatographic-mass spectrometric analysis, and the observed loss of only one deuterium clearly shows that the oxygenase attack occurs on carbons 2 and 3 . An alternative attack at the 5 and 6 carbons followed by a rearrangement leading to the loss of the ortho chlorine would have caused the loss of more than one deuterium . The major metabolite produced from catalytic oxygenation of 2,3,2',3'-CB by LB400 BPDO was identified by NMR as cis-4,5-dihydro-4,5-dihydroxy-2,3,2',3'-tetrachlorobiphenyl . These findings show that LB400 BPDO oxygenates 2,2'-CB principally on carbons 2 and 3 and that BPDO regiospecificity toward 2,2'-CB and 2,3,2,',3'-CB disfavors the dioxygenation of the chlorine-free ortho-meta carbons 5 and 6 for both congeners.

J Biol Chem, 2004 Nov 12, 279(46), 47480 - 8 Epub 2004 Aug 31.
Evolution of the biphenyl dioxygenase BphA from Burkholderia xenovorans LB400 by random mutagenesis of multiple sites in region III; Barriault D et al.; It is now established that several amino acids of region III of the biphenyl dioxygenase (BPDO) alpha subunit are involved in substrate recognition and regiospecificity toward chlorobiphenyls . However, the sequence pattern of the amino acids of that segment of seven amino acids located in the C-terminal portion of the alpha subunit is rather limited in BPDOs of natural occurrence . In this work, we have randomly mutated simultaneously four residues (Thr(335)-Phe(336)-Ile(338)-Ile(341)) of region III of Burkholderia xenovorans LB400 BphA . The library was screened for variants able to oxygenate 2,2'-dichlorobiphenyl (2,2'-CB) . Replacement of Phe(336) with Met or Ile with a concomitant change of Thr(335) to Ala created new variants that transformed 2,2'-CB into 3,4-dihydro-3,4-dihydroxy-2,2'-dichlorobiphenyl, which is a dead end metabolite that was not cleaved by BphC . Replacement of Thr(335)-Phe(336) with Ala(335)-Leu(336) did not cause this type of phenotypic change . Regiospecificity toward congeners other than 2,2'-CB that were oxygenated more efficiently by variant Ala(335)-Met(336) than by LB400 BPDO was similar for both enzymes . Thus structural changes that altered the regiospecificity toward 2,2'-CB did not affect the metabolite profile of other congeners, although it affected the rate of conversion of these congeners . It was especially noteworthy that both LB400 BPDO and the Ala(335)-Met(336) variant generated 2,3-dihydroxy-2',4,4'-trichlorobiphenyl as the sole metabolite from 2,4,2',4'-CB and 4,5-dihydro-4,5-dihydroxy-2,3,2',3'-tetrachlorobiphenyl as the major metabolite from 2,3,2',3'-CB . This shows that 2,4,2',4'-CB is oxygenated principally onto vicinal ortho-meta carbons 2 and 3 and that 2,3,2',3'-CB is oxygenated onto meta-para carbons 4 and 5 by both enzymes . The data suggest that interactions between the chlorine substitutes on the phenyl ring and specific amino acid residues of the protein influence the orientation of the phenyl ring inside the catalytic pocket.

J Bacteriol, 2004 Sep, 186(18), 6015 - 24
Involvement of a plasmid-encoded type IV secretion system in the plant tissue watersoaking phenotype of Burkholderia cenocepacia; Engledow AS et al.; Burkholderia cenocepacia strain K56-2, a representative of the Burkholderia cepacia complex, is part of the epidemic and clinically problematic ET12 lineage . The strain produced plant tissue watersoaking (ptw) on onion tissue, which is a plant disease-associated trait . Using plasposon mutagenesis, mutants in the ptw phenotype were generated . The translated sequence of a disrupted gene (ptwD4) from a ptw-negative mutant showed homology to VirD4-like proteins . Analysis of the region proximal to the transfer gene homolog identified a gene cluster located on the 92-kb resident plasmid that showed homology to type IV secretion systems . The role of ptwD4, ptwC, ptwB4, and ptwB10 in the expression of ptw activity was determined by conducting site-directed mutagenesis . The ptw phenotype was not expressed by K56-2 derivatives with a disruption in ptwD4, ptwB4, or ptwB10 but was observed in a derivative with a disruption in ptwC . Complementation of ptw-negative K56-2 derivatives in trans resulted in complete restoration of the ptw phenotype . In addition, analysis of culture supernatants revealed that the putative ptw effector(s) was a secreted, heat-stable protein(s) that caused plasmolysis of plant protoplasts . A second chromosomally encoded type IV secretion system with complete homology to the VirB-VirD system was identified in K56-2 . Site-directed mutagenesis of key secretory genes in the VirB-VirD system did not affect expression of the ptw phenotype . Our findings indicate that in strain K56-2, the plasmid-encoded Ptw type IV secretion system is responsible for the secretion of a plant cytotoxic protein(s).

J Microbiol Immunol Infect, 2004 Aug, 37(4), 254 - 7
Non-septicemic Burkholderia pseudomallei liver abscess in a young man; Ben RJ et al.; Melioidosis, caused by Burkholderia pseudomallei, has been increasingly recognized in Taiwan recently . Its isolation in liver abscess is rare compared to pulmonary melioidosis . We report a case of liver abscess due to B . pseudomallei in an immunocompetent 27-year-old male soldier admitted due to fever, sore throat and mild non-productive cough for 1 week . Physical examination was unremarkable except for congestion of the pharyngeal wall, moderate enlargement of the tonsils without pus coating, and palpable tender lymphadenopathy over bilateral submental regions . Antibiotic treatment with cefazolin 1 g every 8 hours intravenously was given without response . Left flank pain, followed by right flank pain associated with epigastric tenderness developed . Sonography and computed tomography scan of the abdomen demonstrated liver abscess . Aspiration of the liver abscess was performed and abscess culture yielded B . pseudomallei . Treatment with ceftazidime 2 g every 8 hours intravenously (4 weeks' duration) followed by oral regimens of amoxicillin-clavulanate was given . The patient was free of symptoms at 8 months' follow-up . Early awareness and definite diagnosis as well as institution of proper antimicrobial agents are imperative for successful treatment of melioidosis.

J Chemother, 2004 Aug, 16(4), 404 - 7
Melioidosis in a traveller from Thailand: case report; Maccanti O et al.; A 42-year old Italian male with type 2 diabetes and HCV-related chronic hepatitis spent 6 months in Thailand . After his return in June 2002 he was admitted to the Infectious Diseases Unit of the Hospital of Livorno (Italy) because of fever, chest pain and skin abscesses in the legs . Chest X-rays and CT scan revealed multiple bilateral cavitary lesions in the lungs . Ultrasonography and CT scan showed numerous subcentimetric spleen abscesses . Burkholderia pseudomallei was isolated from the cutaneous lesions and sputum and thus melioidosis was diagnosed . A 6-week course of i.v . ceftazidime plus oral doxycycline was given during the acute phase of the illness . The in vitro susceptibility testing showed that long-term (20 weeks) antimicrobial therapy with doxycycline and moxifloxacin was required . Complete resolution of pulmonary and spleen lesions was obtained within 6 weeks of therapy and of cutaneous abscesses in 10 weeks . No significant side effects were noted during the follow-up period using this scheme of antimicrobial therapy.

J Vet Med B Infect Dis Vet Public Health, 2004 Jun, 51(5), 225 - 30
Experimental Burkholderia pseudomallei infection of pigs; Najdenski H et al.; Experimental infection with Burkholderia pseudomallei was successfully produced after a single intravenous challenge of 2-month-old pigs with a dose of 5.0 x 10(9) bacterial cells . Clinical, paraclinical and morphological findings of the infectious process and post-infectious immunity were examined up to day 30 post infection (p.i.) . A transient and short hyperthermia accompanied by enhanced and longer demonstrated pulse frequency . An increased erythrocyte sedimentation rate and tachypnea were observed too after clinical examination . The infection starts with significant leucopenia, and a reduced number of alveolar and peritoneal macrophages which have been overcome in the latest intervals of infection . In contrast, the phagocytic activity of leucocytes was statistically increased during the course of infection and up to day 15 p.i . in the case of alveolar macrophages . Burkholderia pseudomallei was able to colonize the lungs during the whole experiment and was only present 3 days in the spleen and mesenterial lymph nodes (MLN) . Significant antibody response was developed as early as day 7 p.i . Hyperaemia, haemorrhages and necrotic foci were found in the brain, liver spleen and MLN . Lung tissue was also hyperaemic, with formation of small abscesses and signs of catarrhal pneumonia . Data obtained in this study revealed that B . pseudomallei causes a chronic generalized infection in pigs, even after intravenous challenge.

Biotechnol Bioeng, 2004 Sep 20, 87(6), 779 - 90
Protein engineering of toluene 4-monooxygenase of Pseudomonas mendocina KR1 for synthesizing 4-nitrocatechol from nitrobenzene; Fishman A et al.; After discovering that toluene 4-monooxygenase (T4MO) of Pseudomonas mendocina KR1 oxidizes nitrobenzene to 4-nitrocatechol, albeit at a very low rate, this reaction was improved using directed evolution and saturation mutagenesis . Screening 550 colonies from a random mutagenesis library generated by error-prone PCR of tmoAB using Escherichia coli TG1/pBS(Kan)T4MO on agar plates containing nitrobenzene led to the discovery of nitrocatechol-producing mutants . One mutant, NB1, contained six amino acid substitutions (TmoA Y22N, I84Y, S95T, I100S, S400C; TmoB D79N) . It was believed that position I100 of the alpha subunit of the hydroxylase (TmoA) is the most significant for the change in substrate reactivity due to previous results in our lab with a similar enzyme, toluene ortho-monooxygenase of Burkholderia cepacia G4 . Saturation mutagenesis at this position resulted in the generation of two more nitrocatechol mutants, I100A and I100S; the rate of 4-nitrocatechol formation by I100A was more than 16 times higher than that of wild-type T4MO at 200 microM nitrobenzene (0.13 +/- 0.01 vs . 0.008 +/- 0.001 nmol/min.mg protein) . HPLC and mass spectrometry analysis revealed that variants NB1, I100A, and I100S produce 4-nitrocatechol via m-nitrophenol, while the wild-type produces primarily p-nitrophenol and negligible amounts of nitrocatechol . Relative to wild-type T4MO, whole cells expressing variant I100A convert nitrobenzene into m-nitrophenol with a Vmax of 0.61 +/- 0.037 vs . 0.16 +/- 0.071 nmol/min.mg protein and convert m-nitrophenol into nitrocatechol with a Vmax of 3.93 +/- 0.26 vs . 0.58 +/- 0.033 nmol/min.mg protein . Hence, the regiospecificity of nitrobenzene oxidation was changed by the random mutagenesis, and this led to a significant increase in 4-nitrocatechol production . The regiospecificity of toluene oxidation was also altered, and all of the mutants produced 20% m-cresol and 80% p-cresol, while the wild-type produces 96% p-cresol . Interestingly, the rate of toluene oxidation (the natural substrate of the enzyme) by I100A was also higher by 65% (7.2 +/- 1.2 vs . 4.4 +/- 0.3 nmol/min mg protein) . Homology-based modeling of TmoA suggests reducing the size of the side chain of I100 leads to an increase in the width of the active site channel, which facilitates access of substrates and promotes more flexible orientations .

Biochem J, 2004 Dec 15, 384(Pt 3), 609 - 17
Expression and refolding of Omp38 from Burkholderia pseudomallei and Burkholderia thailandensis, and its function as a diffusion porin; Siritapetawee J et al.; In the present paper, we describe cloning and expression of two outer membrane proteins, BpsOmp38 (from Burkholderia pseudomallei) and BthOmp38 (from Burkholderia thailandensis) lacking signal peptide sequences, using the pET23d(+) expression vector and Escherichia coli host strain Origami(DE3) . The 38 kDa proteins, expressed as insoluble inclusion bodies, were purified, solubilized in 8 M urea, and then subjected to refolding experiments . As seen on SDS/PAGE, the 38 kDa band completely migrated to approximately 110 kDa when the purified monomeric proteins were refolded in a buffer system containing 10% (w/v) Zwittergent 3-14, together with a subsequent heating to 95 degrees C for 5 min . CD spectroscopy revealed that the 110 kDa proteins contained a predominant beta-sheet structure, which corresponded completely to the structure of the Omp38 proteins isolated from B . pseudomallei and B . thailandensis . Immunoblot analysis using anti-BpsOmp38 polyclonal antibodies and peptide mass analysis by MALDI-TOF (matrix-assisted laser-desorption ionization-time-of-flight) MS confirmed that the expressed proteins were BpsOmp38 and BthOmp38 . The anti-BpsOmp38 antibodies considerably exhibited the inhibitory effects on the permeation of small sugars through the Omp38-reconstituted liposomes . A linear relation between relative permeability rates and M(r) of neutral sugars and charged antibiotics suggested strongly that the in vitro re-assembled Omp38 functioned fully as a diffusion porin.

Environ Pollut, 2005 Jan, 133(1), 53 - 62
Influence of plants on the chemical extractability and biodegradability of 2,4-dichlorophenol in soil; Boucard TK et al.; This study investigated the fate and behaviour of {UL-(14)C} 2,4-dichlorophenol (DCP) in planted (Lolium perenne L.) and unplanted soils over 57 days . Extractability of {UL-(14)C} 2,4-DCP associated activity was measured using calcium chloride (CaCl(2)), acetonitrile-water and dichloromethane (DCM) extractions . Biodegradability of {UL-(14)C} 2,4-DCP associated activity was assessed through measurement of (14)CO(2) production by a degrader inoculum (Burkholderia sp.) . Although extractability and mineralisation of {UL-(14)C} 2,4-DCP associated activity decreased significantly in both planted and unplanted soils, plants appeared to enhance the sequestration process . After 57 days, in unplanted soil, 27% of the remaining {UL-(14)C} 2,4-DCP associated activity was mineralised by Burkholderia sp., and 13%, 48%, and 38% of (14)C-activity were extracted by CaCl(2), acetonitrile-water and DCM, respectively . However, after 57 days, in planted soils, only 10% of the {UL-(14)C} 2,4-DCP associated activity was available for mineralisation, whilst extractability was reduced to 2% by CaCl(2), 17% by acetonitrile-water and 11% by DCM . This may be due to the effect of plants on soil moisture conditions, which leads to modification of the soil structure and trapping of the compound . However, the influence of plants on soil biological and chemical properties may also play a role in the ageing process.

Oral Microbiol Immunol, 2004 Oct, 19(5), 303 - 8
Beta-lactamase production and antimicrobial susceptibility of subgingival bacteria from refractory periodontitis; Handal T et al.; This study assessed the extent of beta-lactamase-producing bacteria in subgingival plaque samples obtained from 25 patients with refractory marginal periodontitis in the USA . beta-Lactamase-positive isolates were characterized using commercial diagnostic kits and partial sequencing of the 16S rRNA gene . The susceptibilities to different antimicrobial agents were tested and, in addition, the isolates were screened for the presence of extended spectrum beta-lactamases (ESBLs) . beta-lactamase-producing bacteria were detected in 18 (72%) patients . The most prominent beta-lactamase-producing organisms belonged to the anaerobic genus Prevotella . Other enzyme-producing anaerobic strains were Fusobacterium nucleatum, Propionibacterium acnes and Peptostreptococcus sp . Facultative bacteria, such as Burkholderia spp., Ralstonia pickettii, Capnocytophaga spp., Bacillus spp., Staphylococcus spp . and Neisseria sp., were also detected among the enzyme-producers . Minimum inhibitory concentrations (MICs) of ampicillin and amoxicillin were in the range 1.5-256 micrograms/ml and 4-256 micrograms/ml, respectively, for the isolates of the Prevotella species . All Prevotella isolates were susceptible to amoxicillin/clavulanate and metronidazole, but they showed variable resistance to tetracyclines . Two of the Prevotella isolates had high MICs of cefotaxime and ceftazidime . ESBL activity was not detected in any of the beta-lactamase-producing isolates by the Etest method . Thus, our study demonstrated a wide variety of beta-lactamase-producing bacteria that may play a role in refractory periodontal disease.

Infect Immun, 2004 Sep, 72(9), 5126 - 34
Contribution of Burkholderia cenocepacia flagella to infectivity and inflammation; Urban TA et al.; Burkholderia cenocepacia is an opportunistic pathogen that can cause severe lung infections in cystic fibrosis patients . To understand the contribution of B . cenocepacia flagella to infection, a strain mutated in the major flagellin subunit, fliCII, was constructed in B . cenocepacia K56-2 and tested in a murine agar bead model of lung infection . C57/BL6 mice infected with approximately 10(8) wild-type K56-2 bacteria exhibited 40% mortality after 3 days, whereas no mortality was noted in mice infected with the fliCII mutant . Among the mice surviving the infection with either strain, there was no significant difference in the bacterial loads in the lungs and spleen, bacteremia, weight loss, or infiltration of immune effector cells at 3 days postinfection . Similar results were observed at 24 h, prior to expression of the lethality phenotype . KC, a murine interleukin-8 (IL-8) homolog, was elevated in both the bronchoalveolar lavage fluid and serum of mice infected with the wild type compared to the fliCII mutant at 24 h, suggesting that flagella stimulated host cells . To demonstrate that flagella contributed to these responses, the interaction between B . cenocepacia and Toll-like receptor 5 (TLR5) was investigated . Infection of HEK293 cells with heat-killed wild-type K56-2, but not infection with the fliCII mutant, resulted in both NF-kappaB activation and IL-8 secretion that was dependent upon expression of TLR5 . Together, these results demonstrate that B . cenocepacia flagella contribute to virulence in an in vivo infection model, and that induction of host immune responses through interaction with TLR5 may contribute to its overall pathogenic potential.

Mol Genet Genomics, 2004 Sep, 272(2), 204 - 15 Epub 2004 Aug 13.
Identification of a novel two-partner secretion system from Burkholderia pseudomallei; Brown NF et al.; Two adjacent genes, bpaA and bpaB, whose products display significant similarity to a number of two-partner secretion (TPS) systems have been identified in Burkholderia pseudomallei strain 08, but are absent from the closely related avirulent species B . thailandensis . They possess a number of sequence features characteristic of TPS systems, including the presence of an NPNGI motif in a region of BpaA which strongly resembles a TPS secretion domain . BpaA is a very large protein (~530 kDa) and contains three repeats, each 600-800-amino acids long . Putative membrane-spanning regions in BpaB were identified through alignment with TpsB family members, and this also revealed an N-terminal extension not found in other TpsB proteins . The bpaA gene was found to be absent from the majority of B . pseudomallei strains . It appears that bpaAB are located within a putative genomic island that is inserted in close proximity to a methionine tRNA(CAT)-encoding gene . Expression of BpaA was undetectable in cells grown in laboratory media . However, owing to the similarity of BpaA to known adhesin molecules, a potential role of BpaA in virulence was investigated in cell culture and in an animal model, but no evidence for such a role was found in these test systems.

Res Microbiol, 2004 Sep, 155(7), 587 - 95
Identification and biodegradation potential of tropical aerobic hydrocarbon-degrading microorganisms; Chaillan F et al.; Screening of aerobic culturable hydrocarbon (HC)-degrading microorganisms isolated from petroleum-polluted soils and cyanobacterial mats from Indonesia resulted in the collection of 33 distinct species . Eight bacteria, 21 fungi and 4 yeasts were identified to the specific level by molecular and phenotypic techniques . Bacterial strains belonged to the genera Gordonia, Brevibacterium, Aeromicrobium, Dietzia, Burkholderia and Mycobacterium . Four species are new and not yet described . Fungi belonged to Aspergillus, Penicillium, Fusarium, Amorphoteca, Neosartorya, Paecilomyces, Talaromyces and Graphium . Yeasts were Candida, Yarrowia and Pichia . All strains were cultivated axenically in synthetic liquid media with crude oil as sole carbon and energy source . After incubation, the detailed chemical composition of the residual oil was studied by gravimetric and gas-chromatographic techniques . Thirteen parameters for assessing the biodegradation potential were defined and computed for each strain . Maximum degradation was observed on the saturated HCs (n- and isoalkanes, isoprenoids), whereas aromatic HC degradation was lower and was related to the structural composition of the molecules . A principal components analysis (PCA) permitted grouping and classifying the strains as a function of their degradative capacities . It was shown that the most active strains produced polar metabolites which accumulated in the resins and asphaltene fractions . These fractions are highly resistant to microbial metabolism . No taxonomic trend could be defined between microbial phyla in terms of HC biodegradation activity.

Trans R Soc Trop Med Hyg, 2003 Sep-Oct, 97(5), 577 - 81
A proposed scoring system for predicting mortality in melioidosis; Cheng AC et al.; Melioidosis, due to infection with the environmental organism Burkholderia pseudomallei, continues to be associated with high mortality despite improvements in antibiotic therapy . Using simple clinical findings and baseline laboratory tests available at the time of admission, we attempted to define those patients with acute melioidosis who were at higher risk of death . Using data, collected prospectively from the period October 1989 to June 2002, from patients with acute culture-confirmed melioidosis presenting at the Royal Darwin Hospital, Darwin, Australia, a number of variables were selected that were easily available at the time of admission and reflected organ dysfunction . Mortality was predicted in univariate logistic and multivariate models by the presence of pneumonia, age at diagnosis, serum urea, serum bilirubin, lymphocyte count, and serum bicarbonate . A score was assigned from 0 to 2, based on the degree of abnormality . A melioidosis score was formed from the sum of these scores, with a maximum score of 11 . A score of < or = 3 (n = 140) was associated with a mortality of 8.6%, whereas a score of > or = 4 (n = 112) was associated with a mortality of 44.6% . Although this scoring system requires external validation, it may help identify a suitable target group of patients for intensive intervention such as early admission to an intensive care unit, the early use of meropenem, and goal-directed resuscitation therapies.

J Clin Microbiol, 2004 Aug, 42(8), 3904 - 5
Two novel clinical presentations of Burkholderia cepacia infection; Mukhopadhyay C et al.; We report two cases of multidrug-resistant Burkholderia cepacia (B . cepacia genomovar I) and Burkholderia multivorans causing multiple liver abscesses in a patient with bronchial asthma (case 1) and peritonitis in a patient with cirrhosis and hepatitis C virus disease (case 2), respectively . Both patients were treated successfully.

Appl Environ Microbiol, 2004 Aug, 70(8), 4961 - 70
Biphenyl and benzoate metabolism in a genomic context: outlining genome-wide metabolic networks in Burkholderia xenovorans LB400; Denef VJ et al.; We designed and successfully implemented the use of in situ-synthesized 45-mer oligonucleotide DNA microarrays (XeoChips) for genome-wide expression profiling of Burkholderia xenovorans LB400, which is among the best aerobic polychlorinated biphenyl degraders known so far . We conducted differential gene expression profiling during exponential growth on succinate, benzoate, and biphenyl as sole carbon sources and investigated the transcriptome of early-stationary-phase cells grown on biphenyl . Based on these experiments, we outlined metabolic pathways and summarized other cellular functions in the organism relevant for biphenyl and benzoate degradation . All genes previously identified as being directly involved in biphenyl degradation were up-regulated when cells were grown on biphenyl compared to expression in succinate-grown cells . For benzoate degradation, however, genes for an aerobic coenzyme A activation pathway were up-regulated in biphenyl-grown cells, while the pathway for benzoate degradation via hydroxylation was up-regulated in benzoate-grown cells . The early-stationary-phase biphenyl-grown cells showed similar expression of biphenyl pathway genes, but a surprising up-regulation of C(1) metabolic pathway genes was observed . The microarray results were validated by quantitative reverse transcription PCR with a subset of genes of interest . The XeoChips showed a chip-to-chip variation of 13.9%, compared to the 21.6% variation for spotted oligonucleotide microarrays, which is less variation than that typically reported for PCR product microarrays.

Appl Microbiol Biotechnol . 2004 Jul 28; {Epub ahead of print}
Chemotaxis of Pseudomonas stutzeri OX1 and Burkholderia cepacia G4 toward chlorinated ethenes; Vardar G et al.; The chemotactic responses of Pseudomonas putida F1, Burkholderia cepacia G4, and Pseudomonas stutzeri OX1 were investigated toward toluene, trichloroethylene (TCE), tetrachloroethylene (PCE), cis-1,2-dichloroethylene ( cis-DCE), trans-1,2-dichloroethylene ( trans-DCE), 1,1-dichloroethylene (1,1-DCE), and vinyl chloride (VC) . P . stutzeri OX1 and P . putida F1 were chemotactic toward toluene, PCE, TCE, all DCEs, and VC . B . cepacia G4 was chemotactic toward toluene, PCE, TCE, cis-DCE, 1,1-DCE, and VC . Chemotaxis of P . stutzeri OX1 grown on o-xylene vapors was much stronger than when grown on o-cresol vapors toward some chlorinated ethenes . Expression of toluene- o-xylene monooxygenase (ToMO) from touABCDEF appears to be required for positive chemotaxis attraction, and the attraction is stronger with the touR (ToMO regulatory) gene.

Appl Microbiol Biotechnol . 2004 Jul 28; {Epub ahead of print}
Quorum-sensing antagonist ( 5Z)-4-bromo-5-(bromomethylene)-3-butyl-2( 5H)-furanone influences siderophore biosynthesis in Pseudomonas putida and Pseudomonas aeruginosa; Ren D et al.; Siderophore synthesis of Pseudomonas putida F1 was found to be regulated by quorum sensing since normalized siderophore production (per cell) increased 4.2-fold with cell density after the cells entered middle exponential phase; similarly, normalized siderophore concentrations in Pseudomonas aeruginosa JB2 increased 28-fold, and a 5.5-fold increase was seen for P . aeruginosa PAO1 . Further evidence of the link between quorum sensing and siderophore synthesis of P . putida F1 was that the quorum-sensing-disrupter ( 5Z)-4-bromo-5-(bromomethylene)-3-butyl-2( 5H)-furanone (furanone) from the marine red alga Delisea pulchra was found to inhibit the formation of the siderophore produced by P . putida F1 in a concentration-dependent manner, with 57% siderophore synthesis repressed by 100 microg/ml furanone . In contrast, this furanone did not affect the siderophore synthesis of Burkholderia cepacia G4 at 20-40 microg/ml, and stimulated siderophore synthesis of P . aeruginosa JB2 2.5- to 3.7-fold at 20-100 microg/ml . Similarly, 100 microg/ml furanone stimulated siderophore synthesis in P . aeruginosa PAO1 about 3.5-fold . The furanone appears to interact with the quorum-sensing machinery of P . aeruginosa PAO1 since it stimulates less siderophore synthesis in the P . aeruginosa qscR quorum-sensing mutant (QscR is a negative regulator of LasI, an acylated homoserine lactone synthase).

Appl Microbiol Biotechnol, 2005 Jan, 66(4), 422 - 9 Epub 2004 Jul 31.
Protein engineering of toluene ortho-monooxygenase of Burkholderia cepacia G4 for regiospecific hydroxylation of indole to form various indigoid compounds; Rui L et al.; Previous work showed that random mutagenesis produced a mutant of toluene ortho-monooxygenase (TOM) of Burkholderia cepacia G4 containing the V106A substitution in the hydroxylase alpha-subunit (TomA3) that changed the color of the cell suspension from wild-type brown to green in rich medium . Here, DNA shuffling was used to isolate a random TOM mutant that turned blue due to mutation TomA3 A113V . To better understand the TOM reaction mechanism, we studied the specificity of indole hydroxylation using a spectrum of colored TOM mutants expressed in Escherichia coli TG1 and formed as a result of saturation mutagenesis at TomA3 positions A113 and V106 . Colonies expressing these altered enzymes ranged in color from blue through green and purple to orange; and the enzyme products were identified using thin-layer chromatography, high performance liquid chromatography, and liquid chromatography-mass spectroscopy . Derived from the single TOM template, enzymes were identified that produced primarily isoindigo (wild-type TOM), indigo (A113V), indirubin (A113I), and isatin (A113H and V106A/A113G) . The discovery that wild-type TOM formed isoindigo via C-2 hydroxylation of the indole pyrrole ring makes this the first oxygenase shown to form this compound . Variant TOM A113G was unable to form indigo, indirubin, or isoindigo (did not hydroxylate the indole pyrrole ring), but produced 4-hydroxyindole and unknown yellow compounds from C-4 hydroxylation of the indole benzene ring . Mutations at V106 in addition to A113G restored C-3 indole oxidation, so along with C-2 indole oxidation, isatin, indigo, and indirubin were formed . Other TomA3 V106/A113 mutants with hydrophobic, polar, or charged amino acids in place of the Val and/or Ala residues hydroxylated indole at the C-3 and C-2 positions, forming isatin, indigo, and indirubin in a variety of distributions . Hence, for the first time, a single enzyme was genetically modified to produce a wide range of colors from indole.

Microbiology, 2004 Aug, 150(Pt 8), 2669 - 76
Attenuated virulence and protective efficacy of a Burkholderia pseudomallei bsa type III secretion mutant in murine models of melioidosis; Stevens MP et al.; Melioidosis is a severe infectious disease of animals and humans caused by the Gram-negative intracellular pathogen Burkholderia pseudomallei . An Inv/Mxi-Spa-like type III protein secretion apparatus, encoded by the B . pseudomallei bsa locus, facilitates bacterial invasion of epithelial cells, escape from endocytic vesicles and intracellular survival . This study investigated the role of the Bsa type III secretion system in the pathogenesis of melioidosis in murine models . B . pseudomallei bipD mutants, lacking a component of the translocation apparatus, were found to be significantly attenuated following intraperitoneal or intranasal challenge of BALB/c mice . Furthermore, a bipD mutant was attenuated in C57BL/6 IL-12 p40(-/-) mice, which are highly susceptible to B . pseudomallei infection . Mutation of bipD impaired bacterial replication in the liver and spleen of BALB/c mice in the early stages of infection . B . pseudomallei mutants lacking either the type III secreted guanine nucleotide exchange factor BopE or the putative effectors BopA or BopB exhibited varying degrees of attenuation, with mutations in bopA and bopB causing a significant delay in median time to death . This indicates that bsa-encoded type III secreted proteins may act in concert to determine the outcome of B . pseudomallei infection in mice . Mice inoculated with the B . pseudomallei bipD mutant were partially protected against subsequent challenge with wild-type B . pseudomallei . However, immunization of mice with purified BipD protein was not protective.

J Biol Chem, 2004 Oct 8, 279(41), 43098 - 106 Epub 2004 Jul 26.
Catalase-peroxidases (KatG) exhibit NADH oxidase activity; Singh R et al.; Catalase-peroxidases (KatG) produced by Burkholderia pseudomallei, Escherichia coli, and Mycobacterium tuberculosis catalyze the oxidation of NADH to form NAD+ and either H2O2 or superoxide radical depending on pH . The NADH oxidase reaction requires molecular oxygen, does not require hydrogen peroxide, is not inhibited by superoxide dismutase or catalase, and has a pH optimum of 8.75, clearly differentiating it from the peroxidase and catalase reactions with pH optima of 5.5 and 6.5, respectively, and from the NADH peroxidase-oxidase reaction of horseradish peroxidase . B . pseudomallei KatG has a relatively high affinity for NADH (Km=12 microm), but the oxidase reaction is slow (kcat=0.54 min(-1)) compared with the peroxidase and catalase reactions . The catalase-peroxidases also catalyze the hydrazinolysis of isonicotinic acid hydrazide (INH) in an oxygen- and H2O2-independent reaction, and KatG-dependent radical generation from a mixture of NADH and INH is two to three times faster than the combined rates of separate reactions with NADH and INH alone . The major products from the coupled reaction, identified by high pressure liquid chromatography fractionation and mass spectrometry, are NAD+ and isonicotinoyl-NAD, the activated form of isoniazid that inhibits mycolic acid synthesis in M . tuberculosis . Isonicotinoyl-NAD synthesis from a mixture of NAD+ and INH is KatG-dependent and is activated by manganese ion . M . tuberculosis KatG catalyzes isonicotinoyl-NAD formation from NAD+ and INH more efficiently than B . pseudomallei KatG.

Int J Syst Evol Microbiol, 2004 Jul, 54(Pt 4), 1165 - 72
Burkholderia unamae sp . nov., an N2-fixing rhizospheric and endophytic species; Caballero-Mellado J et al.; It was shown recently that the genus Burkholderia is rich in N2-fixing bacteria that are associated with plants . A group of these diazotrophic isolates with identical or very similar 16S rDNA restriction patterns {designated amplified rDNA restriction analysis (ARDRA) genotypes 13, 14 and 15} was selected and a polyphasic taxonomic study was performed, which included new isolates that were recovered from rhizospheres, rhizoplanes or internal tissues of maize, sugarcane and coffee plants . Morphological, physiological and biochemical features, as well as multi-locus enzyme electrophoresis profiles and whole-cell protein patterns, of 20 strains were analysed . In addition, analysis of cellular fatty acid profiles, 16S rDNA sequence analysis and DNA-DNA reassociation experiments were performed with representative strains . The taxonomic data indicated that the strains analysed belong to a novel diazotrophic Burkholderia species, for which the name Burkholderia unamae sp . nov . is proposed . Strain MTl-641T (=ATCC BAA-744T=CIP 107921T), isolated from the rhizosphere of maize, was designated as the type strain . B . unamae was found as an endophyte of plants grown in regions with climates ranging from semi-hot subhumid to hot humid, but not from plants grown in regions with semi-hot or hot dry climates . Moreover, B . unamae was isolated from rhizospheres and plants growing in soils with pH values in the range 4.5-7.1, but not from soils with pH values higher than 7.5.

Antimicrob Agents Chemother, 2004 Aug, 48(8), 2999 - 3005
Comparison of the susceptibilities of Burkholderia pseudomallei to meropenem and ceftazidime by conventional and intracellular methods; Inglis TJ et al.; The effect of the two antibiotics ceftazidime and meropenem on a collection of 46 Burkholderia pseudomallei isolates representing clinical and environmental sources across northern Australia was investigated by using a series of in vitro test methods . The susceptibility testing methods used included Kirby-Bauer disk diffusion, Etest MIC, broth microdilution MIC, and a modification of the microdilution method in which Acanthamoeba cells were added to simulate the effect of a professional phagocytic cell on test outcome . In a semiquantitative validation coculture series, the majority of bacteria were intracellular up to a multiplicity of infection of 10 bacteria to one ameba . The optical density and bacterial count (log10 CFU/ml) correlated across the range tested (r2 = 0.77; P < 0.0001) . Susceptibility test results were compared against clinical outcomes . The MICs of ceftazidime were consistently higher than those of meropenem by all three methods . The MICs of both agents were significantly higher when Acanthamoeba trophozoites were added to the broth microdilution method . Conventional and intracellular MIC results were consistent for clinical isolates from the Western Australian outbreak cluster despite the wide variety of clinical outcomes . Further development of the intracellular MIC method is expected to help assess the efficacy of antimicrobial agents on this bacterial species in an intracellular setting.

Infect Immun, 2004 Aug, 72(8), 4494 - 502
Immunostimulatory CpG oligodeoxynucleotide confers protection in a murine model of infection with Burkholderia pseudomallei; Wongratanacheewin S et al.; Although CpG oligodeoxynucleotides (CpG ODNs) are known to enhance resistance against infection in a number of animal models, little is known about the CpG-induced protection against acute fatal sepsis such as that associated with the highly virulent bacterium Burkholderia pseudomallei . We previously demonstrated in an in vitro study that immunostimulatory CpG ODN 1826 enhances phagocytosis of B . pseudomallei and induces nitric oxide synthase and nitric oxide production by mouse macrophages . In the present study, CpG ODN 1826 given intramuscularly to BALB/c mice 2 to 10 days prior to B . pseudomallei challenge conferred better than 90% protection . CpG ODN 1826 given 2 days before the bacterial challenge rapidly enhanced the innate immunity of these animals, judging from the elevated serum levels of interleukin-12 (IL-12)p70 and gamma interferon (IFN-gamma) over the baseline values . No bacteremia was detected on day 2 in 85 to 90% of the CpG-treated animals, whereas more than 80% of the untreated animals exhibited heavy bacterial loads . Although marked elevation of IFN-gamma was found consistently in the infected animals 2 days after the bacterial challenge, it was ameliorated by the CpG ODN 1826 pretreatment (P = 0.0002) . Taken together, the kinetics of bacteremia and cytokine profiles presented are compatible with the possibility that protection by CpG ODN 1826 against acute fatal septicemic melioidosis in this animal model is associated with a reduction of bacterial load and interference with the potential detrimental effect of the robust production of proinflammatory cytokines associated with B . pseudomallei multiplication.

Colloids Surf B Biointerfaces, 2004 Jul 15, 36(2), 81 - 90
Bacterial adhesion to glass and metal-oxide surfaces; Li B et al.; Metal oxides can increase the adhesion of negatively-charged bacteria to surfaces primarily due to their positive charge . However, the hydrophobicity of a metal-oxide surface can also increase adhesion of bacteria . In order to understand the relative contribution of charge and hydrophobicity to bacterial adhesion, we measured the adhesion of 8 strains of bacteria, under conditions of low and high-ionic strength (1 and 100 mM, respectively) to 11 different surfaces and examined adhesion as a function of charge, hydrophobicity (water contact angle) and surface energy . Inorganic surfaces included three uncoated glass surfaces and eight metal-oxide thin films prepared on the upper (non-tin-exposed) side of float glass by chemical vapor deposition . The Gram-negative bacteria differed in lengths of lipopolysaccharides on their outer surface (three Escherichia coli strains), the amounts of exopolysaccharides (two Pseudomonas aeruginosa strains), and their known relative adhesion to sand grains (two Burkholderia cepacia strains) . One Gram positive bacterium was also used that had a lower adhesion to glass than these other bacteria (Bacillus subtilis) . For all eight bacteria, there was a consistent increase in adhesion between with the type of inorganic surface in the order: float glass exposed to tin (coded here as Si-Sn), glass microscope slide (Si-m), uncoated air-side float glass surface (Si-a), followed by thin films of (Co(1-y-z)Fe(y)Cr(z))3O4, Ti/Fe/O, TiO2, SnO2, SnO2:F, SnO2:Sb, A1(2)O3, and Fe2O3 (the colon indicates metal doping, a slash indicates that the metal is a major component, while the dash is used to distinguish surfaces) . Increasing the ionic strength from 1 to 100 mM increased adhesion by a factor of 2.0 +/- 0.6 (73% of the sample results were within the 95% CI) showing electrostatic charge was important in adhesion . However, adhesion was not significantly correlated with bacterial charge and contact angle . Adhesion (A) of the eight strains was significantly (P < 10(-25)) correlated with total adhesion free energy (U) between the bacteria and surface (A = 2162e(-1.8U)).Although the correlation was significant, agreement between the model and data was poor for the low energy surfaces (R2 = 0.68), indicating that better models or additional methods to characterize bacteria and surfaces are still needed to more accurately describe initial bacterial adhesion to inorganic surfaces.

Microbiology, 2004 Jul, 150(Pt 7), 2301 - 11
The complex structure of polyhydroxybutyrate (PHB) granules: four orthologous and paralogous phasins occur in Ralstonia eutropha; Potter M et al.; Analysis of the genome sequence of the polyhydroxyalkanoate- (PHA) accumulating bacterium Ralstonia eutropha strain H16 revealed three homologues (PhaP2, PhaP3 and PhaP4) of the phasin protein PhaP1 . PhaP1 is known to constitute the major component of the layer at the surface of poly(3-hydroxybutyrate), poly(3HB), granules . PhaP2, PhaP3 and PhaP4 exhibited 42, 49 and 45 % identity or 61, 62 and 63 % similarity to PhaP1, respectively . The calculated molecular masses of PhaP1, PhaP2, PhaP3 and PhaP4 were 20.0, 20.2, 19.6 and 20.2 kDa, respectively . RT-PCR analysis showed that phaP2, phaP3 and phaP4 were transcribed under conditions permissive for accumulation of poly(3HB) . 2D PAGE of the poly(3HB) granule proteome and analysis of the detected proteins by MALDI-TOF clearly demonstrated that PhaP1, PhaP3 and PhaP4 are bound to the poly(3HB) granules in the cells . PhaP3 was expressed at a significantly higher level in PhaP1-negative mutants . Occurrence of an unknown protein with an N-terminal amino-acid sequence identical to that of PhaP2 in crude cellular extracts of R . eutropha had previously been shown by others . Although PhaP2 could not be localized in vivo on poly(3HB) granules, in vitro experiments clearly demonstrated binding of PhaP2 to these granules . Further analysis of complete or partial genomes of other poly(3HB)-accumulating bacteria revealed the existence of multiple phasin homologues in Ralstonia solanacearum, Burkholderia fungorum and Azotobacter vinelandii . These new and unexpected findings should affect our current models of PHA-granule structure and may also have a considerable impact on the establishment of heterologous production systems for PHAs.

Environ Microbiol, 2004 Aug, 6(8), 842 - 50
From PCBs to highly toxic metabolites by the biphenyl pathway; Camara B et al.; The degradation of polychlorobiphenyls (PCBs) by diverse bacteria, including Burkholderia sp . LB400, is incomplete with a concomitant accumulation of metabolic intermediates . In this study, the toxicity of diverse (chloro)biphenyls and of their biotransformation into the first two metabolic intermediates of the biphenyl pathway, were determined for the model bacterium Escherichia coli . Recombinant E . coli strains expressing different subsets of bph genes of strain LB400 accumulated metabolic intermediates from (chloro)biphenyls . During biotransformation of these compounds into metabolic intermediates, the viability and metabolic kinetics were determined . The toxicity of biotransformation of (chloro)biphenyls into different metabolic intermediates of (chloro)biphenyls varied . Dihydrodiols and dihydroxybiphenyls are very toxic metabolites for bacteria even after short incubation times, affecting the cell viability much more than (chloro)biphenyls . When bacteria transformed 2-CB into dihydrodiol or dihydroxybiphenyl, a great decrease of intact cells and abundant cell lysis was observed by transmission electronic microscopy . Cell viability of Burkholderia sp . LB400 and of E . coli exposed directly to 2,3-dihydroxybiphenyl decreased also drastically . The toxicity of metabolites generated during oxidation of PCBs may partly explain the recalcitrance to biodegradation of these pollutants . Conversion of less toxic compounds into products with increased toxicity resembles the bioactivation of xenobiotics in higher organisms.

Arch Microbiol, 2004 Sep, 182(1), 96 - 101 Epub 2004 Jul 06.
DpsA protects the human pathogen Burkholderia pseudomallei against organic hydroperoxide; Loprasert S et al.; The human pathogen, Burkholderia pseudomalle, is able to survive and multiply in hostile environments such as within macrophages . In an attempt to understand its strategy to cope with oxidative stress, the physiological role and gene regulation of a nonspecific DNA-binding protein (DpsA) was investigated . Expression of dpsA increases in response to oxidative stress through increased transcription from the upstream katG (catalase-peroxidase) promoter, which is OxyR dependent . dpsA is also transcribed from its own promoter, which is activated by osmotic stress in an OxyR-independent manner . DpsA-deficient mutants are hypersensitive to tert-butyl hydroperoxide, while overexpression of DpsA leads to increased resistance to organic oxidants . B . pseudomallei DpsA can also protect Escherichia coli against organic hydroperoxide toxicity . The mechanism of DpsA-mediated resistance to organic hydroperoxides was shown to differ from that of alkyl hydroperoxide reductase.

Appl Environ Microbiol, 2004 Jul, 70(7), 4303 - 17
Detection of genes involved in biodegradation and biotransformation in microbial communities by using 50-mer oligonucleotide microarrays; Rhee SK et al.; To effectively monitor biodegrading populations, a comprehensive 50-mer-based oligonucleotide microarray was developed based on most of the 2,402 known genes and pathways involved in biodegradation and metal resistance . This array contained 1,662 unique and group-specific probes with <85% similarity to their nontarget sequences . Based on artificial probes, our results showed that under hybridization conditions of 50 degrees C and 50% formamide, the 50-mer microarray hybridization can differentiate sequences having <88% similarity . Specificity tests with representative pure cultures indicated that the designed probes on the arrays appeared to be specific to their corresponding target genes . The detection limit was approximately 5 to 10 ng of genomic DNA in the absence of background DNA and 50 to 100 ng of pure-culture genomic DNA in the presence of background DNA or 1.3 x 10(7) cells in the presence of background RNA . Strong linear relationships between the signal intensity and the target DNA and RNA were observed (r(2) = 0.95 to 0.99) . Application of this type of microarray to analyze naphthalene-amended enrichment and soil microcosms demonstrated that microflora changed differently depending on the incubation conditions . While the naphthalene-degrading genes from Rhodococcus-type microorganisms were dominant in naphthalene-degrading enrichments, the genes involved in naphthalene (and polyaromatic hydrocarbon and nitrotoluene) degradation from gram-negative microorganisms, such as Ralstonia, Comamonas, and Burkholderia, were most abundant in the soil microcosms . In contrast to general conceptions, naphthalene-degrading genes from Pseudomonas were not detected, although Pseudomonas is widely known as a model microorganism for studying naphthalene degradation . The real-time PCR analysis with four representative genes showed that the microarray-based quantification was very consistent with real-time PCR (r(2) = 0.74) . In addition, application of the arrays to both polyaromatic-hydrocarbon- and benzene-toluene-ethylbenzene-xylene-contaminated and uncontaminated soils indicated that the developed microarrays appeared to be useful for profiling differences in microbial community structures . Our results indicate that this technology has potential as a specific, sensitive, and quantitative tool in revealing a comprehensive picture of the compositions of biodegradation genes and the microbial community in contaminated environments, although more work is needed to improve detection sensitivity.

Appl Environ Microbiol, 2004 Jul, 70(7), 4012 - 20
Multivariate analyses of Burkholderia species in soil: effect of crop and land use history; Salles JF et al.; The assessment of Burkholderia diversity in agricultural areas is important considering the potential use of this genus for agronomic and environmental applications . Therefore, the aim of this work was to ascertain how plant species and land use management drive the diversity of the genus Burkholderia . In a greenhouse experiment, different crops, i.e., maize, oat, barley, and grass, were planted in pots containing soils with different land use histories, i.e., maize monoculture, crop rotation, and permanent grassland, for three consecutive growth cycles . The diversity of Burkholderia spp . in the rhizosphere soil was assessed by genus-specific PCR-denaturing gradient gel electrophoresis (DGGE) and analyzed by canonical correspondence analysis (CCA) . CCA ordination plots showed that previous land use was the main factor affecting the composition of the Burkholderia community . Although most variation in the Burkholderia community structure was observed between the permanent grassland and agricultural areas, differences between the crop rotation and maize monoculture groups were also observed . Plant species affected Burkholderia community structure to a lesser extent than did land use history . Similarities were observed between Burkholderia populations associated with maize and grass, on the one hand, and between those associated with barley and oat, on the other hand . Additionally, CCA ordination plots demonstrated that these two groups (maize/grass versus barley/oat) had a negative correlation . The identification of bands from the DGGE patterns demonstrated that the species correlated with the environmental variables were mainly affiliated with Burkholderia species that are commonly isolated from soil, in particular Burkholderia glathei, B . caledonica, B . hospita, and B . caribiensis.

Appl Environ Microbiol, 2004 Jul, 70(7), 3814 - 20
Oxidation of benzene to phenol, catechol, and 1,2,3-trihydroxybenzene by toluene 4-monooxygenase of Pseudomonas mendocina KR1 and toluene 3-monooxygenase of Ralstonia pickettii PKO1; Tao Y et al.; Aromatic hydroxylations are important bacterial metabolic processes but are difficult to perform using traditional chemical synthesis, so to use a biological catalyst to convert the priority pollutant benzene into industrially relevant intermediates, benzene oxidation was investigated . It was discovered that toluene 4-monooxygenase (T4MO) of Pseudomonas mendocina KR1, toluene 3-monooxygenase (T3MO) of Ralstonia pickettii PKO1, and toluene ortho-monooxygenase (TOM) of Burkholderia cepacia G4 convert benzene to phenol, catechol, and 1,2,3-trihydroxybenzene by successive hydroxylations . At a concentration of 165 microM and under the control of a constitutive lac promoter, Escherichia coli TG1/pBS(Kan)T4MO expressing T4MO formed phenol from benzene at 19 +/- 1.6 nmol/min/mg of protein, catechol from phenol at 13.6 +/- 0.3 nmol/min/mg of protein, and 1,2,3-trihydroxybenzene from catechol at 2.5 +/- 0.5nmol/min/mg of protein . The catechol and 1,2,3-trihydroxybenzene products were identified by both high-pressure liquid chromatography and mass spectrometry . When analogous plasmid constructs were used, E . coli TG1/pBS(Kan)T3MO expressing T3MO formed phenol, catechol, and 1,2,3-trihydroxybenzene at rates of 3 +/- 1, 3.1 +/- 0.3, and 0.26 +/- 0.09 nmol/min/mg of protein, respectively, and E . coli TG1/pBS(Kan)TOM expressing TOM formed 1,2,3-trihydroxybenzene at a rate of 1.7 +/- 0.3 nmol/min/mg of protein (phenol and catechol formation rates were 0.89 +/- 0.07 and 1.5 +/- 0.3 nmol/min/mg of protein, respectively) . Hence, the rates of synthesis of catechol by both T3MO and T4MO and the 1,2,3-trihydroxybenzene formation rate by TOM were found to be comparable to the rates of oxidation of the natural substrate toluene for these enzymes (10.0 +/- 0.8, 4.0 +/- 0.6, and 2.4 +/- 0.3 nmol/min/mg of protein for T4MO, T3MO, and TOM, respectively, at a toluene concentration of 165 microM).

J Bacteriol, 2004 Jul, 186(14), 4705 - 13
Altering toluene 4-monooxygenase by active-site engineering for the synthesis of 3-methoxycatechol, methoxyhydroquinone, and methylhydroquinone; Tao Y et al.; Wild-type toluene 4-monooxygenase (T4MO) of Pseudomonas mendocina KR1 oxidizes toluene to p-cresol (96%) and oxidizes benzene sequentially to phenol, to catechol, and to 1,2,3-trihydroxybenzene . In this study T4MO was found to oxidize o-cresol to 3-methylcatechol (91%) and methylhydroquinone (9%), to oxidize m-cresol and p-cresol to 4-methylcatechol (100%), and to oxidize o-methoxyphenol to 4-methoxyresorcinol (87%), 3-methoxycatechol (11%), and methoxyhydroquinone (2%) . Apparent Vmax values of 6.6 +/- 0.9 to 10.7 +/- 0.1 nmol/min/ mg of protein were obtained for o-, m-, and p-cresol oxidation by wild-type T4MO, which are comparable to the toluene oxidation rate (15.1 +/- 0.8 nmol/min/mg of protein) . After these new reactions were discovered, saturation mutagenesis was performed near the diiron catalytic center at positions I100, G103, and A107 of the alpha subunit of the hydroxylase (TmoA) based on directed evolution of the related toluene o-monooxygenase of Burkholderia cepacia G4 (K . A . Canada, S . Iwashita, H . Shim, and T . K . Wood, J . Bacteriol . 184:344-349, 2002) and a previously reported T4MO G103L regiospecific mutant (K . H . Mitchell, J . M . Studts, and B . G . Fox, Biochemistry 41:3176-3188, 2002) . By using o-cresol and o-methoxyphenol as model substrates, regiospecific mutants of T4MO were created; for example, TmoA variant G103A/A107S produced 3-methylcatechol (98%) from o-cresol twofold faster and produced 3-methoxycatechol (82%) from 1 mM o-methoxyphenol seven times faster than the wild-type T4MO (1.5 +/- 0.2 versus 0.21 +/- 0.01 nmol/min/mg of protein) . Variant I100L produced 3-methoxycatechol from o-methoxyphenol four times faster than wild-type T4MO, and G103S/A107T produced methylhydroquinone (92%) from o-cresol fourfold faster than wild-type T4MO and there was 10 times more in terms of the percentage of the product . Variant G103S produced 40-fold more methoxyhydroquinone from o-methoxyphenol than the wild-type enzyme produced (80 versus 2%) and produced methylhydroquinone (80%) from o-cresol . Hence, the regiospecific oxidation of o-methoxyphenol and o-cresol was changed for significant synthesis of 3-methoxycatechol, methoxyhydroquinone, 3-methylcatechol, and methylhydroquinone . The enzyme variants also demonstrated altered monohydroxylation regiospecificity for toluene; for example, G103S/A107G formed 82% o-cresol, so saturation mutagenesis converted T4MO into an ortho-hydroxylating enzyme . Furthermore, G103S/A107T formed 100% p-cresol from toluene; hence, a better para-hydroxylating enzyme than wild-type T4MO was formed . Structure homology modeling suggested that hydrogen bonding interactions of the hydroxyl groups of altered residues S103, S107, and T107 influence the regiospecificity of the oxygenase reaction .

J Coll Physicians Surg Pak, 2004 Feb, 14(2), 102 - 4
Control of Burkholderia (Pseudomonas) bacteraemia in the intensive care and paediatric units; Ahmad K et al.; OBJECTIVE: Investigation and control of nosocomial bacteraemia caused by Burkholderia (Pseudomonas) cepacia in the Intensive Care and Paediatric Units of a general care hospital . DESIGN: A cross-sectional analytical study . PLACE AND DURATION OF STUDY: Departments of Pathology, Intensive Care and Paediatrics, Kahota Research Laboratory Hospital, Islamabad from January 1998 to June 2002 . SUBJECTS AND METHODS: Blood cultures from patients admitted to Intensive Care Unit and Paediatric Ward were inoculated into brain heart infusion broth and incubated for upto 10 days . Any Gram-negative rods isolated were characterized by API-20E . Environmental samples were inoculated on blood and MacConkey's agars and isolates, if any, were identified as above . Intensive intervention in the form of hand washing, strict adherence to aseptic practices and standard sterilization techniques were adopted and then cultures were again carried out with similar methodology . RESULTS: Cultures yielded 58 strains of Burkholderia cepacia, 52 from blood cultures and 6 from hospital environment, including 1 from the washbasin of the ICU . Thirty- four of these were isolated before intervention measures were adopted, mainly during 1998 . Findings suggested a strong probability of nosocomial transmission, with washbasin as the common source . After a lapse of about a year, B . cepacia infection re-emerged in a sporadic form but remaining confined to paediatric unit . Only 18 isolates were yielded over the next two and-a-half years . CONCLUSION: The intervention measures for Burkholderia bacteraemia within the hospital, proved effective in stopping the nosocomial transmission leading to disappearance of B . cepacia from blood cultures . We emphasize the crucial role of hand hygienic practices in the hospital setting, especially in critical care units.

Trop Med Int Health, 2004 Jul, 9(7), 795 - 804
Epidemiology of community-acquired and nosocomial bloodstream infections in tropical Australia: a 12-month prospective study; Douglas MW et al.; OBJECTIVES: To define the relative incidence of organisms causing blood stream infections in a tropical setting with a very low prevalence of human immunodeficiency virus infection (<1%) . METHODS: A 12-month prospective study of blood stream infections in 2000 at Royal Darwin Hospital in the tropical north of Australia . RESULTS: Significant isolates were grown from 257 sets of blood cultures . Staphylococcus aureus was the most common isolate overall (28%); 26% of these were methicillin-resistant (MRSA) . Escherichia coli was the most common cause of community-acquired bacteraemia . Burkholderia pseudomallei caused 32% of community acquired, bacteraemic pneumonia; 6% of bacteraemias overall . Vancomycin-resistant enterococci were not isolated . Crude mortality rates (13% overall; 9% attributable mortality) were lower than in most comparable studies . CONCLUSIONS: The major difference between these findings and surveys performed elsewhere is the presence of B . pseudomallei as a significant cause of bacteraemic community-acquired pneumonia . Our results demonstrate the effects of local environmental and patient characteristics on the range of organisms causing blood stream infections, and emphasize the important role of local microbiology laboratories in guiding empiric antibiotic therapy .

Biodegradation, 2004 Jun, 15(3), 161 - 71
Enhancement of 2,4-dinitrotoluene biodegradation by Burkholderia sp . in sand bioreactors using bacterial hemoglobin technology; So J et al.; Continuous flow sand column bioreactor experiments were conducted to investigate the effect of 2,4-dinitrotoluene (DNT) concentration (i.e . DNT loading rate) and influent dissolved oxygen (DO) concentration on aerobic biodegradation of DNT by wild type (DNT) and recombinant (YV1) Burkholderia sp., the latter containing plasmid pSC160 which carries the gene (vgb) encoding the hemoglobin (VHb) from the bacterium Vitreoscilla . The experiments were conducted in two continuous flow packed bed sand column bioreactors, one growing the wild type strain and the other growing YV1 . Under oxygen-rich feed conditions (6.8 mg DO/L in the feed) with an influent DNT concentration of 99.6 mg/L (DNT loading rate approximately = 9.2 mg/m2/day), the effluent DNT concentration from the wild type bioreactor reached 0.7 mg DNT/L in 40 days whereas it was less than 0.2 mg DNT/L for the YV1 bioreactor in about 25 days . When influent DNT concentration was increased to 214 mg/L (DNT loading rate approximately = 20.3 mg/m2/day) while maintaining the same influent DO level of 6.8 mg/L, the effluent DNT concentration increased to about 5 mg/L for the wild type bioreactor whereas it was maintained at less than 0.2 mg/L for the YV1 bioreactor . Additionally, when influent DO was reduced from 6.8 mg/L to 3.1 mg/L while the influent DNT concentration remained at 214 mg/L, the effluent DNT concentration increased to more than 20 mg/L for the wild type bioreactor but up to only 1.7 mg/L for the YV1 bioreactor . A subsequent increase of influent DO back to 6.6 mg/L reduced the effluent DNT concentration to about 5 mg/L for the wild type bioreactor and to 0.10-0.19 mg/L for the YV1 bioreactor . These results confirm the utility of vgb technology to enhance biodegradation of aromatic compounds under hypoxic conditions and also that this enhancement can be maintained over extended periods of time as evidenced by plasmid stability in Burkholderia YV1.

Thorax, 2004 Jul, 59(7), 613 - 7
Inflammatory related changes in bone mineral content in adults with cystic fibrosis; Haworth CS et al.; BACKGROUND: Proinflammatory cytokines stimulate osteoclast activity and this could lead to increased bone resorption in patients with cystic fibrosis . The aim of this study was to determine whether markers of systemic inflammation are related to changes in bone mineral content (BMC) in adults with cystic fibrosis . METHODS: Total body BMC was assessed by dual energy x ray absorptiometry in 100 patients (54 male) of mean (SD) age 25.6 (7.1) years and forced expiratory volume in 1 second (FEV(1)) 61.8 (24.1)% predicted on recruitment to the study and 1 year later . Blood was also taken at these time points to measure markers of systemic inflammation . RESULTS: After 1 year BMC had reduced by 16.1 (62.1) g, p = 0.01; (0.6 (2.8)%) . The change in BMC was related to mean levels of interleukin (IL)-6 (r(s) = -0.39, p<0.001) and C reactive protein (r(s) = -0.34, p = 0.002), intravenous antibiotic use (r(s) = -0.27, p = 0.006) and oral corticosteroid use (r(s) = -0.20, p = 0.045) . Urinary markers of osteoclast activity were also related to IL-6 (r(s) = 0.27, p = 0.02) . Multiple linear regression revealed that IL-6 (coefficient -2.2 (95% CI -3.4 to -1.0) per pg/ml, p = 0.001), colonisation with Burkholderia cepacia (coefficient -46.8 (95% CI -75.5 to -18.1), p = 0.002), and annual change in BMI (coefficient 15.4 (95% CI 3.6 to 27.2) per kg/m(2), p = 0.011) were independently significant predictors of annual change in BMC . CONCLUSIONS: These data suggest a pathophysiological mechanism by which chronic pulmonary infection results in bone loss in patients with cystic fibrosis.

J Ind Microbiol Biotechnol, 2004 Jul, 31(6), 245 - 54 Epub 2004 Jun 22.
Poly-3-hydroxybutyrate (P3HB) production by bacteria from xylose, glucose and sugarcane bagasse hydrolysate; Silva LF et al.; Fifty-five bacterial strains isolated from soil were screened for efficient poly-3-hydroxybutyrate (P3HB) biosynthesis from xylose . Three strains were also evaluated for the utilization of bagasse hydrolysate after different detoxification steps . The results showed that activated charcoal treatment is pivotal to the production of a hydrolysate easy to assimilate . Burkholderia cepacia IPT 048 and B . sacchari IPT 101 were selected for bioreactor studies, in which higher polymer contents and yields from the carbon source were observed with bagasse hydrolysate, compared with the use of analytical grade carbon sources . Polymer contents and yields, respectively, reached 62% and 0.39 g g(-1) with strain IPT 101 and 53% and 0.29 g g(-1) with strain IPT 048 . A higher polymer content and yield from the carbon source was observed under P limitation, compared with N limitation, for strain IPT 101 . IPT 048 showed similar performances in the presence of either growth-limiting nutrient . In high-cell-density cultures using xylose plus glucose under P limitation, both strains reached about 60 g l(-1) dry biomass, containing 60% P3HB . Polymer productivity and yield from this carbon source reached 0.47 g l(-1) h(-1) and 0.22 g g(-1), respectively.

Eur Respir J, 2004 Jun, 23(6), 851 - 6
Epidemiology of Burkholderia cepacia complex colonisation in cystic fibrosis patients; De Boeck K et al.; In Belgian cystic fibrosis (CF) clinics, sputum samples are evaluated on selective MAST medium routinely every 3 months . In this study, in 1993 and 1999, isolates were further examined by recA restriction fragment length polymorphism analysis and pulsed-field gel electrophoresis of genomic DNA restricted with SpeI . In 1993, 12 patients were colonised with Burkholderia cepacia complex (Bcc): B . cenocepacia (n=6), B . multivorans (n=3), B . stabilis (n=3) . Four patients were colonised with the same B . cenocepacia strain; two with the same B . stabilis strain . After 5 yrs, three B . cenocepacia- and one B . multivorans-colonised patients had died . In 1999, Bcc was isolated in 12 patients: B . multivorans (n=9), B . stabilis (n=1) and B . cenocepacia (n=2) . Three patients were colonised by the same B . multivorans strain . Compared to matched controls, the 5 yr outcome was poor; four B . cepacia patients died and none of the control patients died . Lung-function evolution was poor . In conclusion, the rate of colonisation in Belgian cystic fibrosis patients is stable and low . Burkholderia cenocepacia was most prevalent in 1993; Burkholderia multivorans in 1999 . The cross-infection rate is low . Three patients had transient colonisation . The impact of Burkholderia cepacia complex on morbidity in the Belgian cystic fibrosis population is high and not limited to Burkholderia cenocepacia.

Infect Immun, 2004 Jul, 72(7), 4188 - 99
Responses of well-differentiated airway epithelial cell cultures from healthy donors and patients with cystic fibrosis to Burkholderia cenocepacia infection; Sajjan U et al.; Well-differentiated cultures established from airway epithelia of patients with cystic fibrosis (CF cultures) exhibited goblet cell hyperplasia, increased secretion of mucus, and higher basal levels of interleukin-8 than similarly cultured cells from healthy donors . Upon apical infection with low doses (10(4) to 10(5) CFU) of Burkholderia cenocepacia isolate BC7, the two cultures gave different responses . While normal cultures trapped the added bacteria in the mucus layer, killed and/or inhibited bacterial replication, and prevented bacterial invasion of the cells, CF cultures failed to kill and/or supported the growth of bacteria, leading to invasion of underlying epithelial cells, compromised transepithelial permeability, and cell damage . Depletion of the surface mucus layer prior to bacterial infection rendered the normal cultures susceptible to bacterial invasion, but the invading bacteria were mainly confined to vacuoles within the cells and appeared to be nonviable . In contrast, bacteria that invaded cells in CF cultures were found free in the cytoplasm surrounded by intermediate filaments and also between cells . Cultured CF airway epithelium was therefore more susceptible to infection than normal epithelium . This mimics CF tissue in vivo and illustrates differences in the way epithelia in CF patients and normal subjects handle bacterial infection . In addition, we found that the CF and normal cell cultures responded differently not only to isolate BC7 but also to isolates of other B . cepacia complex species . We therefore conclude that this cell culture model is suitable for investigation of B . cepacia complex pathogenesis in CF patients.

Infect Immun, 2004 Jul, 72(7), 4172 - 87
Contribution of gene loss to the pathogenic evolution of Burkholderia pseudomallei and Burkholderia mallei; Moore RA et al.; Burkholderia pseudomallei is the causative agent of melioidosis . Burkholderia thailandensis is a closely related species that can readily utilize l-arabinose as a sole carbon source, whereas B . pseudomallei cannot . We used Tn5-OT182 mutagenesis to isolate an arabinose-negative mutant of B . thailandensis . Sequence analysis of regions flanking the transposon insertion revealed the presence of an arabinose assimilation operon consisting of nine genes . Analysis of the B . pseudomallei chromosome showed a deletion of the operon from this organism . This deletion was detected in all B . pseudomallei and Burkholderia mallei strains investigated . We cloned the B . thailandensis E264 arabinose assimilation operon and introduced the entire operon into the chromosome of B . pseudomallei 406e via homologous recombination . The resultant strain, B . pseudomallei SZ5028, was able to utilize l-arabinose as a sole carbon source . Strain SZ5028 had a significantly higher 50% lethal dose for Syrian hamsters compared to the parent strain 406e . Microarray analysis revealed that a number of genes in a type III secretion system were down-regulated in strain SZ5028 when cells were grown in l-arabinose, suggesting a regulatory role for l-arabinose or a metabolite of l-arabinose . These results suggest that the ability to metabolize l-arabinose reduces the virulence of B . pseudomallei and that the genes encoding arabinose assimilation may be considered antivirulence genes . The increase in virulence associated with the loss of these genes may have provided a selective advantage for B . pseudomallei as these organisms adapted to survival in animal hosts.

Infect Immun, 2004 Jul, 72(7), 4010 - 22
Identification of Burkholderia cenocepacia genes required for bacterial survival in vivo; Hunt TA et al.; Burkholderia cenocepacia (formerly Burkholderia cepacia complex genomovar III) causes chronic lung infections in patients with cystic fibrosis . In this work, we used a modified signature-tagged mutagenesis (STM) strategy for the isolation of B . cenocepacia mutants that cannot survive in vivo . Thirty-seven specialized plasposons, each carrying a unique oligonucleotide tag signature, were constructed and used to examine the survival of 2,627 B . cenocepacia transposon mutants, arranged in pools of 37 unique mutants, after a 10-day lung infection in rats by using the agar bead model . The recovered mutants were screened by real-time PCR, resulting in the identification of 260 mutants which presumably did not survive within the lungs . These mutants were repooled into smaller pools, and the infections were repeated . After a second screen, we isolated 102 mutants unable to survive in the rat model . The location of the transposon in each of these mutants was mapped within the B . cenocepacia chromosomes . We identified mutations in genes involved in cellular metabolism, global regulation, DNA replication and repair, and those encoding bacterial surface structures, including transmembrane proteins and cell surface polysaccharides . Also, we found 18 genes of unknown function, which are conserved in other bacteria . A subset of 12 representative mutants that were individually examined using the rat model in competition with the wild-type strain displayed reduced survival, confirming the predictive value of our STM screen . This study provides a blueprint to investigate at the molecular level the basis for survival and persistence of B . cenocepacia within the airways.

Biotechnol Bioeng, 2004 Jul 5, 87(1), 110 - 8
Enhanced kinetics of genetically engineered Burkholderia cepacia: the role of vgb in the hypoxic metabolism of 2-CBA; Urgun-Demirtas M et al.; Application of Vitreoscilla hemoglobin (VHb) technology to 2-CBA degradation by Burkholderia cepacia strain DNT under hypoxic conditions was studied in continuous culture chemostats . Dechlorination abilities of both recombinant (VHb gene (vgb) containing) and untransformed cells were investigated at various dilution rates to ensure complete degradation of 2-CBA . As the dilution rate increased from 0.025 to 0.25 h(-1), the ratios of chloride release to degraded 2-CBA concentration decreased from 0.95 to 0.72 and from 0.89 to 0.39 for recombinant and untransformed cells, respectively . A nonstoichiometric relationship between chloride release and 2-CBA degradation was more pronounced for untransformed cells . Recombinant cell densities were 0.1-0.2 . g L(-1) greater than untransformed cell densities for a range of dilution rates . As the dilution rate increased, the oxygen uptake rate (OUR) and the substrate utilization rate (SUR) decreased for both strains . The OUR/SUR ratio increased as the dilution rate increased for both strains but was much higher for the recombinant strain compared to untransformed cells . The specific 2-CBA degradation rate of recombinant cells was greater than that of untransformed cells (1.17 vs . 0.46 mg CBA (mg) day(-1), and half-saturation constants for recombinant cells were lower than those of untransformed cells (0.18 and 0.32 mg CBA L(-1), respectively) . The pseudo-first-order degradation constants, k(1CBA) and k(1ACE), were higher for recombinant cells (6.5 L (mg cells)(-1) day(-1) and 95.6 L (mg cells)(-1) day(-1), respectively) than those of untransformed cells (1.44 L (mg cells)(-1) day(-1) and 73.7 L (mg cells)(-1) day(-1), respectively) .

An Pediatr (Barc), 2004 Jun, 60(6), 581 - 2
{Pharyngitis due to Burkholderia cepacia . Person-to-person transmission}; Fajardo Olivares M et al.; Burkholderia cepacia is a Gram-negative bacillus that is widely distributed in nature; it is isolated from the ground, water, plants and vegetables . Generally, it produces nosocomial infection due to contamination of disinfectants, medical equipment, prosthetic material and drugs, such as anesthetics or liquids used in urological irrigation . The most probable mechanism of transmission is through hospital material or through fomites among people after contact for several weeks or months . Recently, it has been considered as an important pathogen in immunocompromised patients, or in those with significant underlying diseases, such as chronic granulomastosis or cystic fibrosis . We present a case of pharyngitis due to B . cepacia and its transmission within a few days in two immunocompetent twin siblings without previous underlying diseases . The infection disappeared after specific treatment for this microorganism was started . We believe that samples should be taken from the pharynx and nasal pits in patients with acute upper respiratory tract processes that do not respond to empiric antibiotic treatment, before classifying them as viral infection without etiologic diagnosis.

J Bacteriol, 2004 Jul, 186(13), 4350 - 60
Mutational analysis and biochemical characterization of the Burkholderia thailandensis DW503 quorum-sensing network; Ulrich RL et al.; Numerous gram-negative bacteria communicate and regulate gene expression through a cell density-responsive mechanism termed quorum sensing (QS), which involves the synthesis and perception of diffusible N-acyl-homoserine lactones (AHL) . In this study we genetically and physiologically characterized the Burkholderia thailandensis DW503 QS network . In silico analysis of the B . thailandensis genome revealed the presence of at least three AHL synthases (AHS) and five transcriptional regulators belonging to the LuxIR family of proteins . Mass spectrometry demonstrated that wild-type B . thailandensis synthesizes N-hexanoyl-homoserine lactone (C6-HSL), N-octanoyl-homoserine lactone (C8-HSL), and N-decanoyl-homoserine lactone (C10-HSL) . Mutation of the btaI1 (luxI) AHS gene prevented accumulation of C8-HSL in culture supernatants, enhanced beta-hemolysis of sheep erythrocytes, increased lipase production, and altered colony morphology on swarming and twitching motility plates . Disruption of the btaI3 (luxI) AHS prevented biosynthesis of C6-HSL and increased lipase production and beta-hemolysis, whereas mutagenesis of the btaI2 (luxI) allele eliminated C10-HSL accumulation and reduced lipase production . Complementation of the btaI1 and btaI3 mutants fully restored the synthesis of C8-HSL and C6-HSL to parental levels . In contrast, mutagenesis of the btaR1, btaR3, btaR4, and btaR5 (luxR) transcriptional regulators had no effect on AHL accumulation, enhanced lipase production, and resulted in extensive beta-hemolysis on sheep blood agar plates . Furthermore, interruption of the btaI1, btaR1, and btaR3 genes altered colony morphology on twitching and swarming motility plates and induced pigmentation . Additionally, phenotypic microarray analysis indicated that QS in B . thailandensis both positively and negatively affects the metabolism of numerous substrates, including citric acid, formic acid, glucose 6-phosphate, capric acid, gamma-hydroxybutyric acid, and d-arabinose . These results demonstrate that mutagenesis of the B . thailandensis QS system affects various cellular processes, including lipase production, swarming and twitching motility, beta-hemolysis of sheep erythrocytes, and carbon metabolism and/or transport.

Biomed Environ Sci, 2004 Mar, 17(1), 21 - 6
Microbial degradation of quinoline: kinetics study with Burkholderia picekttii; Wang JL et al.; OBJECTIVE: To investigate the kinetics of quinoline biodegradation by Burkholderia pickttii, a gram negative rod-shaped aerobe, isolated in our laboratory . METHODS: HPLC (Hewlett-Packard model 5050 with an UV detector) was used for the analysis of quinoline concentration . GC/MS method was used to identify the intermediate metabolites of quinoline degradation . RESULTS: The biodegradation of quinoline was inhibited by quinoline at a high concentration, and the degradation process could be described by the Haldane model . The kinetic parameters based on Haldane substrate inhibition were evaluated . The values were vmax = 0.44 h(-1), K(S) = 166.7 mg/L, Ki = 650 mg/L, respectively . The quinoline concentration to avoid substrate inhibition was inferred theoretically and determined to be 329 mg/L . CONCLUSION: The biodegradation of quinoline conforms to the Haldane inhibition model and the main intermediate metabolite of quinoline biodegradation is 2-hydroxy-quinoline.

Arch Pharm Res, 2004 May, 27(5), 570 - 5
Kinetic property and phylogenic relationship of 2-hydroxymuconic semialdehyde dehydrogenase encoded in tomC gene of Burkholderia cepacia G4; Reddy AM et al.; 2-Hydroxymuconic semialdehyde (2-HMS) dehydrogenase catalyzes the conversion of 2-HMS to 4-oxalocrotonate, which is a step in the meta cleavage pathway of aromatic hydrocarbons in bacteria . A tomC gene that encodes 2-HMS dehydrogenase of Burkholderia cepacia G4, a soil bacterium that can grow on toluene, cresol, phenol, or benzene, was overexpressed into E . coli HB101, and its gene product was characterized in this study . 2-HMS dehydrogenase from B . cepacia G4 has a high catalytic efficiency in terms of Vmax/Km towards 2-hydroxy-5-methylmuconic semialdehyde followed by 2-HMS but has a very low efficiency for 5-chloro-2-hydroxymuconic semialdehyde . However, the enzyme did not utilize 2-hydroxy-6-oxo-hepta-2,4-dienoic acid and 2-hydroxy-6-oxo-6-phenylhexa-2,4-dienoic acid as substrates . The molecular weight of 2-HMS dehydrogenase from B . cepacia G4 was predicted to be 52 kDa containing 485 amino acid residues from the nucleotide sequence of the tomC gene, and it exhibited the highest identity of 78% with the amino acid sequence of 2-HMS dehydrogenase that is encoded in the aphC gene of Comamonas testosteroni TA441 . 2-HMS dehydrogenase from B . cepacia G4 showed a significant phylogenetic relationship not only with other 2-HMS dehydrogenases, but also with different dehydrogenases from evolutionarily distant organisms.

Asian Pac J Allergy Immunol, 2003 Dec, 21(4), 259 - 67
One-step purification of chimeric green fluorescent protein providing metal-binding avidity and protease recognition sequence; Prachayasittikul V et al.; Gene fusion technique was successfully applied as a potential approach to create a metal-binding site to assist one-step purification of green fluorescent protein (GFP) . The chimeric GFP carrying hexapolyhistidine (H6GFPuv) was purified to homogeneous protein via the Immobilized Metal Affinity Chromatography charged with zinc ions . Removal of metal tagger could readily be performed by using enterokinase enzyme . Engineering of the hexahistidine and enterokinase cleavage sites (DDDDK) onto the chimeric protein did not significantly affect the fluorescent property and the binding avidity to Burkholderia pseudomallei protease of a chimeric protease-binding GFP (H6PBGFPuv) . This concludes that engineering of repetitive histidine regions onto interested target protein along with the enterokinase cleavage sites will ease the complication of protein purification.

Exp Lung Res, 2004 Apr-May, 30(3), 163 - 79
Cytokine stimulation by Pseudomonas aeruginosa--strain variation and modulation by pulmonary surfactant; Bufler P et al.; Pulmonary surfactant and its components are part of the first-line immune defense within the lung . Here the authors show that the surfactant protein (SP) SP-D, but not SP-A, agglutinates some clinical isolates of Pseudomonas aeruginosa and Stenotrophomonas maltophilia . No agglutination of Staphylococcus aureus or Burkholderia cepacia was observed . The SP-D-induced agglutination of P . aeruginosa was not dependent on a specific lipopolysaccharide (LPS) serotype . The authors also show that SP-D, but not SP-A, increased the tumor necrosis factor (TNF alpha) release from human monocytic cells in response to a subset of P . aeruginosa and P . aeruginosa LPS . A clinical preparation of surfactant (Alveofact) blocked the TNF alpha release from monocytic cells induced by P . aeruginosa or its LPS . SP-A reversed the inhibitory effect of Alveofact in 6/8 strains of P . aeruginosa and 2/9 preparations of P . aeruginosa LPS . SP-D did not significantly alter the TNF alpha production induced by vital P . aeruginosa in the presence of Alveofact but markedly increased the TNF alpha release induced by a preparation of rough and smooth P . aeruginosa LPS . In summary, this study shows that the immunomodulatory properties of SP-A and SP-D specifically depend on the colonizing strain of P . aeruginosa . In addition, the authors show that the function of SP-A and SP-D is modulated in the presence of surfactant lipids.

Clin Microbiol Infect, 2004 Jun, 10(6), 585 - 7
Exposure to Burkholderia pseudomallei induces cell-mediated immunity in healthy individuals; Govan B et al.; Melioidosis is an emerging tropical infection caused by the intracellular bacterium Burkholderia pseudomallei, and is associated with high mortality rates . Previous studies investigating the prevalence of melioidosis have based conclusions on serological evidence . However, cell-mediated immunity is more relevant for protection against an intracellular pathogen such as B . pseudomallei . This is the first demonstration that exposure to B . pseudomallei may lead to the formation of specific antibodies and the development of cell-mediated immunity in a healthy individual.

Trop Med Int Health, 2004 Jun, 9(6), 715 - 7
Out of hospital treatment of patients with melioidosis using ceftazidime in 24 h elastomeric infusors, via peripherally inserted central catheters; Huffam S et al.; BACKGROUND: In the tropical north of the Northern Territory, Australia, 25-50 patients are admitted to Royal Darwin Hospital (RDH) each year with Burkholderia pseudomallei infection, or melioidosis . Treatment consists of initial intensive therapy with 2-4 weeks of intravenous antibiotics . Clinical improvement may occur early and patients often prefer to be managed out of hospital in the Hospital in the Home (HITH) . OBJECTIVES: To evaluate safety and efficacy of HITH management of patients with melioidosis . METHODS: A prospective observational study of our standard management which consists of 24 h infusions of ceftazidime infused through a peripherally inserted central catheter (PICC) line, plus oral sulphamethoxazole trimethoprim . Treatment is administered in the home, which may be in Darwin, regional areas or remote communities, or in a self-care unit located in the hospital grounds . RESULTS: From February 1998 to December 2001 150 patients were admitted to RDH with culture confirmed B . pseudomallei infection . Of these, 73 patients were treated with 24 h infusions of ceftazidime, of which 70 patients were managed by HITH . Complications of treatment include a PICC line complication rate of 10.6/1000 days in situ . Nine patients had relapse or recrudescence of disease, nearly all as a result of poor adherence to subsequent oral eradication therapy, these patients were all re-treated successfully . One patient remains infected with B . pseudomallei . CONCLUSION: This clinical outcome study suggests that out of hospital management of melioidosis with 24 h infusions of ceftazidime via a PICC line is safe and effective.

Zh Mikrobiol Epidemiol Immunobiol, 2004 Mar-Apr, (2), 38 - 42
{Experimental study on the possibility of using live tularemia vaccine to increase resistance to heterologous infection disease}; Iliukhin VI et al.; In experiments on guinea pigs immunized with Francisella tularensis 15, or live tularemia vaccine (LTV), the level of heterologous protective effect to dangerous infectious diseases caused by Yersinia pestis, Burkholderia pseudomallei, B . mallei, Mycobacterium tuberculosis was studied . The study revealed that during the first 4 weeks after the subcutaneous immunization with LTV the level of resistance of the immunized animals to heterologous infective agent reliably increased as indicated by the survival rate of the animals, as well as by the survival time of those killed by infection, in comparison with the controls . Later (on day 150 after immunization) differences in death rate between the groups perceptibly decreased . Nevertheless, the 1 1/2-fold increase of the survival time of the challenged immunized animals in comparison with the controls proved the possibility of using immunization with LTV for the urgent prophylaxis and treatment not only of tularemia, but also of plague, glanders, melioidosis and tuberculosis.

Zh Mikrobiol Epidemiol Immunobiol, 2004 Mar-Apr, (2), 14 - 20
{Development of the controlled model of persisting infection , caused by Pseudomonas aeruginosa and bacteria of the complex Burkholderia cepacia}; Chernukha MIu et al.; As the result of testing three different variants, the experimental models of persisting infection for P . aeruginosa and B . cepacia have been developed . These doses differ in the time of administration, doses of antibiotics and the infective doses of the microorganisms . The administration of the sub-inhibiting concentration of antibiotics for 5 days and the subsequent infection of laboratory animals (non-inbred mice) B . cepacia strains in a dose of LD50 leads to a considerable increase in the survival rate of mice and to a longer period (up to 20 days) of obtaining inoculative material from the spleen . The isolated cultures are characterized by a sharply slower growth on artificial culture media (up to 5-7 days as compared with 24-48 hours for the initial culture) . The newly developed models have made it possible to control different stages of the infectious process in the induced increase or decrease of the virulent properties of the infective agent and in changes in the immune status of the host . As the result of these studies, in some mice (10%) infected with B . cepacia after the injection of gamma-hydroxybutyric acid lactone the infection has taken the acute form, while in the mice infected with P . aeruginosa no such effect has been observed . On the contrary, in the mice infected with P . aeruginosa and then receiving cyclophosphamide the transition of the infection into the acute form has been observed in 30% of the animals . In the mice infected with B . cepacia no such effect has been noted after the injection of this preparation . Different effects produced by cyclophosphamide and lactone are discussed from the positions of "quorum sensing" in pathogenic bacteria.

J Environ Sci Health B, 2004 May, 39(3), 431 - 41
The response of Escherichia coli, Bacillus subtilis, and Burkholderia cepacia WZ1 to oxidative stress of exposure to quinclorac; Lu Z et al.; The activity response of the antioxidant enzymes superoxide dismutase, catalase, ATP enzyme activities of Escherichia coli (G-), Bacillus subtilis (G+), and Burkholderia cepacia WZ1 (G-) following exposure to quinclorac was investigated . The bacterial strains were treated with the different concentrations of quinclorac (1.65, 16.5, 33.0, 165.0, 330.0, and 500.0 microg L(-1)) . Results obtained indicated that SOD and CAT activities of these bacteria were induced positively and obviously by quinclorac, especially to gram-positive (G+) bacteria treated by lower than 330 microg L(-1) of quinclorac . The inhibition of ATPase in E . coli K12, B . subtilis, and B . cepacia WZ1 appeared stronger with the increase of quinclorac concentration, showing a striking dose response relationship, which can, therefore, be used as an available bioindicator for quinclorac pollution . The concentration of quinclorac applied in this research had significant effects on these three bacteria at the early stage of incubation, but none of which was persistent . Native polyacrylamide gel electrophoresis and activity staining of SOD revealed that quinclorac had effects on isoforms of E . coli and B . subtilis, and on the staining intensities of the isoforms of B . cepacia WZ1 . When E . coli K12 was incubated with 330 microg L(-1) of quinclorac, the upper band of the isoforms of SOD tended to become slightly more apparent at 1 h after the quinclorac treatment, but the staining activity was slightly reduced after the prolonged treatment of quinclorac . No such changes of the isoforms of B . cepacia WZ1 was observed.

J Med Microbiol, 2004 Jul, 53(Pt 7), 663 - 8
Epidemiology of Burkholderia cepacia complex species recovered from cystic fibrosis patients: issues related to patient segregation; McDowell A et al.; Studies of the prevalence of Burkholderia cepacia complex species amongst cystic fibrosis (CF) patients in different geographical regions, and the association between cross-infection and putative transmissibility markers, will further our understanding of these organisms and help to address infection-control issues . In this study, B . cepacia complex isolates from CF patients in different regions of Europe were analysed . Isolates were examined for B . cepacia complex species and putative transmissibility markers {cable pilin subunit gene (cblA) and the B . cepacia epidemic strain marker (BCESM)} . Sporadic and cross-infective strains were identified by random amplification of polymorphic DNA (RAPD) . In total, 79% of patients were infected with Burkholderia cenocepacia (genomovar III), 18% with Burkholderia multivorans (genomovar II) and less than 5% of patients with B . cepacia (genomovar I), Burkholderia stabilis (genomovar IV) or Burkholderia vietnamiensis (genomovar V) . The cblA and BCESM transmissibility markers were only detected in strains of B . cenocepacia . The BCESM was a more sensitive marker for transmissible B . cenocepacia strains than cblA, although sporadic B . cenocepacia strains containing the BCESM, but lacking cblA, were also observed . Furthermore, clusters of cross-infection with transmissibility marker-negative strains of B . multivorans were identified . In conclusion, B . cenocepacia was the greatest cause of cross-infection, and the most widely distributed B . cepacia complex species, within these CF populations . However, cross-infection was not exclusive to B . cenocepacia and cblA and the BCESM were not absolute markers for transmissible B . cenocepacia, or other B . cepacia complex strains . It is therefore suggested that CF centres cohort patients based on the presence or absence of B . cepacia complex infection and not on the basis of transmissibility marker-positive B . cenocepacia as previously suggested.

Appl Environ Microbiol, 2004 Jun, 70(6), 3246 - 52
Saturation mutagenesis of toluene ortho-monooxygenase of Burkholderia cepacia G4 for Enhanced 1-naphthol synthesis and chloroform degradation; Rui L et al.; Directed evolution of toluene ortho-monooxygenase (TOM) of Burkholderia cepacia G4 previously created the hydroxylase alpha-subunit (TomA3) V106A variant (TOM-Green) with increased activity for both trichloroethylene degradation (twofold enhancement) and naphthalene oxidation (six-times-higher activity) . In the present study, saturation mutagenesis was performed at position A106 with Escherichia coli TG1/pBS(Kan)TOMV106A to improve TOM activity for both chloroform degradation and naphthalene oxidation . Whole cells expressing the A106E variant had two times better naphthalene-to-1-naphthol activity than the wild-type cells (V(max) of 9.3 versus 4.5 nmol.min(-1).mg of protein(-1) and unchanged K(m)), and the regiospecificity of the A106E variant was unchanged, with 98% 1-naphthol formed, as was confirmed with high-pressure liquid chromatography . The A106E variant degrades its natural substrate toluene 63% faster than wild-type TOM does (2.12 +/- 0.07 versus 1.30 +/- 0.06 nmol.min(-1).mg of protein(-1) {mean +/- standard deviation}) at 91 microM and has a substantial decrease in regiospecificity, since o-cresol (50%), m-cresol (25%), and p-cresol (25%) are formed, in contrast to the 98% o-cresol formed by wild-type TOM . The A106E variant also has an elevated expression level compared to that of wild-type TOM, as evidenced by sodium dodecyl sulfate-polyacrylamide gel electrophoresis . Another variant, the A106F variant, has 2.8-times-better chloroform degradation activity based on gas chromatography (V(max) of 2.61 versus 0.95 nmol.min(-1).mg of protein(-1) and unchanged K(m)) and chloride release (0.034 +/- 0.002 versus 0.012 +/- 0.001 nmol.min(-1).mg of protein(-1)) . The A106F variant also was expressed at levels similar to those of wild-type TOM and 62%-better toluene oxidation activity than wild-type TOM (2.11 +/- 0.3 versus 1.30 +/- 0.06 nmol.min(-1).mg of protein(-1)) . A shift in regiospecificity of toluene hydroxylation was also observed for the A106F variant, with o-cresol (28%), m-cresol (18%), and p-cresol (54%) being formed . Statistical analysis was used to estimate that 292 colonies must be screened for a 99% probability that all 64 codons were sampled during saturation mutagenesis.

Appl Environ Microbiol, 2004 Jun, 70(6), 3222 - 31
Saturation mutagenesis of Burkholderia cepacia R34 2,4-dinitrotoluene dioxygenase at DntAc valine 350 for synthesizing nitrohydroquinone, methylhydroquinone, and methoxyhydroquinone; Keenan BG et al.; Saturation mutagenesis of the 2,4-dinitrotoluene dioxygenase (DDO) of Burkholderia cepacia R34 at position valine 350 of the DntAc alpha-subunit generated mutant V350F with significantly increased activity towards o-nitrophenol (47 times), m-nitrophenol (34 times), and o-methoxyphenol (174 times) as well as an expanded substrate range that now includes m-methoxyphenol, o-cresol, and m-cresol (wild-type DDO had no detectable activity for these substrates) . Another mutant, V350M, also displays increased activity towards o-nitrophenol (20 times) and o-methoxyphenol (162 times) as well as novel activity towards o-cresol . Products were synthesized using whole Escherichia coli TG1 cells expressing the recombinant R34 dntA loci from pBS(Kan)R34, and the initial rates of product formation were determined at 1 mM substrate by reverse-phase high-pressure liquid chromatography . V350F produced both nitrohydroquinone at a rate of 0.75 +/- 0.15 nmol/min/mg of protein and 3-nitrocatechol at a rate of 0.069 +/- 0.001 nmol/min/mg of protein from o-nitrophenol, 4-nitrocatechol from m-nitrophenol at 0.29 +/- 0.02 nmol/min/mg of protein, methoxyhydroquinone from o-methoxyphenol at 2.5 +/- 0.6 nmol/min/mg of protein, methoxyhydroquinone from m-methoxyphenol at 0.55 +/- 0.02 nmol/min/mg of protein, both methylhydroquinone at 1.52 +/- 0.02 nmol/min/mg of protein and 2-hydroxybenzyl alcohol at 0.74 +/- 0.05 nmol/min/mg of protein from o-cresol, and methylhydroquinone at 0.43 +/- 0.1 nmol/min/mg of protein from m-cresol . V350M produced both nitrohydroquinone at a rate of 0.33 nmol/min/mg of protein and 3-nitrocatechol at 0.089 nmol/min/mg of protein from o-nitrophenol, methoxyhydroquinone from o-methoxyphenol at 2.4 nmol/min/mg of protein, methylhydroquinone at 1.97 nmol/min/mg of protein and 2-hydroxybenzyl alcohol at 0.11 nmol/min/mg of protein from o-cresol . The DDO variants V350F and V350M also exhibited 10-fold-enhanced activity towards naphthalene (8 +/- 2.6 nmol/min/mg of protein), forming (1R,2S)-cis-1,2-dihydro-1,2-dihydroxynaphthalene . Hence, mutagenesis of wild-type DDO through active-site engineering generated variants with relatively high rates toward a previously uncharacterized class of substituted phenols for the nitroarene dioxygenases; seven previously uncharacterized substrates were evaluated for wild-type DDO, and four novel monooxygenase-like products were found for the DDO variants V350F and V350M (methoxyhydroquinone, methylhydroquinone, 2-hydroxybenzyl alcohol, and 3-nitrocatechol).

J Mol Biol, 2004 Jun 25, 340(1), 49 - 65
Burkholderia cenocepacia phage BcepMu and a family of Mu-like phages encoding potential pathogenesis factors; Summer EJ et al.; We have isolated BcepMu, a Mu-like bacteriophage whose host range includes human pathogenic Burkholderia cenocepacia (formally B . cepacia genomovar III) isolates, and determined its complete 36748 bp genomic sequence . Like enteric bacteriophage Mu, the BcepMu genomic DNA is flanked by variable host sequences, a result of transposon-mediated replication . The BcepMu genome encodes 53 proteins, including capsid assembly components related to those of Mu, and tail sheath and tube proteins related to those of bacteriophage P2 . Seventeen of the BcepMu genes were demonstrated to encode homotypic interacting domains by using a cI fusion system . Most BcepMu genes have close homologs to prophage elements present in the two published Salmonella typhi genomes, and in the database sequences of Photorhabdus luminescens, and Chromobacterium violaceum . These prophage elements, designated SalMu, PhotoMu and ChromoMu, respectively, are collinear with BcepMu through nearly their entire lengths and show only limited mosaicism, despite the divergent characters of their hosts . The BcepMu family of Mu-like phages has a number of notable differences from Mu . Most significantly, the critical left end region of BcepMu is inverted with respect to Mu, and the BcepMu family of transposases is clearly of a distinct lineage with different molecular requirements at the transposon ends . Interestingly, a survey of 33 B.cepacia complex strains indicated that the BcepMu prophage is widespread in human pathogenic B.cenocepacia ET12 lineage isolates, but not in isolates from the PHDC or Midwest lineages . Identified members of the BcepMu family all contain a gene possibly involved in bacterial pathogenicity, a homolog of the type-two-secretion component exeA, but only BcepMu also carries a lipopolysaccharide modification acyltransferase which may also contribute a pathogenicity factor.

FEMS Microbiol Lett, 2004 Jun 15, 235(2), 229 - 35
Sequence divergence in type III secretion gene clusters of the Burkholderia cepacia complex; Glendinning KJ et al.; The Burkholderia cepacia complex (BCC) comprises a group of bacteria associated with opportunistic infections, especially in cystic fibrosis patients . B . cenocepacia J2315, of the transmissible ET12 lineage, contains a type III secretion (TTS) gene cluster implicated in pathogenicity . PCR and hybridisation assays indicate that the TTS gene cluster is present in all members of the BCC except B . cepacia (formerly genomovar I) . The TTS gene clusters of B . cenocepacia J2315 and B . multivorans are similar in organisation but have variable levels of gene identity . Nucleotide sequence data obtained for the equivalent region of the B . cepacia genome indicate the absence of TTS structural genes due to a rearrangement likely to involve more than one step.

J Bacteriol, 2004 Jun, 186(12), 3938 - 50
Genomic diversity of Burkholderia pseudomallei clinical isolates: subtractive hybridization reveals a Burkholderia mallei-specific prophage in B . pseudomallei 1026b; DeShazer D; Burkholderia pseudomallei is the etiologic agent of the disease melioidosis and is a category B biological threat agent . The genomic sequence of B . pseudomallei K96243 was recently determined, but little is known about the overall genetic diversity of this species . Suppression subtractive hybridization was employed to assess the genetic variability between two distinct clinical isolates of B . pseudomallei, 1026b and K96243 . Numerous mobile genetic elements, including a temperate bacteriophage designated phi1026b, were identified among the 1026b-specific suppression subtractive hybridization products . Bacteriophage phi1026b was spontaneously produced by 1026b, and it had a restricted host range, infecting only Burkholderia mallei . It possessed a noncontractile tail, an isometric head, and a linear 54,865-bp genome . The mosaic nature of the phi1026b genome was revealed by comparison with bacteriophage phiE125, a B . mallei-specific bacteriophage produced by Burkholderia thailandensis . The phi1026b genes for DNA packaging, tail morphogenesis, host lysis, integration, and DNA replication were nearly identical to the corresponding genes in phiE125 . On the other hand, phi1026b genes involved in head morphogenesis were similar to head morphogenesis genes encoded by Pseudomonas putida and Pseudomonas aeruginosa bacteriophages . Consistent with this observation, immunogold electron microscopy demonstrated that polyclonal antiserum against phiE125 reacted with the tail of phi1026b but not with the head . The results presented here suggest that B . pseudomallei strains are genetically heterogeneous and that bacteriophages are major contributors to the genomic diversity of this species . The bacteriophage characterized in this study may be a useful diagnostic tool for differentiating B . pseudomallei and B . mallei, two closely related biological threat agents.

J Bacteriol, 2004 Jun, 186(12), 3826 - 36
Genetic characterization of a multicomponent signal transduction system controlling the expression of cable pili in Burkholderia cenocepacia; Tomich M et al.; Cable pili are peritrichous organelles expressed by certain strains of Burkholderia cenocepacia, believed to facilitate colonization of the lower respiratory tract in cystic fibrosis patients . The B . cenocepacia cblBACDS operon encodes the structural and accessory proteins required for the assembly of cable pili, as well as a gene designated cblS, predicted to encode a hybrid sensor kinase protein of bacterial two-component signal transduction systems . In this study we report the identification of two additional genes, designated cblT and cblR, predicted to encode a second hybrid sensor kinase and a response regulator, respectively . Analyses of the deduced amino acid sequences of the cblS and cblT gene products revealed that both putative sensor kinases have transmitter and receiver domains and that the cblT gene product has an additional C-terminal HPt domain . Mutagenesis of the cblS, cblT, or cblR gene led to a block in expression of CblA, the major pilin subunit, and a severe decrease in cblA transcript abundance . Using transcriptional fusion analyses, the decrease in the abundance of the cblA transcript in the cblS, cblT, and cblR mutants was shown to be due to a block in transcription from the cblB-proximal promoter, located upstream of the cblBACDS operon . Furthermore, ectopic expression of either cblS or cblR in wild-type B . cenocepacia strain BC7 led to a significant increase, while ectopic expression of cblT resulted in a dramatic decrease, in abundance of the CblA major pilin and the cblA transcript . Our results demonstrate that the B . cenocepacia cblS, cblT, and cblR genes are essential for cable pilus expression and that their effect is exerted at the level of transcription of the cblBACDS operon . These findings are consistent with the proposed function of the cblSTR gene products as a multicomponent signal transduction pathway controlling the expression of cable pilus biosynthetic genes in B . cenocepacia.

Ann Clin Microbiol Antimicrob . 2004 Jun 2;3(1):8.
Infection control and the significance of sputum and other respiratory secretions from adult patients with cystic fibrosis; Moore JE et al.; BACKGROUND: There is limited data available on the environmental and public health impact of the microbiological hazards associated with sputa from patients with cystic fibrosis {CF} . Pseudomonas aeruginosa, Burkholderia cenocepacia (formerly Burkholderia cepacia genomovar III), Staphylococcus aureus and Stenotrophomonas maltophilia are bacterial pathogens which are commonly found in the sputum of CF patients . A study was performed to ascertain the amount of sputum produced relating to microbial loading, as well as the diversity of bacteria present in a population of adult patients, with particular attention to pathogenic organisms . METHODS: Sputum from adult {>18 years old} CF patients {n = 20}, chosen randomly from a population of 138 CF patients, was collected over a 24 h period on admission to the in-patient CF unit and enumerated quantitatively, as well as the sputa from 138 adult CF patients was examined qualitatively for the presence of infecting microflora . In addition, all different phenotypes from the sputum of each patient were identified phenotypically employing a combination of conventional identification methods {e.g . oxidase}, as well as the API Identification schemes {API 20 NE, API 20 E} . RESULTS: This study demonstrated that patients with cystic fibrosis generate large numbers of bacteria in their sputum, approximating to 109 organisms per patient per day . Although these organisms are introduced to the environment from the respiratory tract mainly via sputum, relatively few represent true bacterial pathogens and therefore are not clinically important to the general public who are immunocompotent . The greatest risk of such environmental microbial loading is to other patients with CF and therefore CF patients should be made aware of the hazards of acquiring such organisms from the environment, as well as socializing with other CF patients with certain transmissible types, such as Pseudomonas aeruginosa and Burkholderia cenocepacia . CONCLUSIONS: Environmental health professionals should therefore be aware that CF patients are a greater risk to their peer grouping rather than to the general public or health care workers and that good personal hygiene practices with CF patients should be encouraged to minimize environmental contamination and potential acquistion.

Rapid Commun Mass Spectrom, 2004, 18(12), 1341 - 4
Direct analysis of selected N-acyl-L-homoserine lactones by gas chromatography/mass spectrometry; Cataldi TR et al.; A rapid, simple and selective method involving direct separation by gas chromatography (GC) with electron ionization mass spectrometry (EI-MS) was employed to determine some N-acylhomoserine lactones (AHLs) . Using GC/EI-MS, simultaneous separation and characterization of AHLs were possible without prior derivatization . Informative fragmentation patterns were obtained to identify the structures of N-acyl chains of AHLs . Electron ionization resulted in a common fragmentation pattern with the most abundant ion at m/z 143 and other minor peaks at m/z 71, 57, and 43 . The presence of AHLs in extracts of Burkholderia cepacia strains was achieved in selected ion monitoring mode by using the prominent fragment at m/z 143 .

Thorax, 2004 Jun, 59(6), 526 - 8
Prevalence and clonality of Burkholderia cepacia complex genomovars in UK patients with cystic fibrosis referred for lung transplantation; De Soyza A et al.; BACKGROUND: It has previously been reported that patients infected with Burkholderia cenocepacia (genomovar III) before lung transplantation have a poorer outcome than those with other B . cepacia complex infections . METHODS: An extensive study was conducted to determine the prevalence and clonality of B . cepacia complex genomovars isolated from patients referred for transplant assessment between 1989 to the present and, where appropriate, whether strain type was related to transplant outcome . RESULTS: Isolates from 29 patients were identified as B . cepacia complex organisms by molecular analysis . Thirteen patients (45%) were infected with the highly transmissible ET-12 strain of B . cenocepacia recA lineage III-A, while all remaining patients were infected with genetically unique B . cenocepacia, B . multivorans, and B . vietnamiensis strains . All previously reported deaths following transplantation were associated with ET-12 infection . CONCLUSIONS: The ET-12 strain is the predominant cause of B . cenocepacia infections in patients with cystic fibrosis referred to our pulmonary transplant centre and is associated with poor transplant outcomes using standard treatment regimens.

Can J Microbiol, 2003 Dec, 49(12), 781 - 7
Identification of indole-3-acetic acid producing freshwater wetland rhizosphere bacteria associated with Juncus effusus L; Halda-Alija L; Production of indole-3-acetic acid (IAA), a key physiological feature of culturable, O2-tolerant bacteria associated with the freshwater macrophyte Juncus effusus L., was examined over a period of 2 years . Up to 74% of rhizobacteria identified and tested produced IAA . The number of indoleacetic acid producers decreased in winter . IAA was produced even when L-tryptophan, a precursor of IAA, was not added to the medium . Most of the IAA-producing strains were dominated by strains that were not identifiable to species level on the basis of API testing . Based on 16S rRNA gene sequencing and fatty acid analysis, it was found that IAA-producing rhizosphere bacteria associated with the freshwater wetland plant Juncus effusus L . are representatives of several families, including the Enterobacteriaceae, Pseudomonadaceae, Aeromonadaceae, Burkholderiaceae, and Bacillaceae . This study identifies numerous potentially important bacterial physiological groups of freshwater wetlands . Additionally, the study provides a baseline for monitoring and assessing the mutualistic relationships of wetland plants with rhizosphere bacteria in freshwater wetlands.

Water Res, 2004 May, 38(10), 2529 - 36
The use of isotopic and lipid analysis techniques linking toluene degradation to specific microorganisms: applications and limitations; Fang J et al.; Phospholipid fatty acid (PLFA) analysis combined with (13)C-labeled tracers has been used recently as an environmental forensics tool to demonstrate microbial degradation of pollutants . This study investigated the effectiveness and limitations of this approach, applied to the biodegradation of toluene by five reference strains that express different aerobic toluene degradation pathways: Pseudomonas putida mt-2, P . putida F1, Burkholderia cepacia G4, B . pickettii PKO1, and P . mendocina KR1 . The five strains were grown on mineral salts base medium amended with either 10 mM natural or {(13)C-ring}-labeled toluene . PLFA analysis showed that all five strains incorporated the toluene carbon into membrane fatty acids, as demonstrated by increases in the mass of fatty acids and their mass-spectrometry fragments for cells grown on (13)C-labeled toluene . Because of its ubiquitous presence and high abundance in bacteria, C16:0 fatty acid might be a useful biomarker for tracking contaminant degradation and (13)C flow . On the other hand, the (13)C-label (which was supplied at relatively high concentrations) generally exerted an inhibitory effect on fatty acid biosynthesis . Differences in fatty acid concentrations between cells grown on natural versus (13)C-labeled toluene would affect the interpretation of lipid profiles for microbial community analysis as indicated by principal component analysis of fatty acids . Therefore, caution should be exercised in linking lipid data with microbial population shifts in biodegradation experiments with (13)C-labeled tracers.

Am J Trop Med Hyg, 2004 May, 70(5), 580 - 2
C-reactive protein in the diagnosis of melioidosis; Cheng AC et al.; Previous work suggested that C-reactive protein (CRP) may be a useful test in the diagnosis of melioidosis, the infection caused by Burkholderia pseudomallei . We reviewed patients with culture-confirmed melioidosis to define the role of this inflammatory marker in the diagnosis of melioidosis . In 175 patients, we found that the admission CRP level may be normal or only mildly elevated, including patients with severe sepsis, fatal cases, and in relapsed melioidosis . In a multivariate analysis, sepsis and bacteremia were more strongly associated with mortality than CRP . Admission levels of CRP are not a sensitive marker for the presence of melioidosis and a normal level cannot be used to exclude acute, chronic, or relapsed melioidosis in febrile patients in or from endemic regions.

Assist Inferm Ric, 2004 Jan-Mar, 23(1), 14 - 20
{Prevention and control of respiratory tract infections in the network of Italian Centers for Cystic Fibrosis}; Festini F et al.; Infections caused by respiratory pathogens such as Burkholderia cepacia and Pseudomonas aeruginosa are associated with an increased morbidity and mortality in people affected by cystic fibrosis, the most common lethal genetic disease in Caucasian populations . Preventing the acquisition of these pathogens is paramount for these patients . The goal of this survey was to assess the distribution and the prevalence of the measures adopted for the prevention and control of infections caused by respiratory pathogens in the 28 italian centres for cystic fibrosis . 21 questionnaires were returned and some important differences can be observed in the adoption of segregation measures . Although results may be influenced by other factors, specific segregation policies appear to be more directly associated than other measures (e.g., intensive disinfection; behavioural rules to minimise patient' contacts) with lower prevalence of Pseudomonas aeruginosa (OR 0.36 CI95% 0.31-0.42), of multidrug-resistant Pseudomonas aeruginosa (OR 0.30 CI95% 0.22-0.40), and of methicillin-resistant Staphylococcus aureus (OR 0.67 CI95% 0.48-0.94).

Int J Med Microbiol, 2004 Apr, 293(7-8), 549 - 55
Exploitation of host cells by Burkholderia pseudomallei; Stevens MP et al.; Intracellular bacterial pathogens have evolved mechanisms to enter and exit eukaryotic cells using the power of actin polymerisation and to subvert the activity of cellular enzymes and signal transduction pathways . The proteins deployed by bacteria to subvert cellular processes often mimic eukaryotic proteins in their structure or function . Studies on the exploitation of host cells by the facultative intracellular pathogen Burkholderia pseudomallei are providing novel insights into the pathogenesis of melioidosis, a serious invasive disease of animals and humans that is endemic in tropical and subtropical areas . B . pseudomallei can invade epithelial cells, survive and proliferate inside phagocytes, escape from endocytic vesicles, form actin-based membrane protrusions and induce host cell fusion . Here we review current understanding of the molecular mechanisms underlying these processes.

Methods Mol Biol, 2004, 266, 323 - 52
Exploring the concept of clonality in bacteria; Spratt BG; Isolates of bacterial species that are indistinguishable in genotype are assigned as a clone, with the implication that they are descended from the same recent ancestor . Clones are difficult to define with precision since bacteria are not truly asexual, and recombinational replacements result in diversification of the ancestral genotype of a clone, to produce a cluster of increasingly diverse genotypes (a clonal complex) . The rate at which clonal diversification occurs depends on the extent of recombination, which varies among bacteria, so that some species have rather stable clones (e.g., Salmonella enterica), whereas in other species (e.g., Helicobacter pylori) clones may be so transient that they cannot readily be discerned . Clones and clonal complexes need to be assigned by indexing genetic variation that is selectively neutral, and currently this is best achieved using multilocus sequence typing . Some species of bacterial pathogens are very diverse, whereas others are genetically uniform, and some are, in essence, a single clone of a mother species that has been raised to species status due to the distinctiveness of the disease it causes (e.g., Yersinia pestis, Salmonella typhi, or Burkholderia mallei) . The population structures of bacteria depend on the rate of recombination, and comparative measures of the extent of recombination during clonal diversification can be obtained from multilocus sequence typing data, as can measures of the longer-term impact of recombination . These studies show a wide range of recombination rates among bacterial species, and indicate that recombination in many bacteria has been sufficiently extensive that a reliable evolutionary history of the species cannot be inferred.

BMC Microbiol . 2004 May 17;4(1):21.
Multi-pathogens sequence containing plasmids as positive controls for universal detection of potential agents of bioterrorism; Charrel RN et al.; BACKGROUND: The limited circulation of many of the agents that are likely to be used in a bioterrorism attack precludes the ready availability of positive controls . This means that only specialized laboratories can screen for the presence of these agents by nucleic amplification assays . Calibrated controls are also necessary for quantitative measurements . Primers and probes to be used in both conventional and real-time PCR assays were designed for the detection of agents likely to be used by a bioterrorist . Three plasmids, each of which contains 4 to 6 specific sequences from agents on the CDC Category A and B list (excluding RNA viruses) were constructed . Two plasmids incorporate the sequences of Category A and B agents, respectively . The third plasmid incorporates sequences from Variola major and organisms that cause rash-like illnesses that may be clinically confused with smallpox . An "exogenic sequence", introducing a NotI restriction site was incorporated in the native sequences of the bioterrorism agents inserted in plasmids . The designed molecular system for detection of bioterrorism agents was tested on each of these agents (except Monkeypox virus, Smallpox virus and 2 Burkholderia species for which no native DNA was available) and a collection of 50 isolates of C . burnetii using constructed plasmids as positive controls . RESULTS: Designed primers and probes allowed molecular detection, in either single or multiplex assays, of agent-specific targets with analytical sensitivities of between 1 and 100 DNA copies . The plasmids could be used as positive controls . False-positive results due to contamination by the positive control were easily detected by sequencing and eliminated by digestion with NotI . CONCLUSION: Plasmid A and B can be used as positive controls in molecular assays for the detection of bioterrorism agents in clinical specimens or environmental samples . Plasmid C can be used as a positive control in differentiation of vesicular rashes . It is also possible to avoid or to ensure immediate detection of false positive results due to contamination by positive controls using these plasmids . These plasmids and the corresponding primers and probes are immediately available for all clinical microbiology laboratories provided they have molecular amplification equipment.

Int J Syst Evol Microbiol, 2004 May, 54(Pt 3), 857 - 64
Collimonas fungivorans gen . nov., sp . nov., a chitinolytic soil bacterium with the ability to grow on living fungal hyphae; de Boer W et al.; A polyphasic approach was used to describe the phylogenetic position of 22 chitinolytic bacterial isolates that were able to grow at the expense of intact, living hyphae of several soil fungi . These isolates, which were found in slightly acidic dune soils in the Netherlands, were strictly aerobic, Gram-negative rods . Cells grown in liquid cultures were flagellated and possessed pili . A wide range of sugars, alcohols, organic acids and amino acids could be metabolized, whereas several di- and trisaccharides could not be used as substrates . The major cellular fatty acids were C(16 : 0), C(16 : 1)omega7c and C(18 : 1)omega7c . DNA G+C contents were 57-62 mol% . Analysis of nearly full-length 16S rDNA sequences showed that the isolates were related closely to each other (>98.6 % sequence similarity) and could be assigned to the beta-Proteobacteria, family 'Oxalobacteraceae', order 'Burkholderiales' . The most closely related species belonged to the genera Herbaspirillum and Janthinobacterium, exhibiting 95.9-96.7 % (Herbaspirillum species) and 94.3-95.6 % (Janthinobacterium species) 16S rDNA sequence similarity to the isolates . Several physiological and biochemical properties indicated that the isolates could be distinguished clearly from both of these genera . Therefore, it is proposed that the isolates described in this study are representatives of a novel genus, Collimonas gen . nov . Genomic fingerprinting (BOX-PCR), detailed analysis of 16S rDNA patterns and physiological characterization (Biolog) of the isolates revealed the existence of four subclusters . The name Collimonas fungivorans gen . nov., sp . nov . has been given to one subcluster (four isolates) that appears to be in the centre of the novel genus; isolates in the other subclusters have been tentatively named Collimonas sp . The type strain of Collimonas fungivorans gen . nov., sp . nov . is Ter6(T) (=NCCB 100033(T)=LMG 21973(T)).

Int J Syst Evol Microbiol, 2004 May, 54(Pt 3), 689 - 91
Proposal to accommodate Burkholderia cepacia genomovar VI as Burkholderia dolosa sp . nov; Vermis K et al.; Phenotypic and genotypic studies revealed new tools for differentiating Burkholderia cepacia genomovar VI from Burkholderia multivorans and other B . cepacia-complex species . Hence, the name Burkholderia dolosa sp . nov . is proposed, with LMG 18943(T) (=CCUG 47727(T)) as the type strain . B . dolosa can be differentiated from other B . cepacia-complex bacteria by its inability to assimilate tryptamine, azelaic acid and salicin and by its failure to grow on the B . cepacia-selective medium PCAT . Both 16S rDNA and recA RFLP analysis revealed unique B . dolosa restriction patterns . In addition, new 16S rDNA- and recA-based PCR assays allowed its specific identification.

Res Microbiol, 2004 May, 155(4), 238 - 44
Quorum sensing in the Burkholderia cepacia complex; Venturi V et al.; Quorum sensing is a cell-density-dependent regulatory mechanism which, in Gram-negative bacteria, usually involves the production and detection of N-acyl homoserine lactones (HSLs) . In the last four years HSL-dependent quorum sensing has been identified in members of the Burkholderia cepacia complex, and this mini-review summarizes initial findings and discusses future perspectives.

J Appl Microbiol, 2004, 96(6), 1306 - 16
Cloning and expression of a novel esterase gene cpoA from Burkholderia cepacia; Kim CH et al.; AIMS: To screen and clone a novel enzyme with specific activity for the resolution of (R)-beta-acetylmercaptoisobutyrate (RAM) from (R,S)-beta-acetylmercaptoisobutyrate {(R,S)-ester} . METHODS AND RESULTS: A micro-organism that produces a novel esterase was isolated and identified as the bacterium Burkholderia cepacia by using the analysis of cellular fatty acids, Biolog automated microbial identification/characterization system, and 16S rRNA gene sequence analysis . A novel esterase gene was cloned from the chromosomal DNA of B . cepacia and was designated as cpoA . The cpoA encodes a polypeptide of 273 amino acids which shows a strong sequence homology with many bacterial nonhaeme chloroperoxidases . In addition, a typical serine-hydrolase motif, Gly-X-Ser-X-Gly, and the highly conserved catalytic triad, Ser95, Asp224, and His253, were identified in the deduced amino acid sequence of cpoA by multiple sequence alignment . CONCLUSION: The cpoA cloned from B . cepacia encodes a novel esterase which is highly related to the nonhaeme chloroperoxidases . SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first report that describes the isolation and cloning of a serine esterase gene from B . cepacia, which is useful in the chiral resolution of (R,S)-ester . The cloned gene will allow additional research on the bifunctionality of the enzyme with esterase and chloroperoxidase activity at the structural and functional levels.

Arch Pediatr, 2004 Apr, 11(4), 360 - 6
{Epidemiology of infections associated to "Burkholderia cepacia complex" in the course of cystic fibrosis}; Meghdas I et al.; Within a few years bacteriological knowledge on Burkholderia cepacia species has progressed considerably . Within bacterial classification (taxonomy), B . cepacia gathers eight species and one species on standby of nomenclature (genomovar VI); the whole of these species constitutes the "B . cepacia complex" or B . cepacia "sensu lato" and the denomination B . cepacia "sensu stricto" is attributed to the genomovar I . These new data call into question the knowledge on the clinic and the epidemiology of B . cepacia "sensu lato" infection in the course of cystic fibrosis . Among these newly described species, B . cenocepacia (formerly genomovar III) and B . multivorans (formerly genomovar II) are the most frequent species and together they represent more than 90% of infections associated to "B . cepacia complex" in the course of cystic fibrosis . B . cenocepacia is often associated to the "cepacia syndrome" which is characterized as a fatal necrotizing pneumonia with bacteremia . The progress of molecular epidemiology allowed the description of bacterial clones of which some are highly transmissible from person-to-person . Their distribution varies according to the species and the geography . The identification of these new species appears particularly difficult and, by the fact, the data on taxonomy and molecular epidemiology can be provided only by highly specialized reference centers.

Toxicology, 2004 May 20, 198(1-3), 229 - 38
Microbial contamination of drinking water and disease outcomes in developing regions; Ashbolt NJ; Drinking water is a major source of microbial pathogens in developing regions, although poor sanitation and food sources are integral to enteric pathogen exposure . Gastrointestinal disease outcomes are also more severe, due to under-nutrition and lack of intervention strategies in these regions . Poor water quality, sanitation and hygiene account for some 1.7 million deaths a year world-wide (3.1% of all deaths and 3.7% of all DALY's), mainly through infectious diarrhoea . Nine out of 10 such deaths are in children and virtually all of the deaths are in developing countries . Major enteric pathogens in these children include: rotavirus, Campylobacter jejuni, enterotoxigenic Escherichia coli, Shigella spp . and Vibrio cholerae O1, and possibly enteropathogenic E . coli, Aeromonas spp . V . cholerae O139, enterotoxigenic Bacteroides fragilis, Clostridium difficile and Cryptosporidium parvum . All except the latter are easily control by chlorination of water, but recontamination of treated water is a huge problem . Emerging environmental pathogens, such as Helicobacter pylori and Burkholderia pseudomallei, may well be of significance in some regions . In adults, much less is understood of various sequellae such as myocarditis, diabetes, reactive arthritis and cancers some months-years after initial infections . So in addition to the traditional pathogens (helminths, Entamoeba histolytica, Giardia lamblia hepatitis A and E) various enteroviruses, C . jejuni and H . pylori are emerging issues in adults.

FEMS Microbiol Lett, 2004 May 15, 234(2), 281 - 7
Age influences resistance of Caenorhabditis elegans to killing by pathogenic bacteria; Laws TR et al.; Caenorhabditis elegans has previously been proposed as an alternative host for models of infectious disease caused by human pathogens . When exposed to some human pathogenic bacteria, the life span of nematodes is significantly reduced . We have shown that mutations in the age-1, and/or age-2 genes of C . elegans, that normally enhance life expectancy, can also increase resistance to killing by the bacterial pathogens Pseudomonas aeruginosa, Salmonella enterica var . Typhimurium, Burkholderia cepacia or Yersinia pseudotuberculosis . We also found that the rate at which wild-type C . elegans was killed by the bacterial pathogens tested increased as nematodes aged . In the case of P . aeruginosa infection, the difference in life span of wild type and age-1 mutants of C . elegans was not due to differences in the level of bacterial colonisation of the gut .

J Clin Microbiol, 2004 May, 42(5), 2239 - 40
Further evaluation of a rapid diagnostic test for melioidosis in an area of endemicity; O'Brien M et al.; Immunochromatographic test (ICT) kits for the rapid detection of immunoglobulin G (IgG) and IgM antibodies to Burkholderia pseudomallei were compared to the indirect hemagglutination (IHA) assay . In 138 culture-confirmed melioidosis cases, sensitivities were 80, 77, and 88% for IHA, ICT IgG, and ICT IgM, respectively . In a prospective study of 160 consecutive sera samples sent for melioidosis serology, respective specificities were 91, 90, and 69, positive predictive values were 41, 32, and 18, and negative predictive values were 99, 98, and 100% . ICT IgM kits are unreliable for diagnosis of melioidosis, but ICT IgG kits may be useful for diagnosing travelers presenting with possible melioidosis who return from regions where melioidosis is endemic.

J Clin Microbiol, 2004 May, 42(5), 2227 - 30
Outbreak of Burkholderia cepacia bacteremia in a pediatric hospital due to contamination of lipid emulsion stoppers; Doit C et al.; We describe a 7-month outbreak of nosocomial Burkholderia cepacia bacteremia involving eight children in a pediatric hospital and the results of epidemiological investigations . A B . cepacia strain genotypically identical to the blood isolates was recovered from the upper surface of capped rubber stoppers of bottles of a commercial lipid emulsion used for parenteral nutrition.

Clin Infect Dis, 2004 May 1, 38(9), 1243 - 50 Epub 2004 Apr 15.
Virulence associated with outbreak-related strains of Burkholderia cepacia complex among a cohort of patients with bacteremia; Woods CW et al.; The Burkholderia cepacia complex includes 9 genomovars . The relative virulence of each is unknown . Host and pathogen features associated with mortality were evaluated among patients with B . cepacia complex bacteremia . Cases were ascertained through review of blood culture results for the period of May 1996 through May 2002 . Isolates were identified to species level with 16S rDNA and recA-based species-specific polymerase chain reaction analyses and recA restriction fragment-length polymorphism . Strain typing was performed with pulsed-field gel electrophoresis . Fifty-three patients with B . cepacia complex bacteremia were identified; only 9 (17%) had cystic fibrosis . Twenty-five patients (47%) died within 14 days of bacteremia . After controlling for comorbid conditions and therapeutic interventions, 2 outbreak-related strains of Burkholderia cenocepacia (genomovar III) were associated with 14-day mortality (odds ratio, 5.5; 95% confidence interval, 1.20-25.02) . B . cenocepacia is an emerging nosocomial pathogen . Certain strains are associated with an enhanced capacity for interpatient spread and poor outcome.

J Bacteriol, 2004 May, 186(10), 3117 - 23
Toluene 3-monooxygenase of Ralstonia pickettii PKO1 is a para-hydroxylating enzyme; Fishman A et al.; Oxygenases are promising biocatalysts for performing selective hydroxylations not accessible by chemical methods . Whereas toluene 4-monooxygenase (T4MO) of Pseudomonas mendocina KR1 hydroxylates monosubstituted benzenes at the para position and toluene ortho-monooxygenase (TOM) of Burkholderia cepacia G4 hydroxylates at the ortho position, toluene 3-monooxygenase (T3MO) of Ralstonia pickettii PKO1 was reported previously to hydroxylate toluene at the meta position, producing primarily m-cresol (R . H . Olsen, J . J . Kukor, and B . Kaphammer, J . Bacteriol . 176:3749-3756, 1994) . Using gas chromatography, we have discovered that T3MO hydroxylates monosubstituted benzenes predominantly at the para position . TG1/pBS(Kan)T3MO cells expressing T3MO oxidized toluene at a maximal rate of 11.5 +/- 0.33 nmol/min/mg of protein with an apparent Km value of 250 microM and produced 90% p-cresol and 10% m-cresol . This product mixture was successively transformed to 4-methylcatechol . T4MO, in comparison, produces 97% p-cresol and 3% m-cresol . Pseudomonas aeruginosa PAO1 harboring pRO1966 (the original T3MO-bearing plasmid) also exhibited the same product distribution as that of TG1/pBS(Kan)T3MO . TG1/pBS(Kan)T3MO produced 66% p-nitrophenol and 34% m-nitrophenol from nitrobenzene and 100% p-methoxyphenol from methoxybenzene, as well as 62% 1-naphthol and 38% 2-naphthol from naphthalene; similar results were found with TG1/pBS(Kan)T4MO . Sequencing of the tbu locus from pBS(Kan)T3MO and pRO1966 revealed complete identity between the two, thus eliminating any possible cloning errors . 1H nuclear magnetic resonance analysis confirmed the structural identity of p-cresol in samples containing the product of hydroxylation of toluene by pBS(Kan)T3MO.

Scand J Infect Dis, 2004, 36(3), 174 - 8
Investigation of Burkholderia cepacia nosocomial outbreak with high fatality in patients suffering from diseases other than cystic fibrosis; Shehabi AA et al.; Over a 1-y period, 26 inpatients at the Jordan University Hospital in Amman were detected with bacteraemia (23 cases) or respiratory tract colonized with B . cepacia (3 cases) . A combination of genetic identification and molecular typing has proved that all cases were caused by a single epidemic strain of B . cepacia genomovar IIIa . Nosocomial infections could be documented in 21/26 (81%) patients, mostly with severe underlying or malignant diseases other than cystic fibrosis, but the source of infection was undetected . The overall mortality related to infection with B . cepacia was 42% . All B . cepacia isolates were resistant to ampicillin, amikacin, carbenicillin and gentamicin; and mostly susceptible to piperacillin, chloramphenicol, cotri-moxazole, tetracycline, ceftazidime, and tazocin (62-88%) . This study demonstrates the nosocomial and high fatality of B . cepacia genomovar IIIa in Jordanian patients suffering from diseases other than cystic fibrosis.

J Thorac Cardiovasc Surg, 2004 May, 127(5), 1493 - 501
Twenty-year experience of lung transplantation at a single center: Influence of recipient diagnosis on long-term survival; de Perrot M et al.; OBJECTIVES: The objective of this study was to examine the long-term patient outcomes of lung transplantation in a single center . METHODS: Between 1983 and 2003, 521 lung transplants were performed in 501 patients . Major indications were cystic fibrosis (n = 124), chronic obstructive pulmonary disease (n = 88), alpha-1 antitrypsin deficiency (n = 63), pulmonary fibrosis (n = 97), primary pulmonary hypertension (n = 35), Eisenmenger syndrome (n = 21), and miscellaneous end-stage lung diseases (n = 93) . RESULTS: The 5-, 10-, and 15-year survivals for all recipients were 55.1% (95% confidence interval: +/-5%), 35.3% (+/-6%), and 26.5% (+/-11%), respectively . The most common causes of death were sepsis and bronchiolitis obliterans syndrome . Despite an increased postoperative mortality rate, patients with primary pulmonary hypertension achieved the best long-term survival (10-year survival: 59%) . Recipients with cystic fibrosis without Burkholderia cepacia infection achieved significantly better long-term survival (10-year survival: 52%) than those with Burkholderia cepacia infection (10-year survival: 15%) . The 10-year survival was also significantly better in recipients with chronic obstructive pulmonary disease (43%) than in recipients with alpha-1 antitrypsin deficiency (23%) . Although the incidence of bronchiolitis obliterans syndrome was similar between recipients with chronic obstructive pulmonary disease (39%) and alpha-1 antitrypsin deficiency (46%), recipients with alpha-1 antitrypsin deficiency died of sepsis more frequently than recipients with chronic obstructive pulmonary disease (27% vs 6%, respectively; P =.0003) . CONCLUSIONS: Although bronchiolitis obliterans syndrome and sepsis still limit the durability of the benefit, lung transplantation returns many patients with end-stage lung disease to active and productive lives . Differences in the complications and long-term survival show the important contribution of the recipient diagnosis to the success of lung transplantation.

Indian J Med Res, 2004 Mar, 119(3), 101 - 6
Detection of virulence attributes of Burkholderia pseudomallei; Balaji V et al.; BACKGROUND & OBJECTIVES: Melioidosis caused by Burkholderia pseudomallei is an emerging disease in India . This study examined the toxin activity of bacteria-free culture filtrate in three different cell lines (cytotoxic assay) and its effect on Caenorhabditis elegans (nematode toxicity assay) . Endotoxic activity of the viable bacteria was also studied in C . elegans (co-culture killing assay) . METHODS: For toxin studies, serial doubling dilutions of unheated, heated crude and ultra filtrate of bacteria-free culture supernatants of B . pseudomallei were tested in 96-well microtitre plate containing confluent mono layers of McCoy, Hep-2 and HeLa cell lines . For the effects on C . elegans, the worms were exposed to heated and unheated bacteria-free culture supernatants in 24-well microtitre plate for 24h and then transferred to OP50 Escherichia coli lawn culture . The endotoxic activity of the live bacterium was studied by feeding the worms in the lawn culture of B . pseudomallei . RESULTS: All the clinical isolates (n=38) produced cytotoxic changes in all the cell lines . No difference was observed in the cytotoxicity of unheated, heated and ultra-filtered culture supernatant . The septicaemic isolates were observed to produce cytotoxic changes in high dilutions (1:160) of culture filtrate . None of the unheated and heated crude filtrate had deleterious effect on C . elegans, while all the live bacteria were found to be lethal to the nematode . INTERPRETATION & CONCLUSION: The culture supernatants, though produced cytopathic effect in various tissue cultures, failed to have any deleterious effect on the worms . However, live bacteria were lethal to the worms B . pseudomallei . Use of C . elegans model to detect virulence attributes of B . pseudomallei is recommended as an alternative to tissue culture methods as this can be carried out in laboratories where a tissue culture set up is not available.

Southeast Asian J Trop Med Public Health, 2003 Dec, 34(4), 810 - 21
Searching for virulence Burkholderia pseudomallei genes by immunoscreening the lambda ZAPII expressed genomic library; Jitsurong S et al.; The lambdaZAP II expressed genomic library of B . pseudomallei was screened with pooled melioidosis serum preabsorbed with E . coli host cell . The positive clones were detected by using protein A-CDP-star chemiluminescence . All of 14 positive clones reacted with only the pooled absorbed melioidosis serum and not the pooled absorbed normal serum when tested with the plaque dot blot analysis . The expressed genes were detected by using a combination of immunoscreening, bioinformatics and molecular biology . At least six in vivo expressed genes were identified by this approach . Two were well known virulent genes, gmhA (a capsule biosynthetic gene) and bipD (type III secretion protein gene) . Another two were genes coded for conserved hypothetical protein . The last two isolated genes were groEL (a chaperonine protein gene), and a gene encoding transmembrane protein.

Pediatr Pulmonol, 2004 Jun, 37(6), 537 - 47
Identification of Pseudomonas aeruginosa, Burkholderia cepacia complex, and Stenotrophomonas maltophilia in respiratory samples from cystic fibrosis patients using multiplex PCR; da Silva Filho LV et al.; A multiplex PCR method was developed to identify P . aeruginosa, B . cepacia complex, and S . maltophilia directly in sputum and oropharyngeal samples from CF patients . One hundred and six patients (53 male, and 53 female) attending our pulmonology clinic were studied from September 2000-April 2001 . Two hundred and fifty-seven samples were cultured in selective media and submitted to multiplex PCR reactions, using three primer pairs targeting specific genomic sequences of each species, with an additional primer pair targeting a stretch of ribosomal 16S DNA, universal for bacteria, to act as a control . P . aeruginosa was isolated by culture in 56% of samples, B . cepacia complex in 4.3%, and S . maltophilia in 2.7%, while multiplex PCR identified P . aeruginosa in 78.7%, B . cepacia complex in 3.9%, and S . maltophilia in 3.1% of samples . Multiplex PCR results were verified by PCR reactions using different species-specific primers described in the literature and DNA sequencing of amplicons from a few samples . Comparing to culture results, the sensitivity and specificity values of multiplex PCR for bacterial identification were, respectively, 97.2% and 45.5% for P . aeruginosa, 45.5% and 97.9% for B . cepacia complex, and 40% and 97.6% for S . maltophilia . All 10 multiplex PCR-positive results for B . cepacia complex were confirmed using other species-specific primers described in the literature, while this approach confirmed results for S . maltophilia identification in 7/8 samples (87.5%) . Sequencing of amplicons from samples culture-negative but multiplex PCR-positive for P . aeruginosa and B . cepacia complex confirmed their identity, while minor nucleotide differences among amplicons ruled out the hypothesis of PCR contamination .

Theor Appl Genet, 2004 Jun, 109(1), 71 - 9 Epub 2004 Apr 28.
The Rxo1/ Rba1 locus of maize controls resistance reactions to pathogenic and non-host bacteria; Zhao BY et al.; Infiltration of different maize lines with a variety of bacterial pathogens of maize, rice and sorghum identified qualitative differences in resistant reactions . Isolates from two bacterial species induced rapid hypersensitive reactions (HR) in some maize lines, but not others . All isolates of the non-host pathogen Xanthomonas oryzae pv . oryzicola (bacterial leaf streak disease of rice) and some isolates of the pathogenic bacterium Burkholderia andropogonis induced HR when infiltrated into maize line B73, but not Mo17 . Genetic control of the HR to both bacteria segregated as a single dominant gene . Surprisingly, both phenotypes mapped to the same locus, indicating they are either tightly linked or controlled by the same gene . The locus maps on the short arm of maize chromosome six near several other disease-resistance genes . Results indicate the same type of genes may contribute to both non-host resistance and resistance to pathogens.

Infect Control Hosp Epidemiol, 2004 Apr, 25(4), 291 - 6
Burkholderia cepacia infections associated with intrinsically contaminated ultrasound gel: the role of microbial degradation of parabens; Hutchinson J et al.; OBJECTIVE: To describe an outbreak of serious nosocomial Burkholderia cepacia infections occurring after transrectal prostate biopsy associated with ultrasound gel intrinsically contaminated with paraben-degrading microorganisms . METHODS: A retrospective chart review prompted by a blood culture isolate of B . cepacia . Identification of microorganisms in ultrasound gel in two Canadian centers and characterization by pulsed-field gel electrophoresis and assays for paraben degradation . SETTING: Two Canadian university-affiliated, tertiary-care centers in Newfoundland and Alberta . RESULTS: Six serious B . cepacia infections were identified at the two centers . Isolates of B . cepacia recovered from the blood of patients from both centers and the ultrasound gel used during the procedures were identical, confirming intrinsic contamination . Strains of Enterobacter cloacae isolated from ultrasound gel at the two centers were also identical . The ability to degrade parabens was proven for both B . cepacia and E . cloacae strains recovered from the ultrasound gel . CONCLUSIONS: Ultrasound gel is a potential source of infection . Contamination occurs at the time of manufacture, with organisms that degrade parabens, which are commonly used as stabilizing agents . There are far-reaching implications for the infection control community.

Antimicrob Agents Chemother, 2004 May, 48(5), 1763 - 5
Outcomes of patients with melioidosis treated with meropenem; Cheng AC et al.; Melioidosis, an infection due to Burkholderia pseudomallei, is endemic in southeast Asia and northern Australia . We reviewed our experience with meropenem in the treatment of severe melioidosis in 63 patients over a 6-year period . Outcomes were similar to those of ceftazidime-treated patients (n = 153) despite a deliberate selection bias to more-unwell patients receiving meropenem . The mortality among meropenem-treated patients was 19% . One patient had a possible drug fever associated with the use of meropenem . We conclude that meropenem (1 g or 25 mg/kg every 8 h intravenously for >/=14 days) is an alternative to ceftazidime and imipenem in the treatment of melioidosis . The use of meropenem may be associated with improved outcomes in patients with severe sepsis associated with melioidosis.

Infect Immun, 2004 May, 72(5), 2850 - 7
Importance of the ornibactin and pyochelin siderophore transport systems in Burkholderia cenocepacia lung infections; Visser MB et al.; Previously, orbA, the gene encoding the outer membrane receptor for ferric-ornibactin, was identified in Burkholderia cenocepacia K56-2, a strain which produces ornibactin, salicylic acid, and negligible amounts of pyochelin . A K56-2 orbA mutant was less virulent than the parent strain in a rat agar bead infection model . In this study, an orbA mutant of B . cenocepacia Pc715j which produces pyochelin in addition to ornibactin and salicylic acid was constructed . The gene encoding the outer membrane receptor for ferric-pyochelin (fptA) was also identified . An fptA mutant was constructed in Pc715j and shown to be deficient in {(59)Fe}pyochelin uptake . A 75-kDa iron-regulated protein was identified in outer membrane preparations of Pc715j that was absent in outer membrane preparations of Pc715jfptA::tp . Pc715jfptA::tp and Pc715jorbA::tp produced smaller amounts of their corresponding siderophores . Both Pc715jorbA::tp and Pc715jfptA::tp were able to grow in iron starvation conditions in vitro . In the agar bead model, the Pc715jorbA::tp mutant was cleared from the lung, indicating that the pyochelin uptake system does not compensate for the absence of a functional ornibactin system . Pc715jfptA::tp persisted in rat lung infections in numbers similar to those of the parent strain, indicating that the ferric-ornibactin uptake system could compensate for the defect in ferric-pyochelin uptake in vivo . These studies suggest that the ornibactin uptake system is the most important siderophore-mediated iron transport system in B . cenocepacia lung infections.

Trans R Soc Trop Med Hyg, 2004 Jun, 98(6), 337 - 41
The effect of quicklime (calcium oxide) as an inhibitor of Burkholderia pseudomallei; Na-ngam N et al.; Measurement of in vitro activity of quicklime against Burkholderia pseudomallei revealed that quicklime at concentrations of 10% or more was bactericidal for up to 35 d . The effect of quicklime as an inhibitor of B . pseudomallei in soil from a rice field was studied in a laboratory setting . The soil, collected from a rice field in north-eastern Thailand, was mixed with B . pseudomallei . In experiment 1, quicklime was mixed with the soil in different amounts . In experiment 2, quicklime was spread over the soil surface . In experiment 3, quicklime solution was poured onto the soil . It was found that the pH of the soil in experiment 1 was much higher than that in experiments 2 and 3 . Only quicklime mixed with soil at a concentration of 40% or more (weight/weight) was effective in inhibiting the growth of B . pseudomallei for up to six weeks.

J Med Microbiol, 2004 May, 53(Pt 5), 389 - 98
Conservation of the opcL gene encoding the peptidoglycan-associated outer-membrane lipoprotein among representatives of the Burkholderia cepacia complex; Plesa M et al.; Members of the Burkholderia cepacia complex are Gram-negative beta-proteobacteria that are classified into nine genomic species or genomovars . Some representatives of this group of bacteria, such as Burkholderia multivorans (genomovar II) and Burkholderia cenocepacia (genomovar III), are considered to be dangerous pathogens for cystic fibrosis (CF) patients because of their capacity to colonize CF lungs . The opcL gene, which encodes the peptidoglycan-associated outer-membrane lipoprotein (PAL), was detected in the genome of Burkholderia sp . LB 400 by a similarity search that was based on the sequence of the Pseudomonas aeruginosa PAL, OprL . Primers that could amplify part of opcL from B . multivorans LMG 13010(T) were designed . This PCR fragment was used as a probe for screening of a B . multivorans genomic bank, allowing cloning of the complete opcL gene . The complete opcL gene could be PCR-amplified from DNA from all genomovars . The sequences of these opcL genes showed a high degree of conservation (> 95 %) among different species of the B . cepacia complex . OpcL protein that was purified from B . multivorans LMG 13010(T) was used to generate mouse polyclonal antisera against OpcL . The OpcL protein could be produced in Escherichia coli and detected in outer-membrane fractions by Western blot . Burkholderia cells were labelled by immunofluorescence staining using antibodies against OpcL, but only after treatment with EDTA and SDS . The opcL gene could be amplified directly from the sputa of 15 CF patients who were known to be colonized by B . cepacia; sequence data derived from the amplicons identified the colonizing strains as B . cenocepacia (genomovar III, n = 14) and B . multivorans (n = 1).

Clin Exp Immunol, 2004 May, 136(2), 277 - 83
Induction of iNOS expression and antimicrobial activity by interferon (IFN)-beta is distinct from IFN-gamma in Burkholderia pseudomallei-infected mouse macrophages; Utaisincharoen P et al.; Burkholderia pseudomallei is a causative agent of melioidosis . This Gram-negative bacterium is able to survive and multiple inside both phagocytic and nonphagocytic cells . We previously reported that exogenous interferons (both type I and type II) enhanced antimicrobial activity of the macrophages infected with B . pseudomallei by up-regulating inducible nitric oxide synthase (iNOS) . This enzyme thus plays an essential role in controlling intracellular growth of bacteria . In the present study we extended our investigation, analysing the mechanism(s) by which the two types of interferons (IFNs) regulate antimicrobial activity in the B . pseudomallei-infected macrophages . Mouse macrophage cell line (RAW 264.7) that was exposed simultaneously to B . pseudomallei and type I IFN (IFN-beta) expressed high levels of iNOS, leading to enhanced intracellular killing of the bacteria . However, neither enhanced iNOS expression nor intracellular bacterial killing was observed when the macrophages were preactivated with IFN-beta prior to being infected with B . pseudomallei . On the contrary, the timing of exposure was not critical for the type II IFN (IFN-gamma) because when the cells were either prestimulated or co-stimulated with IFN-gamma, both iNOS expression and intracellular killing capacity were enhanced . The differences by which these two IFNs regulate antimicrobial activity may be related to the fact that IFN-gamma was able to induce more sustained interferon regulatory factor-1 (IRF-1) expression compared with the cells activated with IFN-beta.

Microb Ecol, 2004 Jul, 48(1), 90 - 102 Epub 2004 Apr 19.
Chlorobenzoate-degrading bacteria in similar pristine soils exhibit different community structures and population dynamics in response to anthropogenic 2-, 3-, and 4-chlorobenzoate levels; Gentry TJ et al.; A study was conducted to determine the diversity of 2-, 3-, and 4-chlorobenzoate (CB) degraders in two pristine soils with similar physical and chemical characteristics . Surface soils were collected from forested sites and amended with 500 microg of 2-, 3-, or 4-CB g(-1) soil . The CB levels and degrader numbers were monitored throughout the study . Degraders were isolated, grouped by DNA fingerprints, identified via 16S rDNA sequences, and screened for plasmids . The CB genes in selected degraders were isolated and/or sequenced . In the Madera soil, 2-CB and 4-CB degraded within 11 and 42 d, respectively, but 3-CB did not degrade . In contrast, 3-CB and 4-CB degraded in the Oversite soil within 14 and 28 d, respectively, while 2-CB did not degrade . Approximately 10(7) CFU g(-1) of degraders were detected in the Madera soil with 2-CB, and the Oversite soil with 3- and 4-CB . No degraders were detected in the Madera soil with 4-CB even though the 4-CB degraded . Nearly all of the 2-CB degraders isolated from the Madera soil were identified as a Burkholderia sp . containing chromosomally encoded degradative genes . In contrast, several different 3- and 4-CB degraders were isolated from the Oversite soil, and their populations changed as CB degradation progressed . Most of these 3-CB degraders were identified as Burkholderia spp . while the majority of 4-CB degraders were identified as Bradyrhizobium spp . Several of the 3-CB degraders contained the degradative genes on large plasmids, and there was variation between the plasmids in different isolates . When a fresh sample of Madera soil was amended with 50, 100, or 200 microg 3-CB g(-1), 3-CB degradation occurred, suggesting that 500 microg 3-CB g(-1) was toxic to the degraders . Also, different 3-CB degraders were isolated from the Madera soil at each of the three lower levels of 3-CB . No 2-CB degradation was detected in the Oversite soil even at lower 2-CB levels . These results indicate that the development of 2-, 3-, and 4-CB degrader populations is site-specific and that 2-, 3-, and 4-CB are degraded by different bacterial populations in pristine soils . These results also imply that the microbial ecology of two soils that develop under similar biotic and abiotic environments can be quite different.

Nat Biotechnol, 2004 May, 22(5), 583 - 8 Epub 2004 Apr 11.
Engineered endophytic bacteria improve phytoremediation of water-soluble, volatile, organic pollutants; Barac T et al.; Phytoremediation of highly water soluble and volatile organic xenobiotics is often inefficient because plants do not completely degrade these compounds through their rhizospheres . This results in phytotoxicity and/or volatilization of chemicals through the leaves, which can cause additional environmental problems . We demonstrate that endophytic bacteria equipped with the appropriate degradation pathway improve the in planta degradation of toluene . We introduced the pTOM toluene-degradation plasmid of Burkholderia cepacia G4 into B . cepacia L.S.2.4, a natural endophyte of yellow lupine . After surface-sterilized lupine seeds were successfully inoculated with the recombinant strain, the engineered endophytic bacteria strongly degraded toluene, resulting in a marked decrease in its phytotoxicity, and a 50-70% reduction of its evapotranspiration through the leaves . This strategy promises to improve the efficiency of phytoremediating volatile organic contaminants.

J Clin Microbiol, 2004 Apr, 42(4), 1491 - 7
Epidemiology and clinical course of Burkholderia cepacia complex infections, particularly those caused by different Burkholderia cenocepacia strains, among patients attending an Italian Cystic Fibrosis Center; Manno G et al.; In this study, the epidemiology of Burkholderia cepacia complex (Bcc) recovered from the sputum of 75 patients attending the Genoa Cystic Fibrosis (CF) Center at the Gaslini Children's Hospital (Genoa, Italy) was investigated, and the clinical course of the CF patients infected with the different species and genomovars of Bcc was evaluated . All isolates were analyzed for genomovar status by recA gene polymorphism and subsequently random amplified polymorphic DNA fingerprinting . Burkholderia cenocepacia is the predominant species recovered from the CF patients infected with Bcc at the Genoa CF Center . Of the other eight species comprising the Bcc, only a few isolates belonging to B . cepacia genomovar I, Burkholderia stabilis, and Burkholderia pyrrocinia were found . Of the four recA lineages of B . cenocepacia, most patients were infected by epidemic strains belonging to lineages IIIA and IIID, whereas only a few patients harbored IIIB strains . Patient-to-patient spread of Bcc among CF patients was mostly associated with B . cenocepacia, in particular with strains belonging to recA lineages IIIA and IIID . The mortality of CF patients infected with Bcc at the Genoa CF Center was significantly higher than mortality among CF patients not infected with Bcc . All of the deaths were associated with the presence of B . cenocepacia, except the case of a patient infected with B . cepacia genomovar I . Within B . cenocepacia, infection with epidemic strains belonging to lineages IIIA and IIID was associated with higher rates of mortality than was infection with lineage IIIB strains . No significant differences in lung function, body weight, and mortality rate were observed between patients infected with epidemic strains belonging to either B . cenocepacia IIIA or B . cenocepacia IIID.

Biodegradation, 2004 Apr, 15(2), 111 - 23
Identification of two new sets of genes for dibenzothiophene transformation in Burkholderia sp . DBT1; Di Gregorio S et al.; A novel genotype for the initial steps of the oxidative degradation of dibenzothiophene (DBT) is described in a Burkholderia sp . strain isolated from a drain receiving oil refinery wastewater . The strain is capable of transforming DBT with significant efficiency when compared to other microorganisms . Its genotype was discovered by investigating insertional mutants of genes involved in DBT degradation by the Kodama pathway . The cloned dbt genes show a novel genomic organization when compared to previously described genes capable of DBT catabolism in that they constitute two distinct operons and are not clustered in a single transcript . Sequence analysis suggests the presence of a sigma54-dependent positive transcriptional regulator that may be involved in the control of the transcription of the two operons, both activated by DBT . The achieved results suggest the possibility of novel features of DBT biotransformation in nature.

Appl Environ Microbiol, 2004 Apr, 70(4), 2110 - 8
Root nodule Bradyrhizobium spp . harbor tfdAalpha and cadA, homologous with genes encoding 2,4-dichlorophenoxyacetic acid-degrading proteins; Itoh K et al.; The distribution of tfdAalpha and cadA, genes encoding 2,4-dichlorophenoxyacetate (2,4-D)-degrading proteins which are characteristic of the 2,4-D-degrading Bradyrhizobium sp . isolated from pristine environments, was examined by PCR and Southern hybridization in several Bradyrhizobium strains including type strains of Bradyrhizobium japonicum USDA110 and Bradyrhizobium elkanii USDA94, in phylogenetically closely related Agromonas oligotrophica and Rhodopseudomonas palustris, and in 2,4-D-degrading Sphingomonas strains . All strains showed positive signals for tfdAalpha, and its phylogenetic tree was congruent with that of 16S rRNA genes in alpha-Proteobacteria, indicating evolution of tfdAalpha without horizontal gene transfer . The nucleotide sequence identities between tfdAalpha and canonical tfdA in beta- and gamma-Proteobacteria were 46 to 57%, and the deduced amino acid sequence of TfdAalpha revealed conserved residues characteristic of the active site of alpha-ketoglutarate-dependent dioxygenases . On the other hand, cadA showed limited distribution in 2,4-D-degrading Bradyrhizobium sp . and Sphingomonas sp . and some strains of non-2,4-D-degrading B . elkanii . The cadA genes were phylogenetically separated between 2,4-D-degrading and nondegrading strains, and the cadA genes of 2,4-D degrading strains were further separated between Bradyrhizobium sp . and Sphingomonas sp., indicating the incongruency of cadA with 16S rRNA genes . The nucleotide sequence identities between cadA and tftA of 2,4,5-trichlorophenoxyacetate-degrading Burkholderia cepacia AC1100 were 46 to 53% . Although all root nodule Bradyrhizobium strains were unable to degrade 2,4-D, three strains carrying cadA homologs degraded 4-chlorophenoxyacetate with the accumulation of 4-chlorophenol as an intermediate, suggesting the involvement of cadA homologs in the cleavage of the aryl ether linkage . Based on codon usage patterns and GC content, it was suggested that the cadA genes of 2,4-D-degrading and nondegrading Bradyrhizobium spp . have different origins and that the genes would be obtained in the former through horizontal gene transfer.

J Hosp Infect, 2004 Apr, 56(4), 277 - 82
The impact of pan-resistant bacterial pathogens on survival after lung transplantation in cystic fibrosis: results from a single large referral centre; Dobbin C et al.; Reported actuarial one-year survival for patients with cystic fibrosis (CF) after lung transplant is 55-91% . Infection is the most common cause of early death . Colonization with Burkholderia cepacia complex is associated with reduced survival and international lung transplant referral guidelines support individual unit assessment policies for patients colonized with other pan-resistant bacteria . We examined local data on survival after transplant for CF to determine the impact of colonization with pan-resistant bacteria . A retrospective review of all CF patients from Royal Prince Alfred Hospital (RPAH), Sydney, who underwent lung transplantation at St Vincent's Hospital, Sydney, 1989-2002, was performed . Sixty-five patients were listed for lung transplantation with 54 (male: female=29:25) receiving transplants . Of the 11 patients (17%) who died on the waiting list, six were colonized with pan-resistant Pseudomonas aeruginosa . Thirty of the 54 transplanted patients had at least one pan-resistant organism before transplant . In 28 this included P . aeruginosa . Overall one-year survival was 92% with a median survival of 67 months . Overall survival for the pan-resistant group (N = 30) was not significantly different to survival in those with sensitive organisms (N = 24) (Logrank chi square = 1.6, P = 0.2) . Three patients colonized with B . cepacia complex pre-transplant survive at 11, 40 and 60 months post-transplant . Infection contributed to 11 of the 18 post-transplant deaths, with pre-transplant-acquired bacterial pathogens responsible in two cases . Patients continued to acquire multiresistant bacteria post-transplantation . Lung transplant survival at St Vincent's Hospital for CF adults from RPAH compares favourably with international benchmarks . Importantly, colonization with pan-resistant bacteria pre-transplant did not appear to adversely affect survival post-transplant.

J Infect, 2004 May, 48(4), 334 - 8
Bacteraemic melioidosis pneumonia: impact on outcome, clinical and radiological features; Mukhopadhyay A et al.; OBJECTIVES: Melioidosis is an endemic disease in South-east (SE) Asia and bacteraemia in melioidosis is associated with high mortality . We describe some clinical and radiological features of bacteraemic pneumonia due to Burkholderia pseudomallei as well as a comparison with bacteraemic patients without pneumonia . METHODS: Patients with positive blood cultures for B . pseudomallei from October 1997 to November 2001 were included . Patients were grouped as 'Pneumonia' and 'Non-pneumonia' according to clinical and radiological features . RESULTS: Eighteen (60%) out of total 30 patients were in the pneumonia group . There was no significant difference in age, WBC count, platelet counts and bilirubin levels between the groups . However the 'Pneumonia' group had higher incidences of hyponatraemia, acidosis, diabetes with poor control, renal impairment and shorter length of stay . Twelve (66%) of 18 patients in the pneumonia group required ICU admission compared to none in the non-pneumonia group; all required mechanical ventilation . Only 13/30 (43%) patients had initial empiric antibiotic therapy that is appropriate for melioidosis . The pneumonia group also had significantly higher mortality (13/18, 72%) rate than the non-pneumonia group (3/12, 25%, P=0.03) . Chest radiographs were non-specific . 7/18 (38%) had unilobar involvement of the lung, mostly left sided; the rest had multilobar or bilateral involvement . Six (33%) had pleural effusion . No patient had cavitary lung disease . Visceral abscesses (spleen, liver and prostate) were also common in ultrasound and CT scans in both groups . CONCLUSION: (1) Bacteraemic melioidosis with pneumonia carries high mortality with most patients dying early . (2) Radiological features of melioidosis pneumonia are non-specific . (3) Clinicians who treat patients from SE Asia need to be aware of this condition to institute early and appropriate antibiotic therapy.

Biochim Biophys Acta, 2004 Apr 8, 1698(1), 111 - 9
Biophysical characterization of the catalytic domain of guanine nucleotide exchange factor BopE from Burkholderia pseudomallei; Upadhyay A et al.; BopE is a type III secreted protein from Burkholderia pseudomallei, the aetiological agent of melioidosis . Like its Salmonella homologues SopE and SopE2, BopE is a guanine nucleotide exchange factor for Rho GTPases . It is thought that, in order to be secreted by the type III system, proteins must be unfolded or only partially folded . As part of a study of B . pseudomallei virulence proteins, we have expressed, purified and characterized the catalytic domain of BopE (amino acids 78-261) . Analytical ultracentrifugation experiments in conjunction with analytical size exclusion chromatography show that BopE(78-261) is monomeric in aqueous solution . CD spectroscopy indicates that the protein is predominantly alpha-helical, with predicted secondary structure composition of 59% alpha-helix and 7% beta-strand . NMR spectroscopy confirms that BopE(78-261) adopts a single, stable conformation . In differential scanning calorimetry experiments, thermal denaturation of BopE(78-261) (T(m) 52 degrees C) is reversible . Also, the secondary structure composition of BopE(78-261) changes little over a range of pH values from 3.5 to 10.5 . BopE may therefore fold spontaneously to a functional form upon secretion into the host cell cytoplasm, and retains a native or native-like fold in varied environments . These properties are likely to be advantageous for a secreted bacterial effector protein.

Infect Control Hosp Epidemiol, 2004 Mar, 25(3), 231 - 9
Outbreak of Burkholderia cepacia bacteremia traced to contaminated hospital water used for dilution of an alcohol skin antiseptic; Nasser RM et al.; OBJECTIVE: To identify the source of an epidemic of Burkholderia cepacia bloodstream infections during 7 years (411 episodes in 361 patients) . DESIGN: Outbreak investigation . SETTING: A 250-bed university hospital in Beirut, Lebanon . METHODS: Matched case-control and retrospective cohort studies, and microbiological surveillance and polymerase chain reaction-restriction fragment length ascertainment were employed . Special media and filtration techniques were used to isolate organisms from water and diluted alcohol solutions . RESULTS: In a group of 50 randomly selected case-matched patients from 1999, the positive blood cultures were concomitant with fever in 98%, intravenous phlebitis in 44%, and recurrent bacteremia in 20% . Fever disappeared approximately 6 hours after intravenous catheter removal . Polymerase chain reaction-restriction fragment length polymorphism revealed strain homogeneity in patient, water, and alcohol isolates . Contaminated tap water had been used to dilute alcohol for skin antisepsis and for decontamination of the caps of heparin vials . Only sporadic cases directly attributable to breach of protocol were reported after single-use alcohol swabs were substituted . CONCLUSION: This is potentially the largest single-source nosocomial bloodstream infection outbreak ever reported, and the first report of an alcohol skin antiseptic contaminated by tap water as a source for nosocomial bacteremia.

J Bacteriol, 2004 Apr, 186(8), 2376 - 84
Burkholderia spp . alter Pseudomonas aeruginosa physiology through iron sequestration; Weaver VB et al.; Pseudomonas aeruginosa and members of the Burkholderia cepacia complex often coexist in both the soil and the lungs of cystic fibrosis patients . To gain an understanding of how these different species affect each other's physiology when coexisting, we performed a screen to identify P . aeruginosa genes that are induced in the presence of Burkholderia: A random gene fusion library was constructed in P . aeruginosa PA14 by using a transposon containing a promoterless lacZ gene . Fusion strains were screened for their ability to be induced in the presence of Burkholderia strains in a cross-streak assay . Three fusion strains were induced specifically by Burkholderia species; all three had transposon insertions in genes known to be iron regulated . One of these fusion strains, containing a transposon insertion in gene PA4467, was used to characterize the inducing activity from Burkholderia: Biochemical and genetic evidence demonstrate that ornibactin, a siderophore produced by nearly all B . cepacia strains, can induce P . aeruginosa PA4467 . Significantly, PA4467 is induced early in coculture with an ornibactin-producing but not an ornibactin-deficient B . cepacia strain, indicating that ornibactin can be produced by B . cepacia and detected by P . aeruginosa when the two species coexist.

J Bacteriol, 2004 Apr, 186(8), 2288 - 94
The PmlI-PmlR quorum-sensing system in Burkholderia pseudomallei plays a key role in virulence and modulates production of the MprA protease; Valade E et al.; Burkholderia pseudomallei is the causative agent of melioidosis, an often fatal infection of humans and animals . The virulence of this pathogen is thought to depend on a number of secreted proteins, including the MprA metalloprotease . We observed that MprA is produced upon entry into the stationary phase, when the cell density is high, and this prompted us to study cell density-dependent regulation in B . pseudomallei . A search of the B . pseudomallei genome led to identification of a quorum-sensing system involving the LuxI-LuxR homologs PmlI-PmlR . PmlI directed the synthesis of an N-acylhomoserine lactone identified as N-decanoylhomoserine lactone . A B . pseudomallei pmlI mutant was significantly less virulent than the parental strain in a murine model of infection by the intraperitoneal, subcutaneous, and intranasal routes . Inactivation of pmlI resulted in overproduction of MprA at the onset of the stationary phase . A wild-type phenotype was restored following complementation with pmlI or addition of cell-free culture supernatant . In contrast, there was no significant difference between the virulence of a B . pseudomallei mprA mutant and the virulence of the wild-type strain . These results suggest that the PmlI-PmlR quorum-sensing system of B . pseudomallei is essential for full virulence in a mouse model and downregulates the production of MprA at a high cell density.

Curr Microbiol, 2004 Apr, 48(4), 300 - 4
Identification and expression of a Burkholderia pseudomallei collagenase in Escherichia coli; Rainbow L et al.; Extracellular protein profiles were compared for broth-grown cultures of Burkholderia pseudomallei and its avirulent close relative Burkholderia thailandensis . A number of protein bands were present in the B . pseudomallei profile but absent or less abundant in the B . thailandensis profile . Four such prominent secreted proteins were identified by using N-terminal sequencing coupled to searches of the B . pseudomallei genome sequence database . The genes for two proteins with similarity to carbohydrate-binding proteins, and a further protein homologous to known bacterial collagenases, were present in both B . pseudomallei and B . thailandensis . The putative collagenase gene was cloned and expressed as a fusion protein in Escherichia coli . Cell lysates from Escherichia coli containing the recombinant protein exhibited detectable gelatinase and collagenase activities.

Rev Inst Med Trop Sao Paulo, 2004 Jan-Feb, 46(1), 51 - 4 Epub 2004 Mar 29.
Burkholderia pseudomallei: a case report of a human infection in Ceará, Brazil; Miralles IS et al.; Burkholderia pseudomallei has rarely been isolated from environmental and clinical specimens in South America, particularly, in Brazil . This report describes a case of melioidosis with fulminant sepsis in a 10 year old boy, from rural area, in Tejucuoca, State of Ceara, Brazil . Blood samples were positive and, through the analysis of results from biochemical tests and of drugs susceptibility profile, identified this gram-negative bacillus as B . pseudomallei . The contamination source remains obscure in this case, as soil and water tanks samples submitted to microbiological analyses did not indicate the presence of B . pseudomallei.

Biosci Biotechnol Biochem, 2004 Mar, 68(3), 650 - 5
Purification and characterization of alkaline serine proteinase from photosynthetic bacterium, Rubrivivax gelatinosus KDDS1; Oda K et al.; In order to reduce the protein content of wastewater, photosynthetic bacteria producing proteinases were screened from wastewater of various sources and stocked in culture . An isolated strain, KDDS1, was identified as Rubrivivax gelatinosus, a purple nonsulfur bacterium that secretes proteinase under micro-aerobic conditions under light at 35 degrees C . Molecular weight of the purified enzyme was estimated to be 32.5 kDa . The enzyme showed the highest activity at 45 degrees C and pH 9.6, and the activity was completely inhibited by phenylmethyl sulfonyl fluoride (PMSF), but not by EDTA . The amino-terminal 24 amino acid sequence of the enzyme showed about 50% identity to those of serine proteinases from Pseudoalteromonas piscicida strain O-7 and Burkholderia pseudomallei . Thus, the enzyme from Rvi . gelatinosus KDDS1 was thought to be a serine-type proteinase . This was the first serine proteinase characterized from photosynthetic bacteria.

Can J Microbiol, 2004 Jan, 50(1), 29 - 39
Mutagenic DNA repair potential in Pseudomonas spp., and characterization of the rulABPc operon from the highly mutable strain Pseudomonas cichorii 302959; Zhang S et al.; We assessed the tolerance to ultraviolet B (UVB; 290-320 nm) radiation and UVB-induced mutability in 28 Pseudomonas spp . and four Burkholderia cepacia strains . The UVB survival of 23 (72%) of the strains was elevated (>46% survival following irradiation with a 2250 J m-2 dose), and 17 (53%) strains were defined as mutable by UVB . A mutagenic DNA repair determinant was cloned and characterized from the highly mutable strain P . cichorii 302959 and shown by sequence analysis to be an allele of rulAB, a mutagenic DNA repair determinant previously characterized from Pseudomonas syringae . Phylogenetic analyses of RulA- and RulB-related sequences indicated that the sequences identified in environmental bacteria shared a common ancestor with UmuDC-like sequences from enteric bacteria but were considerably diverged . The dynamics of UVB-induced mutability to nalidixic acid resistance (NalR) and rifampicin resistance (RifR) were studied in replicate populations of P . cichorii 302959 subjected to a daily UVB dose of 2250 J m-2 for 14 consecutive days . While there was an initial spike in the frequency of NalR and RifR mutants recovered on Days 1 and 2 of two separate experiments, the frequencies were sharply reduced and then fluctuated throughout the duration of both experiments . These experimental results are intriguing because they point to the possibility that P . cichorii possesses additional mechanisms to curtail the induction of spontaneous mutants following repeated episodes of UVB irradiation.

Mol Cell Probes, 2004 Apr, 18(2), 97 - 101
PCR-RFLP based differentiation of Burkholderia mallei and Burkholderia pseudomallei; Tanpiboonsak S et al.; Burkholderia mallei and Burkholderia pseudomallei manifest a high similarity with regard to clinical syndromes, glanders and melioidosis . Phenotypic and genotypic characters are also highly similar . In an attempt to differentiate the two organisms, the molecular method was applied . This study aimed to identify the different DNA fragment in B . mallei, as compared with B . pseudomallei . The Sau3AI-digested genomic DNA patterns of B . mallei and B . pseudomallei are distinctive, especially the DNA fragments between 0.9-1.5 Kb in size . A 900-bp specific DNA fragment of B . mallei was cloned and sequenced . Using the specific DNA fragment as a probe, Southern blot hybridization was performed to differentiate the two species . The results of hybridization patterns are effective in to elucidating the genetic dissimilarities among these two Burkholderia species . Furthermore, polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) digested with Sau3AI was developed to allow a more reliable and rapid identification of the two species . A 650-bp PCR-RFLP product of B . mallei was detected, while two fragments of 250 and 400-bp PCR-RFLP products of B . pseudomallei were visualized . The results suggest that the specific DNA fragment in our study should be of considerable use as a genetic marker for ensuring identification of the two species.

Environ Microbiol, 2004 May, 6(5), 491 - 500
Metabolic pathway engineering to enhance aerobic degradation of chlorinated ethenes and to reduce their toxicity by cloning a novel glutathione S-transferase, an evolved toluene o-monooxygenase, and gamma-glutamylcysteine synthetase; Rui L et al.; Aerobic, co-metabolic bioremediation of trichloroethylene (TCE), cis-1,2-dichloroethylene (cis-DCE) and other chlorinated ethenes with monooxygenase-expressing microorganisms is limited by the toxic epoxides produced as intermediates . A recombinant Escherichia coli strain less sensitive to the toxic effects of cis-DCE, TCE and trans-1,2-dichloroethylene (trans-DCE) degradation has been created by engineering a novel pathway consisting of eight genes including a DNA-shuffled toluene ortho-monooxygenase from Burkholderia cepacia G4 (TOM-Green), a newly discovered glutathione S-transferase (GST) from RhodococcusAD45 (IsoILR1), found to have activity towards epoxypropane and cis-DCE epoxide, and an overexpressed E . coli mutant gamma-glutamylcysteine synthetase (GSHI*) . Along with IsoILR1, another new RhodococcusAD45 GST, IsoILR2, was cloned that lacks activity towards cis-DCE epoxide and differs from IsoILR1 by nine amino acids . The recombinant strain in which TOM-Green and IsoILR1 were co-expressed on separate plasmids degraded 1.9-fold more cis-DCE compared with a strain that lacked IsoILR1 . In the presence of IsoILR1 and TOM-Green, the addition of GSH1* resulted in a sevenfold increase in the intracellular GSH concentration and a 3.5-fold improvement in the cis-DCE degradation rate based on chloride released (2.1 +/- 0.1 versus 0.6 +/- 0.1 nmol min(-1) mg(-1) protein at 540 microM), a 1.8-fold improvement in the trans-DCE degradation rate (1.29 +/- 0.03 versus 0.71 +/- 0.04 nmol x min(-1) mg(-1) protein at 345 microM) and a 1.7-fold improvement in the TCE degradation rate (6.8 +/- 0.24 versus 4.1 +/- 0.16 nmol x min(-1) mg(-1) protein at 339 microM) . For cis-DCE degradation with TOM-Green (based on substrate depletion), V(max) was 27 nmol x min(-1) mg(-1) protein with both IsoILR1 and GSHI* expressed compared with V(max) = 10 nmol x min(-1) mg(-1) protein for the GST(-)GSHI*(-) strain . In addition, cells expressing IsoILR1 and GSHI* grew 78% faster in rich medium than a strain lacking these two heterologous genes.

Thorax, 2004 Apr, 59(4), 334 - 6
Increased morbidity associated with chronic infection by an epidemic Pseudomonas aeruginosa strain in CF patients; Al-Aloul M et al.; BACKGROUND: Chronic pulmonary infection with transmissible Pseudomonas aeruginosa strains in individuals with cystic fibrosis (CF) has been reported, raising issues of cross infection and patient segregation . The first such strain to be described (the Liverpool epidemic strain, LES) is now widespread in many UK CF centres . However, whether such infection carries a worse prognosis is unknown . To address this, the clinical course of a group of CF patients chronically infected by LES was compared with that in patients harbouring unique strains . METHODS: Using P aeruginosa strain genotyping, two cohorts of CF patients attending the Liverpool CF service were identified who were LES positive or negative in 1998 and remained so until 2002 . From these, two groups of 12 patients were matched in 1998 for age, spirometric parameters, and nutritional state and their clinical course was followed for 5 years . Patients chronically infected with Burkholderia cepacia were excluded . RESULTS: Patients chronically infected with LES had a greater annual loss of lung function than those not chronically infected by LES (mean difference between groups -4.4% (95% CI -8.1 to -0.9; p<0.02)), and by 2002 their percentage predicted forced expiratory volume in 1 second (FEV1) was worse (mean 65.0% v 82.6%, p<0.03) . Their nutritional state also deteriorated over the study period (mean difference between groups in body mass index -0.7 (95% CI -1.2 to -0.2; p<0.01)), such that by 2002 they were malnourished compared with LES negative patients (mean BMI 19.4 v 22.7, p<0.02) . CONCLUSIONS: Chronic infection with the Liverpool epidemic P aeruginosa strain in CF patients confers a worse prognosis than infection with unique strains alone, confirming the need for patient segregation . Since this strain is common in many CF units, strain identification in all CF centres is essential . This can only be carried out using genomic typing methods.

Antimicrob Agents Chemother, 2004 Apr, 48(4), 1128 - 35
BpeAB-OprB, a multidrug efflux pump in Burkholderia pseudomallei; Chan YY et al.; Burkholderia pseudomallei, the causative agent of melioidosis, is intrinsically resistant to a wide range of antimicrobial agents, including beta-lactams, aminoglycosides, macrolides, and polymyxins . An operon, bpeR-bpeA-bpeB-oprB, which encodes a putative repressor, a membrane fusion protein, an inner membrane protein, and an outer membrane protein, respectively, of a multidrug efflux pump of the resistance-nodulation-division family was identified in B . pseudomallei . The divergently transcribed bpeR gene encodes a putative repressor protein of the TetR family which probably regulates the expression of the bpeAB-oprB gene cluster . Comparison of the MICs and minimal bactericidal concentrations of antimicrobials for bpeAB deletion mutant KHW Delta bpeAB and its isogenic wild-type parent, KHW, showed that the B . pseudomallei BpeAB-OprB pump is responsible for the efflux of the aminoglycosides gentamicin and streptomycin, the macrolide erythromycin, and the dye acriflavine . Antibiotic efflux by the BpeAB-OprB pump was dependent on a proton gradient and differs from that by the AmrAB-OprA pump in that it did not efflux the aminoglycoside spectinomycin or the macrolide clarithromycin . The broad-spectrum efflux pump inhibitor MC-207,110 did not potentiate the effectiveness of the antimicrobials erythromycin and streptomycin in B . pseudomallei.

Am J Respir Crit Care Med, 2004 Jul 1, 170(1), 70 - 7 Epub 2004 Mar 24.
Burkholderia cenocepacia lipopolysaccharide, lipid A, and proinflammatory activity; De Soyza A et al.; Organisms from the Burkholderia cepacia complex are important pathogens in cystic fibrosis and are associated with increased rates of sepsis and death . These organisms comprise nine closely related species known as genomovars . B . cenocepacia (genomovar III) is the most prevalent and appears the most virulent . We investigated the biological activity of a reference panel of strains using whole-cell lysates to induce septic-shock related cytokines from differentiated human monocytic cells . We found varying biological activity within and between genomovars, with B . cenocepacia strains possessing the greatest cytokine induction activity . This activity was CD-14 dependent, suggesting that LPS was responsible for the cytokine induction . Cytokine induction was not simply related to the expression of rough or smooth LPS . We purified LPS from two strains, B . cenocepacia LMG 12614 and B . multivorans LMG 14273, each possessing rough LPS . Divergence in biological activity of the two genomovars was preserved when human monocytic cells were stimulated with purified LPS . Lipid A purified from LMG 14273 and LMG 12614 were analyzed by matrix-assisted laser desorption ionization/time of flight mass spectrometry . Lipid A from the less effective cytokine inducer LMG 14273 was found to be missing a beta-hydroxymyristate (3-OH C14:0) relative to the lipid A of B . cenocepacia LMG 12614.

Biochem Biophys Res Commun, 2004 Apr 16, 316(4), 1101 - 6
Structure-function studies of the Vitreoscilla hemoglobin D-region; Lee SY et al.; The D-region connecting helices C and E of Vitreoscilla hemoglobin (VHb) appears disordered in the crystal structure . Six site-directed mutants in this region were made to investigate its possible functions . The mutant VHb's were analyzed using UV-visible and FTIR spectroscopy, using primarily the CO liganded forms, and their heme/protein ratios were determined . The results implicate Asp44, Arg47, and Glu49 as especially important in heme-globin interactions and ligand binding, and enabled construction of a model in which the D-region forms a loop that protrudes upward over the heme . Interactions between VHb (wild type and the D-region mutants) with the flavin domain of 2,4-DNT dioxygenase from Burkholderia were tested using bacterial two-hybrid screening . There was a correlation between the extent of the D-loop perturbation predicted for each mutant and the amount of the reduction in VHb-flavin domain interaction, suggesting that this region may be more generally involved in binding of VHb to flavoproteins.

Microb Pathog, 2004 May, 36(5), 287 - 92
Virulent Burkholderia pseudomallei is more efficient than avirulent Burkholderia thailandensis in invasion of and adherence to cultured human epithelial cells; Kespichayawattana W et al.; Burkholderia pseudomallei, a causative agent of melioidosis, is a facultative intracellular gram-negative bacillus that is closely related to its avirulent counterpart, Burkholderia thailandensis . However, pathogenic mechanisms and virulence factors of B . pseudomallei remain elusive . In the present study, we compared the invasiveness, adherence, and replication of B . pseudomallei and B . thailandensis in human respiratory epithelial cells A549 . Invasion was determined after 4 h of coculturing using antibiotic protection assay . Adherence was demonstrated by coculturing the cells with fluorescein-labeled bacteria for 1 h and the number of positive cells was analyzed by flow cytometry . The results obtained with this in vitro study demonstrated that compared with its avirulent counterpart, B . pseudomallei is significantly more efficient (P<0.01) in invasion, adherence and inducing cellular damage, as represented by plaque formation.

J Environ Sci Health A Tox Hazard Subst Environ Eng, 2004, 39(1), 61 - 75
Biodegradation of 1,3,5-trinitro-1,3,5-triazine (RDX); Lee SY et al.; Two bacteria were isolated from 1,3,5-trinitro-1,3,5-triazine (RDX) contaminated soil at Picatinny Arsenal, New Jersey . These organisms were subsequently identified as Rhiziobium rhizogenes BL and Burkholderia sp.BL by the Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (DSMZ, German Collection of Microorganisms and Cell Cultures) . In addition a fungus, identified as Cladosporium cladosporioides by DSMZ, was found to be growing on water wet RDX . All of these organisms were found to degrade RDX . The two bacteria were found to represent new species that have not been reported before . It was found that these organisms along with an added carbon source could degrade RDX to simple gaseous products . Data are presented that elucidate the mechanisms of RDX biodegradation for these organisms.

J Bacteriol, 2004 Apr, 186(7), 2173 - 8
Multiple formaldehyde oxidation/detoxification pathways in Burkholderia fungorum LB400; Marx CJ et al.; Burkholderia species are free-living bacteria with a versatile metabolic lifestyle . The genome of B . fungorum LB400 is predicted to encode three different pathways for formaldehyde oxidation: an NAD-linked, glutathione (GSH)-independent formaldehyde dehydrogenase; an NAD-linked, GSH-dependent formaldehyde oxidation system; and a tetrahydromethanopterin-methanofuran-dependent formaldehyde oxidation system . The other Burkholderia species for which genome sequences are available, B . mallei, B . pseudomallei, and B . cepacia, are predicted to contain only the first two of these pathways . The roles of the three putative formaldehyde oxidation pathways in B . fungorum LB400 have been assessed via knockout mutations in each of these pathways, as well as in all combinations of knockouts . The resulting mutants have the expected loss of enzyme activities and exhibit defects of varying degrees of severity during growth on choline, a formaldehyde-producing substrate . Our data suggest that all three pathways are involved in formaldehyde detoxification and are functionally redundant under the tested conditions.

Comp Med, 2004 Feb, 54(1), 93 - 9
Outbreak of otitis media caused by Burkholderia gladioli infection in immunocompromised mice; Foley PL et al.; An athymic nude mouse with severe head tilt due to otitis media was identified . Within weeks of identification of this first case, immune-deficient mice of various genotypes from the same facility were similarly affected, and cases from other facilities were found within two months . Culture of ear exudate specimens from affected mice yielded bacteria that were initially identified as Burkholderia cepacia, a plant pathogen considered an important opportunistic pathogen in persons with cystic fibrosis or chronic granulomatous disease . Several of these isolates, however, were subsequently identified as B . gladioli on the basis of results of biochemical analysis and a species-specific polymerase chain reaction (PCR) assay . Genotyping analysis revealed clonality among the isolates, indicating a shared strain among affected mice . A 16S rDNA-based PCR assay specific for the genera Burkholderia and Ralstonia, and a selective culture medium were used in efforts to characterize the epidemiology of this outbreak . In addition to culture of specimens from the oropharyngeal cavity of affected mice, samples were obtained from the environment, feces, sipper tubes, drinking water, and soiled bedding from cages of affected individuals . Burkholderia gladioli was most consistently detected in oropharyngeal swab specimens from affected mice . The PCR assay was equivalent to selective culture in identifying mice in the carrier state that did not have clinical signs of infection . However, neither detection method had sufficient sensitivity to reliably identify all carrier mice, causing the organism to persist at low levels unless entire colonies of immune-deficient mice were removed . The organism was highly resistant to antibiotic therapy . The source and epidemiology of this organism remain unknown . This epizootic serves as an important reminder that immunocompromised rodent colonies may harbor important human opportunistic pathogens.

J Steroid Biochem Mol Biol, 2004 Jan, 88(1), 91 - 100
Identification of a novel steroid inducible gene associated with the beta hsd locus of Comamonas testosteroni; Pruneda-Paz JL et al.; Comamonas testosteroni is a soil bacterium, which can use a variety of steroids as carbon and energy source . Even if it can be estimated that the complete degradation of the steroid nucleus requires more than 20 enzymatic reactions, the complete molecular characterization of the genes encoding these steroid degradative enzymes as well as the genetic organization of them remain to be elucidated . We have previously reported the cloning and nucleotide sequence of two steroid-inducible genes, beta hsd and stdC encoding 3 beta-17 beta-hydroxysteroid dehydrogenase and a hypothetical protein respectively, located in both ends of a 3.2kb HindIII fragment . Herein, we report the cloning and characterization of another steroid-inducible gene, called sip48 (steroid inducible protein), located between these two genes . The analysis of Sip48 amino acid sequence predicts a protein of 438 amino acids with a molecular mass of 48.5 kDa . This protein bears high homology with conserved hypothetical proteins of unknown function described in Pseudomonas aeruginosa, Pseudomonas syringae, Pseudomonas putida, Burkholderia fungorum, Shewanella oneidensis, Pseudomonas fluorescens and Thauera aromatica . The predicted protein shows a typical structure of a leader peptide at its N-terminus . A 48.5 kDa protein encoded by the recombinant plasmid was detected by SDS-PAGE analysis of in vitro {35S}-methionine labeled polypeptides . Analysis of gene expression indicates that Sip48 is tightly controlled at the transcriptional level by several steroid compounds . In addition, transcriptional analysis of sip48 and beta hsd in a sip48 mutant strain, indicates that both genes are transcribed as a polycistronic mRNA . lacZ transcriptional fusions integrated into the chromosome of C . testosteroni demonstrate that a steroid-inducible promoter located upstream of sip48 regulates the expression of both genes.

Mol Gen Mikrobiol Virusol, 2004, (1), 12 - 7
{Use of PCR for identification of Burkholderia mallei}; Antonov VA et al.; Stimuli of glanders belong to the potential agents of biological terror . The possibility to use various primers in the identification of B . mallei was investigated and the significance of polymerase chain reaction (PCR) was defined within the scheme of laboratory glanders diagnosis in the offered paper . The constructed amplifying test-systems can be used to detect the glanders both in the environmental objects contaminated with B . mallei and in experimental clinical material.

Curr Opin Infect Dis, 2004 Apr, 17(2), 131 - 6
Recent development in melioidosis; Leelarasamee A; PURPOSE OF REVIEW: Burkholderia pseudomallei, the causative agent of melioidosis and a potential biological weapon, is still unfamiliar in some areas where sporadic cases are being reported among travelers . This review highlights findings in 2002-2003 and is an extension of a recent review by Dance . RECENT FINDINGS: The allele profiles of B . pseudomallei are distinguishable from avirulent Burkholderia thailandensis, but Burkholderia mallei is a clone of B . pseudomallei . Capsule and a type III protein secretion apparatus enable B . pseudomallei to survive intracellular killing and facilitate intercellular spread . A strong antibody response to infection is useful for monitoring disease activity . A mutant that is auxotrophic in the branched chain amino acid biosynthetic pathway has been found to be attenuated and protective . A new selective media is useful for isolation from contaminated specimens and the environment . Molecular techniques have been developed to distinguish B . pseudomallei from B . thailandensis and B . mallei as well as for serological diagnosis . Classification of the clinical manifestation is proposed to facilitate global communication, and will be useful to compare the efficacies of new regimens and adjunctive immunomodulatory therapies, such as granulocyte colony-stimulating factor and activated protein C for septicemic melioidosis . SUMMARY: Study of pathogenesis and intracellular survival of B . pseudomallei is advancing and may lead to better methods of therapy and vaccine production . New antimicrobial agents and immunomodulators are being studied to shorten the duration of treatment in the acute and maintenance phases, reduce the high mortality rate in septicemic melioidosis, and prevent relapses.

J Microbiol Methods, 2004 Apr, 57(1), 107 - 14
Use of breakpoint combination sensitivity testing as a simple and convenient method to evaluate the combined effects of ceftazidime and tobramycin on Pseudomonas aeruginosa and Burkholderia cepacia complex isolates in vitro; Tunney MM et al.; An in vitro method of determining the activity of antibiotics in combination which is simple and convenient to perform and which could be used routinely in clinical microbiology laboratories is desirable . We investigated the activity, against Pseudomonas aeruginosa and Burkholderia cepacia complex clinical isolates, of ceftazidime and tobramycin in combination using a broth macrodilution sensitivity method based on breakpoint minimum inhibitory concentrations and compared the results obtained using this method with those obtained using the microtitre checkerboard method . There was good agreement in interpretation of results between the two methods for both P . aeruginosa (90%) and B . cepacia complex isolates (70%) with tobramycin and for P . aeruginosa isolates (70%) with ceftazidime . As the breakpoint combination sensitivity testing method employs only four tubes and does not require initial determination of individual antibiotic minimum inhibitory concentrations, it is simpler and more convenient for determining the activity of antibiotics in combination than the microtitre checkerboard method . The use of this method in routine microbiology laboratories to determine the activity of antibiotic combinations against clinical isolates should optimise treatment of infection by ensuring that appropriate antibiotic combinations are prescribed.

Scand J Infect Dis, 2004, 36(1), 68 - 70
Mycotic aneurysm caused by Burkholderia pseudomallei with negative blood cultures; Tanyaowalak W et al.; We describe a case of bacterial aortitis caused by Burkholderia pseudomallei . This patient presented with prolonged fever and hoarseness of voice . Aneurysm removal with Dacron graft replacement was performed, followed by a prolonged course of antibiotics . The patient has progressed satisfactorily without recurrence of symptoms . Previous case reports are summarized.

Eur J Clin Microbiol Infect Dis, 2004 Mar, 23(3), 205 - 7 Epub 2004 Feb 19.
Granulocyte colony-stimulating factor and an in vitro whole blood model of melioidosis; Cheng AC et al.; The study reported here was conducted in order to explore the mechanism of action of granulocyte colony-stimulating factor (G-CSF) in the treatment of Burkholderia pseudomallei infections (otherwise known as melioidosis) . Use of G-CSF as an adjunct to antibiotics has been associated with decreasing mortality among patients with melioidosis in the tropical region of the Northern Territory of Australia . However, using an in vitro whole blood assay, no significant difference was detected in the bactericidal activity of samples obtained from dialysis patients, patients with type 2 diabetes mellitus and healthy controls, and there was no improvement following coincubation with G-CSF.

Paediatr Respir Rev, 2004, 5 Suppl A, S367 - 9
Microbiology of early CF lung disease; Saiman L; Recent bronchoscopy studies using assays to measure inflammation and molecular typing techniques have facilitated an increased understanding of the early events that occur within the lungs of young children with cystic fibrosis and provided additional insights into the natural history of lung disease in children . In 2000, the US CF National Patient Registry data showed that among 1000 infants <2 years of age, the first bacterial pathogens detected are Haemophilus influenzae, Staphylococcus aureus, and Pseudomonas aeruginosa and the prevalence of these pathogens in these young infants are 19%, 42%, and 29%, respectively . In addition, 7% harbour Stenotrophomonas maltophilia and <1% harbour Burkholderia cepacia complex . Several investigators have performed bronchoscopy studies on young infants to further examine the natural history of lung disease . In one such study of 40 CF infants, 65%, 63% and 70% of children at 1, 2, and 3 years of age harboured at least one CF pathogen . H . influenzae was most common (38%) in infants at 1 year of age, and S . aureus was most common in 2 (37%) and 3 (36%) year olds . P . aeruginosa increased from 18% at 1 year of age to 33% at 3 years of age and was usually present in high numbers, i.e., > or =10(5) CFU/ml of BAL fluid . Investigators have studied the microbiology of young CF infants using specimens derived from the upper airway (deep throat) compared with the lower airway (broncheoalveolar lavage specimens) to determine if the upper airway is predictive of pathogens in the lower airway . In general, these studies have shown that a negative oropharyngeal culture indicated that isolation of P . aeruginosa from the lower airway was unlikely, but a positive culture did not predict lower airway infection . Similar findings were noted for H . influenzae and S . aureus.

Infect Immun, 2004 Mar, 72(3), 1537 - 47
The Burkholderia cepacia epidemic strain marker is part of a novel genomic island encoding both virulence and metabolism-associated genes in Burkholderia cenocepacia; Baldwin A et al.; The Burkholderia cepacia epidemic strain marker (BCESM) is a useful epidemiological marker for virulent B . cenocepacia strains that infect patients with cystic fibrosis . However, there was no evidence that the original marker, identified by random amplified polymorphic DNA fingerprinting, contributed to pathogenicity . Here we demonstrate that the BCESM is part of a novel genomic island encoding genes linked to both virulence and metabolism . The BCESM was present on a 31.7-kb low-GC-content island that encoded 35 predicted coding sequences (CDSs): an N-acyl homoserine lactone (AHL) synthase gene (cciI) and corresponding transcriptional regulator (cciR), representing the first time cell signaling genes have been found on a genomic island; fatty acid biosynthesis genes; an IS66 family transposase; transcriptional regulator CDSs; amino acid metabolism genes; and a group of hypothetical genes . Mutagenesis of the AHL synthase, amidase (amiI), and porin (opcI) genes on the island was carried out . Testing of the isogenic mutants in a rat model of chronic lung infection demonstrated that the amidase played a role in persistence, while the AHL synthase and porin were both involved in virulence . The island, designated the B . cenocepacia island (cci), is the first genomic island to be defined in the B . cepacia complex and its discovery validates the original epidemiological correlation of the BCESM with virulent CF strains . The features of the cci, which overlap both pathogenicity and metabolism, expand the concept of bacterial pathogenicity islands and illustrate the diversity of accessory functions that can be acquired by lateral gene transfer in bacteria.

Biodegradation, 2004 Feb, 15(1), 19 - 28
Trichloroethylene degradation by toluene-oxidizing bacteria grown on non-aromatic substrates; Yeager CM et al.; The potential of trichloroethylene (TCE) to induce and non-aromatic growth substrates to support TCE degradation in five strains (Pseudomonas mendocina KR1, Ralstonia pickettii PKO1, Pseudomonas putida F1, Burkholderia cepacia G4, B . cepacia PR1) of toluene-oxidizing bacteria was examined . LB broth and acetate did not support TCE degradation in any of the wild-type strains . In contrast, fructose supported the highest specific levels of TCE oxidation observed in each of the strains tested, except B . cepacia G4 . We discuss the potential mechanisms and implications of this observation . In particular, cells of P . mendocina KR1 degraded significant amounts of TCE during cell growth on non-aromatic substrates . Apparently, TCE degradation was not completely constrained by any given factor in this microorganism, as was observed with P . putida F1 (TCE was an extremely poor substrate) or B . cepacia G4 (lack of oxygenase induction by TCE) . Our results indicate that multiple physiological traits are required to enable useful TCE degradation by toluene-oxidizing bacteria in the absence of aromatic cosubstrates . These traits include oxygenase induction, effective TCE turnover, and some level of resistance to TCE mediated toxicity.

Appl Microbiol Biotechnol, 2004 Jun, 64(6), 763 - 81 Epub 2004 Feb 14.
Bacterial lipases: an overview of production, purification and biochemical properties; Gupta R et al.; Lipases, triacylglycerol hydrolases, are an important group of biotechnologically relevant enzymes and they find immense applications in food, dairy, detergent and pharmaceutical industries . Lipases are by and large produced from microbes and specifically bacterial lipases play a vital role in commercial ventures . Some important lipase-producing bacterial genera include Bacillus, Pseudomonas and Burkholderia . Lipases are generally produced on lipidic carbon, such as oils, fatty acids, glycerol or tweens in the presence of an organic nitrogen source . Bacterial lipases are mostly extracellular and are produced by submerged fermentation . The enzyme is most commonly purified by hydrophobic interaction chromatography, in addition to some modern approaches such as reverse micellar and aqueous two-phase systems . Most lipases can act in a wide range of pH and temperature, though alkaline bacterial lipases are more common . Lipases are serine hydrolases and have high stability in organic solvents . Besides these, some lipases exhibit chemo-, regio- and enantioselectivity . The latest trend in lipase research is the development of novel and improved lipases through molecular approaches such as directed evolution and exploring natural communities by the metagenomic approach.

J Appl Microbiol, 2004, 96(3), 430 - 6
Efficiency of chlorocatechol metabolism in natural and constructed chlorobenzoate and chlorobiphenyl degraders; Brenner V et al.; AIMS: A possibility for the complementation of both ortho- and meta-cleavage pathway for chlorocatechols in one strain and its impact on degradation of chlorobenzoates accumulated during degradation of polychlorinated biphenyls was investigated . METHODS AND RESULTS: Genes responsible for ortho-cleavage of chlorocatechols were subcloned into two biphenyl degraders and the activities of chlorocatechol dioxygenases responsible for ortho- and meta-cleavage in these hybrid strains were monitored spectrophotometrically and also electrochemically by ion-selective electrode . CONCLUSIONS: While strain Pseudomonas fluorescens S12/C apparently gained metabolic advantage from this gene manipulation, strain Burkholderia cepacia P166/C did not express better degradation features in comparison with the parental strain . SIGNIFICANCE AND IMPACT OF THE STUDY: This approach has the potential to enhance chlorocatechol metabolism in selected biphenyl degraders.

Microbiology, 2004 Feb, 150(Pt 2), 463 - 72
Characterization of a gene cluster encoding the maleylacetate reductase from Ralstonia eutropha 335T, an enzyme recruited for growth with 4-fluorobenzoate; Seibert V et al.; A gene cluster containing a gene for maleylacetate reductase (EC 1.3.1.32) was cloned from Ralstonia eutropha 335(T) (DSM 531(T)), which is able to utilize 4-fluorobenzoate as sole carbon source . Sequencing of this gene cluster showed that the R . eutropha 335(T) maleylacetate reductase gene, macA, is part of a novel gene cluster, which is not related to the known maleylacetate-reductase-encoding gene clusters . It otherwise comprises a gene for a hypothetical membrane transport protein, macB, possibly co-transcribed with macA, and a presumed regulatory gene, macR, which is divergently transcribed from macBA . MacA was found to be most closely related to TftE, the maleylacetate reductase from Burkholderia cepacia AC1100 (62 % identical positions) and to a presumed maleylacetate reductase from a dinitrotoluene catabolic gene cluster from B . cepacia R34 (61 % identical positions) . By expressing macA in Escherichia coli, it was confirmed that macA encodes a functional maleylacetate reductase . Purification of maleylacetate reductase from 4-fluorobenzoate-grown R . eutropha 335(T) cells allowed determination of the N-terminal sequence of the purified protein, which was shown to be identical to that predicted from the cloned macA gene, thus proving that the gene is, in fact, recruited for growth of R . eutropha 335(T) with this substrate.

J Bacteriol, 2004 Feb, 186(4), 1009 - 20
Transcriptional and posttranscriptional control of cable pilus gene expression in Burkholderia cenocepacia; Tomich M et al.; Burkholderia cenocepacia is an important member of the Burkholderia cepacia complex, a group of closely related bacteria that inhabits a wide variety of environmental niches in nature and that also colonizes the lungs of compromised humans . Certain strains of B . cenocepacia express peritrichous adherence organelles known as cable pili, thought to be important in the colonization of the lower respiratory tract . The genetic locus required for cable pilus biogenesis is comprised of at least five genes, designated cblB, cblA, cblC, cblD, and cblS . In this study a transcriptional analysis of cbl gene expression was undertaken . The principal promoter, located upstream of the cbl locus, was identified and characterized . By using lacZ transcriptional fusions, the effects of multiple environmental cues on cbl gene expression were examined . High osmolarity, temperature of 37 degrees C, acidic pH, and low iron bioavailability were found to induce cbl gene expression . Northern hybridization analysis of the cbl locus identified a single, stable transcript corresponding to cblA, encoding the major pilin subunit . Transcriptional fusion studies combined with reverse transcription-PCR analysis indicated that the stable cblA transcript is the product of an mRNA processing event . This event may ensure high levels of expression of the major pilin, relative to other components of the assembly pathway . Our findings lend further insight into the control of cable pilus biogenesis in B . cenocepacia and provide evidence for regulation of cbl gene expression on both the transcriptional and posttranscriptional levels.

Chembiochem, 2004 Feb 6, 5(2), 152 - 61
Lipase-specific foldases; Rosenau F et al.; Lipases represent the most important class of enzymes used in biotechnology . Many bacteria produce and secrete lipases but the enzymes originating from Pseudomonas and Burkholderia species seem to be particularly useful for a wide variety of different biocatalytic applications . These enzymes are usually encoded in an operon together with a second gene which codes for a lipase-specific foldase, Lif, which is necessary to obtain enzymatically active lipase . A detailed analysis based on amino acid homology has suggested the classification of Lif proteins into four different families and also revealed the presence of a conserved motif, Rx1x2FDY(F/C)L(S/T)A . Recent experimental evidence suggests that Lifs are so-called steric chaperones, which exert their physiological function by lowering energetic barriers during the folding of their cognate lipases, thereby providing essential steric information needed to fold lipases into their enzymatically active conformation.

J Clin Invest, 2004 Feb, 113(3), 464 - 73
Salicylate induces an antibiotic efflux pump in Burkholderia cepacia complex genomovar III (B . cenocepacia); Nair BM et al.; An antibiotic efflux gene cluster that confers resistance to chloramphenicol, trimethoprim, and ciprofloxacin has been identified in Burkholderia cenocepacia (genomovar III), an important cystic fibrosis pathogen . Five open reading frames have been identified in the cluster . There is apparently a single transcriptional unit, with llpE encoding a lipase-like protein, ceoA encoding a putative periplasmic linker protein, ceoB encoding a putative cytoplasmic membrane protein, and opcM encoding a previously described outer membrane protein . A putative LysR-type transcriptional regulatory gene, ceoR, is divergently transcribed upstream of the structural gene cluster . Experiments using radiolabeled chloramphenicol and salicylate demonstrated active efflux of both compounds in the presence of the gene cluster . Salicylate is an important siderophore produced by B . cepacia complex isolates, and both extrinsic salicylate and iron starvation appear to upregulate ceoR promoter activity, as does chloramphenicol . These results suggest that salicylate is a natural substrate for the efflux pump in B . cenocepacia and imply that the environment of low iron concentration in the cystic fibrosis lung can induce efflux-mediated resistance, even in the absence of antibiotic selective pressure.

Infect Immun, 2004 Feb, 72(2), 1150 - 4
Type III secretion: a virulence factor delivery system essential for the pathogenicity of Burkholderia mallei; Ulrich RL et al.; By creating mutations in the Burkholderia mallei ATCC 23344 animal pathogen-like type III secretion system (TTSS), this study analyzes the correlation between type III secretion and the pathogenicity of ATCC 23344 in vivo . Mutagenesis demonstrated that a functional TTSS was required for the full pathogenicity of ATCC 23344 in the BALB/c mouse and Syrian hamster models of infection . However, vaccination with each mutant failed to elicit a protective immunity against challenge with wild-type ATCC 23344.

J Bacteriol, 2004 Feb, 186(3), 767 - 76
Direct glutaminyl-tRNA biosynthesis and indirect asparaginyl-tRNA biosynthesis in Pseudomonas aeruginosa PAO1; Akochy PM et al.; The genomic sequence of Pseudomonas aeruginosa PAO1 was searched for the presence of open reading frames (ORFs) encoding enzymes potentially involved in the formation of Gln-tRNA and of Asn-tRNA . We found ORFs similar to known glutamyl-tRNA synthetases (GluRS), glutaminyl-tRNA synthetases (GlnRS), aspartyl-tRNA synthetases (AspRS), and trimeric tRNA-dependent amidotransferases (AdT) but none similar to known asparaginyl-tRNA synthetases (AsnRS) . The absence of AsnRS was confirmed by biochemical tests with crude and fractionated extracts of P . aeruginosa PAO1, with the homologous tRNA as the substrate . The characterization of GluRS, AspRS, and AdT overproduced from their cloned genes in P . aeruginosa and purified to homogeneity revealed that GluRS is discriminating in the sense that it does not glutamylate tRNA(Gln), that AspRS is nondiscriminating, and that its Asp-tRNA(Asn) product is transamidated by AdT . On the other hand, tRNA(Gln) is directly glutaminylated by GlnRS . These results show that P . aeruginosa PAO1 is the first organism known to synthesize Asn-tRNA via the indirect pathway and to synthesize Gln-tRNA via the direct pathway . The essential role of AdT in the formation of Asn-tRNA in P . aeruginosa and the absence of a similar activity in the cytoplasm of eukaryotic cells identifies AdT as a potential target for antibiotics to be designed against this human pathogen . Such novel antibiotics could be active against other multidrug-resistant gram-negative pathogens such as Burkholderia and Neisseria as well as all pathogenic gram-positive bacteria.

Clin Microbiol Rev, 2004 Jan, 17(1), 57 - 71
Infection control in cystic fibrosis; Saiman L et al.; Over the past 20 years there has been a greater interest in infection control in cystic fibrosis (CF) as patient-to-patient transmission of pathogens has been increasingly demonstrated in this unique patient population . The CF Foundation sponsored a consensus conference to craft recommendations for infection control practices for CF care providers . This review provides a summary of the literature addressing infection control in CF . Burkholderia cepacia complex, Pseudomonas aeruginosa, and Staphylococcus aureus have all been shown to spread between patients with CF . Standard precautions, transmission-based precautions including contact and droplet precautions, appropriate hand hygiene for health care workers, patients, and their families, and care of respiratory tract equipment to prevent the transmission of infectious agents serve as the foundations of infection control and prevent the acquisition of potential pathogens by patients with CF . The respiratory secretions of all CF patients potentially harbor clinically and epidemiologically important microorganisms, even if they have not yet been detected in cultures from the respiratory tract . CF patients should be educated to contain their secretions and maintain a distance of >3 ft from other CF patients to avoid the transmission of potential pathogens, even if culture results are unavailable or negative . To prevent the acquisition of pathogens from respiratory therapy equipment used in health care settings as well as in the home, such equipment should be cleaned and disinfected . It will be critical to measure the dissemination, implementation, and potential impact of these guidelines to monitor changes in practice and reduction in infections.

Emerg Infect Dis, 2003 Nov, 9(11), 1484 - 5
Cutaneous melioidosis and necrotizing fasciitis caused by Burkholderia pseudomallei; Wang YS et al.; In areas where melioidosis is endemic, stress on the healthcare system is substantial . Because clinical manifestations are protean, the illness is difficult to diagnose, and cutaneous Burkholderia pseudomallei infections can progress to necrotizing fasciitis . While it is and uncommon complication of cutaneous melioidosis, necrotizing fasciitis is potentially fatal and requires aggressive management, including early diagnosis, appropriate antibiotics selection and operative debridement.

Chemosphere, 2004 Apr, 55(1), 27 - 33
Haloalkane and haloacid dehalogenases from aerobic bacterial isolates indigenous to contaminated sites in Africa demonstrate diverse substrate specificities; Olaniran AO et al.; Five bacteria were isolated from contaminated sites in Nigeria and South Africa using the culture enrichment technique . They were subjected to standard cultural, biochemical and microbiological techniques and identified to be species of Bacillus, Burkholderia, Corynebacterium, Micrococcus and Pseudomonas . Axenic cultures of the bacterial isolates utilized 1,2-dichloroethane (1,2-DCE) as the sole carbon source up to a final substrate concentration of 10 mM . Their mean generation time in 1,2-DCE ranged significantly (P<0.05) from 9.77 to 15.72 h with the maximum chloride release ranging between 59% and 86% . All the bacterial isolates produced two different dehalogenases, viz . one which is heat labile and specific for halogenated alkanes with optimum activity at a pH of 7.5 and the other which is more heat stable with a higher pH optimum of 9.0 and specific for halogenated alkanoic acids . However, the two enzyme types when tested demonstrated wide substrate specificities . It is therefore adjudged that these organisms may play a vital role in the bioremediation of sites polluted with chlorinated hydrocarbons.

Emerg Infect Dis, 2003 Dec, 9(12), 1538 - 42
Intensity of rainfall and severity of melioidosis, Australia; Currie BJ et al.; In a 12-year prospective study of 318 culture-confirmed cases of melioidosis from the Top End of the Northern Territory of Australia, rainfall data for individual patient locations were correlated with patient risk factors, clinical parameters, and outcomes . Median rainfall in the 14 days before admission was highest (211 mm) for those dying with melioidosis, in comparison to 110 mm for those surviving (p=0.0002) . Median 14-day rainfall was also significantly higher for those with pneumonia . On univariate analysis, a prior 14-day rainfall of 125 mm was significantly correlated with pneumonia (odds ratio {OR} 1.70 {confidence interval {CI} 1.09 to 2.65}), bacteremia (OR 1.93 {CI 1.24 to 3.02}), septic shock (OR 1.94 {CI 1.14 to 3.29}), and death (OR 2.50 {CI 1.36 to 4.57}) . On multivariate analysis, rainfall in the 14 days before admission was an independent risk factor for pneumonia (p=0.023), bacteremic pneumonia (p=0.001), septic shock (p=0.005), and death (p<0.0001) . Heavy monsoonal rains and winds may cause a shift towards inhalation of Burkholderia pseudomallei.

Vet Q, 2003 Dec, 25(4), 165 - 9
First cases of animal diseases published since 2000 . 5 . Sheep; Elsinghorst TA; In this fifth article of a series of papers listing first case reports of animal diseases published since 2000, the following five cases of sheep diseases are discussed: Ependymoma . Mastitis caused by Burkholderia cepacia infection . Meningoencephalitis associated with Globicatella sanguinis infection . Neospora caninum infection in an adult sheep and her twin fetuses . Plasma cell tumor . After a short introduction, the bibliographical data, the abstract of the author(s), and some additional information derived from the article are given . The article will be regularly updated adding overlooked as well as new first reports.

J Bacteriol, 2004 Jan, 186(2), 270 - 7
Isolation and characterization of Burkholderia cenocepacia mutants deficient in pyochelin production: pyochelin biosynthesis is sensitive to sulfur availability; Farmer KL et al.; The opportunistic pathogen Burkholderia cenocepacia produces the yellow-green fluorescent siderophore, pyochelin . To isolate mutants which do not produce this siderophore, we mutagenized B . cenocepacia with the transposon mini-Tn5Tp . Two nonfluorescent mutants were identified which were unable to produce pyochelin . In both mutants, the transposon had integrated into a gene encoding an orthologue of CysW, a component of the sulfate/thiosulfate transporter . The cysW gene was located within a putative operon encoding other components of the transporter and a polypeptide exhibiting high homology to the LysR-type regulators CysB and Cbl . Sulfate uptake assays confirmed that both mutants were defective in sulfate transport . Growth in the presence of cysteine, but not methionine, restored the ability of the mutants to produce pyochelin, suggesting that the failure to produce the siderophore was the result of a depleted intracellular pool of cysteine, a biosynthetic precursor of pyochelin . Consistent with this, the wild-type strain did not produce pyochelin when grown in the presence of lower concentrations of sulfate that still supported efficient growth . We also showed that whereas methionine and certain organosulfonates can serve as sole sulfur sources for this bacterium, they do not facilitate pyochelin biosynthesis . These observations suggest that, under conditions of sulfur depletion, cysteine cannot be spared for production of pyochelin even under iron starvation conditions.

Microbiol Immunol, 2003, 47(12), 915 - 25
Need to differentiate lethal toxin-producing strains of Burkholderia gladioli, which cause severe food poisoning: description of B . gladioli pathovar cocovenenans and an emended description of B . gladioli; Jiao Z et al.; Burkholderia cocovenenans produces a lethal toxin (Bongkrekic acid) that leads to high fatality in food poisoning cases . However, B . cocovenenans was combined in Burkholderia gladioli in 1999 . B . gladioli was originally described as a phytopathogenic bacteria that sometimes causes pneumonia in humans and that acts as an opportunistic pathogen . We thought that it was clinically dangerous to describe these two species without considering their pathogenicities . From our data of 16S rRNA sequence analysis, DNA-DNA hybridization, and fatty acid analysis, we could confirm that B . cocovenenans and B . gladioli should be categorized as a single species . However the species really weaved lethal toxin-producing strains with non-lethal strains . To emphasize that B . gladioli contains two different pathogens, we describe a new pathovar, B . gladioli pathovar cocovenenans, for the lethal toxin-producing strains . We provide characteristics that differentiate this lethal toxin-producing pathovar from other phytopathogenic pathovars within B . gladioli, together with an emended description of B . gladioli.

Biochem Biophys Res Commun, 2004 Jan 9, 313(2), 245 - 57
Expression of L-ornithine Ndelta-oxygenase (PvdA) in fluorescent Pseudomonas species: an immunochemical and in silico study; Putignani L et al.; Omega-amino acid monooxygenases (EC 1.14.13.-), catalysing the formation of hydroxamate precursors of microbial siderophores (e.g., pyoverdine), have so far eluded structural and biochemical characterisation . Here, the expression of recombinant L-ornithine-Ndelta-oxygenase (PvdA) from Pseudomonas aeruginosa PAO1 is reported . A library of eight monoclonal antibodies (MAbs) directed against PvdA has been generated . Two MAb families recognising the N- and C-terminal regions of PvdA were identified . The MAbs made it possible to demonstrate that 45-48 kDa PvdA homologues are expressed in response to iron limitation by different species and strains of fluorescent pseudomonads . Despite the different degrees in sequence similarity between P . aeruginosa PvdA and putative homologues from Pseudomonas fluorescens, Pseudomonas putida, Pseudomonas syringae, Burkholderia cepacia, and Ralstonia solanacearum, in silico domain scanning predicts an impressive conservation of putative cofactor and substrate binding domains . The MAb library was also used to monitor PvdA expression during the transition of P . aeruginosa from iron-sufficient to iron-deficient growth.

Indian J Med Res, 2003 Aug, 118, 68 - 70
Indirect immunofluorescent antibody test for the rapid diagnosis of melioidosis; Mathai E et al.; Culture is the only reliable method available at present for the diagnosis of melioidosis . Though serological tests have been described, their value in routine diagnosis is controversial . All indirect immunofluorescent assay (IFA) was therefore evaluated to determine its use in the diagnosis of melioidosis . Whole cell antigen prepared from a laboratory isolate of Burkholderia pseudomallei was used to assay IgG and IgM antibodies . Fourteen of the 22 (63.6%) culture proven cases had IgM antibodies while only 10 (45.5%) had IgG antibodies . Negative predictive value of IgM assay was 92 per cent . Positive predictive value was 100 per cent if both IgM and IgG were considered together . The present study done on a limited number of samples suggests that IFA may be useful in routine diagnosis of melioidosis.

Mikrobiologiia, 2003 Sep-Oct, 72(5), 666 - 71
{The use of the {13C}/{12C} ratio for the assay of the microbial oxidation of hydrocarbons}; Ziakun AM et al.; The study deals with a comparative analysis of the relative abundances of the carbon isotopes 12C and 13C in the metabolites and biomass of the Burkholderia sp . BS3702 and Pseudomonas putida BS202-p strains capable of utilizing aliphatic (n-hexadecane) and aromatic (naphthalene) hydrocarbons as sources of carbon and energy . The isotope composition of the carbon dioxide, biomass, and exometabolites produced during the growth of Burkholderia sp . BS3702 on n-hexadecane (delta 13C = -44.6 +/- 0.2@1000) were characterized by the isotope effects delta 13CCO2 = -50.2 +/- 0.4@1000, delta 13Cbiom = -46.6 +/- 0.4@1000 and delta 13Cexo = -41.5 +/- 0.4@1000, respectively . The isotope composition of the carbon dioxide, biomass, and exometabolites produced during the growth of the same bacterial strain on naphthalene (delta 13C = -21 +/- 0.4@1000) were characterized by the isotope effects delta 13CCO2 = -24.1 +/- 0.4@1000, delta 13Cbiom = -19.2 +/- 0.4@1000 and delta 13Cexo = -19.1 +/- 0.4@1000, respectively . The possibility of using the isotope composition of metabolic carbon dioxide for the rapid monitoring of the microbial degradation of petroleum hydrocarbons in the enviroment is discussed.

Clin Infect Dis, 2004 Jan 1, 38(1), 32 - 7 Epub 2003 Dec 04.
Adjunctive granulocyte colony-stimulating factor for treatment of septic shock due to melioidosis; Cheng AC et al.; Melioidosis, caused by the intracellular pathogen Burkholderia pseudomallei, is endemic in northern Australia and Southeast Asia . Risk factors for this infection have also been associated with functional neutrophil defects . Because of this, granulocyte colony-stimulating factor (G-CSF) was adopted for use in patients with septic shock due to melioidosis in December 1998 . We compared the mortality rates from before and after the introduction of G-CSF therapy at the Royal Darwin Hospital (Darwin, Australia) during the period of 1989-2002 . The mortality rate decreased from 95% to 10% after the introduction of G-CSF . Risk factors, the duration of illness before presentation, and the severity of illness were similar in both groups . A smaller decrease in mortality among patients in the intensive care unit who did not have melioidosis was observed, suggesting that other changes in management did not account for the magnitude of the benefit seen . We conclude that G-CSF may have contributed to the reduction in the mortality rate among patients with septic shock due to melioidosis.

Proc Natl Acad Sci U S A, 2003 Dec 23, 100(26), 15364 - 9 Epub 2003 Dec 12.
Protection of Cftr knockout mice from acute lung infection by a helper-dependent adenoviral vector expressing Cftr in airway epithelia; Koehler DR et al.; We developed a helper-dependent adenoviral vector for cystic fibrosis lung gene therapy . The vector expresses cystic fibrosis transmembrane conductance regulator (Cftr) using control elements from cytokeratin 18 . The vector expressed properly localized CFTR in cultured cells and in the airway epithelia of mice . Cftr RNA and protein were present in whole lung and bronchioles, respectively, for 28 days after a vector dose . Acute inflammation was minimal to moderate . To test the therapeutic potential of the vector, we challenged mice with a clinical strain of Burkholderia cepacia complex (Bcc) . Cftr knockout mice (but not Cftr+/+ littermates) challenged with Bcc developed severe lung histopathology and had high lung bacteria counts . Cftr knockout mice receiving gene therapy 7 days before Bcc challenge had less severe histopathology, and the number of lung bacteria was reduced to the level seen in Cftr+/+ littermates . These data suggest that gene therapy could benefit cystic fibrosis patients by reducing susceptibility to opportunistic pathogens.

Carbohydr Res, 2003 Nov 14, 338(23), 2687 - 95
Exopolysaccharides produced by a clinical strain of Burkholderia cepacia isolated from a cystic fibrosis patient; Cescutti P et al.; Burkholderia cepacia is an opportunistic pathogen involved in pulmonary infections related to cystic fibrosis . A clinical strain, BTS13, was isolated and the production of exopolysaccharides was tested growing the bacteria on two different media, one of which was rich in mannitol as carbon source . The primary structure of the polysaccharides was determined using mostly mass spectrometry and NMR spectroscopy . On both media an exopolysaccharide having the following repeating unit was produced: -->5)-beta-Kdop-(2-->3)-beta-D-Galp2Ac-(1-->4)-alpha-D-Galp-(1-->3)-beta-D-Galp-(1--> This polysaccharide has already been described as the biosynthetic product of another Burkholderia species, B . pseudomallei, the microbial agent causing melioidosis . In addition to this, when grown on the mannitol-rich medium, B . cepacia strain BTS13 produced another polysaccharide that was established to be levan: -->6)-beta-D-Fruf-(2--> . The content of levan was about 20% (w/w) of the total amount of polymers . The ability of B . cepacia to produce these two exopolysaccharides opens new perspectives in the investigation of the role of polysaccharides in lung infections.

Carbohydr Res, 2003 Nov 14, 338(23), 2659 - 66
Structure of the core-oligosaccharide with a characteristic D-glycero-alpha-D-talo-oct-2-ulosylonate-(2-->4)-3-deoxy-D-manno-oct-2-ulosonate {alpha-Ko-(2-->4)-Kdo} disaccharide in the lipopolysaccharide from Burkholderia cepacia; Isshiki Y et al.; The core oligosaccharide in the lipopolysaccharide (LPS) of Burkholderia cepacia GIFU 645(T) was investigated . After mild acid hydrolysis of the LPS, a heptasaccharide was isolated and identified by chemical analyses, GLC-MS, FABMS, and NMR spectroscopy as follows: {carbohydrate structure: see text} where L-alpha-D-Hep stands for L-glycero-alpha-D-manno-heptose, Ko for D-glycero-D-talo-oct-2-ulosonic acid, and Kdo for 3-deoxy-D-manno-oct-2-ulosonic acid.

Free Radic Res, 2003 Sep, 37(9), 909 - 17
Lipid peroxidation of lung surfactant due to reactive oxygen species released from phagocytes stimulated by bacteria from children with cystic fibrosis; Bouhafs RK et al.; We used Pseudomonas aeruginosa, Burkholderia cepacia and Stenotrophomonas maltophilia, live or heat-killed, isolated from the airways of children with Cystic Fibrosis, to stimulate human neutrophils (PMN) and rat alveolar macrophages (AM) to produce reactive oxygen metabolites in the presence or absence of Curosurf, a natural porcine lung surfactant . We determined: (1) the amount of lipid peroxidation (LPO) as assessed by the amounts of malondialdehyde (MDA) and 4-hydroxyalkenals (4-HNE) using the LPO 586 test kit; (2) the production by AM of superoxide with the nitroblue tetrazolium test and (3) of nitric oxide (NO) with the Griess reaction . Stimulation of PMN or AM increases LPO of Curosurf and cell wall lipids . In both types of phagocytes, B . cepacia induced the highest LPO levels followed by P . aeruginosa and S . maltophilia . PMN, stimulated by live bacteria, induced higher LPO than those stimulated by heat-killed bacteria . B . cepacia stimulated AM to produce more superoxide and NO than did P . aeruginosa and S . maltophilia . The high phagocyte-stimulating ability of B . cepacia and its higher surfactant LPO than those of the other bacteria used in this in vitro study may play a role in vivo in the serious clinical condition known as the "Cepacia syndrome".

Anal Bioanal Chem, 2004 Feb, 378(4), 1014 - 20 Epub 2003 Dec 11.
A simple and robust set-up for on-column sample preconcentration--nano-liquid chromatography--electrospray ionization mass spectrometry for the analysis of N-acylhomoserine lactones; Frommberger M et al.; A simple method for the simultaneous, rapid and sensitive determination of N-acylhomoserine lactone signaling molecules in bacterial isolates, without prior sample preconcentration and with minimal sample cleanup, is presented . The analysis relies on the combination of analyte preconcentration and separation on a single device: a relatively large sample volume (1-5 microL) is directly loaded onto a laboratory-made, miniaturized (75 microm i . d.) reverse phase nano-liquid chromatography column, connected on-line to a microelectrospray-ionization ion trap mass spectrometer . In a first step the analyte is adsorbed (and so concentrated) at the beginning of the column, and is eluted and selectively separated in a second step by the organic mobile phase . Sample preconcentration follows the mechanisms of solid phase extraction on a nano-scale, while separation takes place according to classical liquid chromatography separation principles . The columns can be manufactured easily, are simply connected, and used with minimal solvent amounts; this makes this method extremely robust and cost-effective . The analytical setup was found to be routinely quantitative down to a concentration of 10 ng/mL (corresponding to a total analyte amount of 10 pg or ca . 50 fmol) . The limit of detection was reached at 1 ng/mL (1 pg, ca . 5 fmol) . Compared to the classical AHL analysis of bacterial cultures with biosensors, where selectivity and sensitivity is often limited, this rapid analytical technique is a substantial qualitative and quantitative improvement . Two unsubstituted N-acylhomoserine lactones could be identified and quantified from a Burkholderia cepacia culture supernatant in a chloroform extract.

Syst Appl Microbiol, 2003 Nov, 26(4), 595 - 600
Isolation of Burkholderia cepacia complex genomovars from waters; Vermis K et al.; The aim of this study was to develop a selective enrichment broth as an aid for the isolation of Burkholderia cepacia complex (Bcc) bacteria from water . To allow growth of all nine genomovars, mixtures of two carbon sources had to be used, i.e . L-arabinose/D-cellobiose or L-arabinose/L-threonine . Selectivity was provided by polymyxin B and 9-chloro-9-(4-diethylaminophenyl)-10-phenylacridan (C-390) . Following enrichment, Bcc bacteria were isolated on a diagnostic O/F agar supplemented with gentamicin . A preliminary bio-diversity study on 28 surface waters yielded five different genomovars, i.e . B . cepacia (genomovar I), B . multivorans, B . cenocepacia, B . vietnamiensis and B . anthina . Drinking waters did not contain Bcc bacteria . However, the genomovar pattern from a given sample varied with the enrichment broth used.

Chemosphere, 2004 Feb, 54(8), 1255 - 65
Effect of temperature and dissolved oxygen on the growth kinetics of Pseudomonas putida F1 growing on benzene and toluene; Alagappan G et al.; Batch experiments were conducted to determine the effect of temperature and dissolved oxygen concentration on the rates of growth and substrate (benzene and toluene) degradation by the toluene degrading strain, Pseudomonas putida F1 . Over a range of temperature from 15 to 35 degrees C the maximum specific growth rate followed the Topiwala-Sinclair relationship when either benzene or toluene served as the sole carbon and energy source . Oxygen limited growth followed Monod saturation kinetics with the specific growth rate given as a function of the dissolved oxygen concentration . The oxygen half-saturation coefficient was found to be approximately 1 mg/l regardless of whether benzene or toluene was the substrate . Similar experiments with Burkholderia (Ralstonia) pickettii PKO1 for grown on toluene revealed an oxygen half-saturation coefficient of 0.7 mg/l.

Mol Gen Mikrobiol Virusol, 2003, (4), 15 - 20
{Clinical strains of Burkholderia cepacia: characteristic and detection of the components in the quorum sensing regulatory system}; Shaginian IA et al.; Described in the paper are characteristics of B . cepacia clinical strains isolated from patients at Moscow hospitals . The strains were investigated for the presence of proteolytic, chitinolytic, hemolytic and lipase activities as well as for presence of components of the "Quorum sensing" gene activity regulatory system by using biological test-systems and in the polymerase chain reaction with primers to genes cepI and cepR.

Can J Microbiol, 2003 Oct, 49(10), 613 - 24
Putative virulence factors are released in association with membrane vesicles from Burkholderia cepacia; Allan ND et al.; Like many other Gram-negative bacteria, Burkholderia cepacia naturally releases membrane vesicles (n-MVs) during normal growth . Through filtration and differential centrifugation, n-MVs from clinical isolates of the IIIa and V genomovars were isolated and their characteristics compared . Electron microscopy revealed that they were spherical, 30-220 nm in diameter, and bilayered . Virulence factors thought to play a role in pathogenicity (e.g., lipase, phospholipase-N, and protease, including a metalloprotease) were found associated with n-MVs, while peptidoglycan zymogram analysis also revealed 26, 28, 36, and 66 kDa peptidoglycan-degrading enzymes . n-MVs were often contaminated with flagella and pili when isolated by traditional methods, and a new strategy using a linear isopycnic sucrose gradient was utilized . For better characterization, this was applied to a representative genomovar IIIa strain (C5424) and showed that n-MVs consisted of a subset of specific outer membrane and periplasmic proteins as well as lipopoly saccharide possessing only a putative minor O-side chain polymer . This finding suggests that certain components are selected by B . cepacia during n-MV formation, and since some are putative virulence factors, this property could help deliver the factors to tissue, thereby aiding infection.

Microbiology, 2003 Dec, 149(Pt 12), 3649 - 58
The CepIR quorum-sensing system contributes to the virulence of Burkholderia cenocepacia respiratory infections; Sokol PA et al.; The cepIR genes encode an N-acyl homoserine lactone (AHL)-dependent quorum-sensing system consisting of an AHL synthase that directs the synthesis of N-octanoyl-L-homoserine lactone (ohl) and n-hexanoyl-L-homoserine lactone and a transcriptional regulator . The virulence of cepIR mutants was examined in two animal models . Rats were infected with agar beads containing Burkholderia cenocepacia K56-2, K56-I2 (cepI : : Tp(r)) or K56-R2 (cepR : : Tn5-OT182) . At 10 days post-infection, the extent of lung histopathological changes was significantly lower in lungs infected with K56-I2 or K56-R2 compared to the parent strain . Intranasal infections were performed in Cftr((-/-)) mice and their wild-type siblings . K56-2 was more virulent in both groups of mice . K56-I2 was the least virulent strain and was not invasive in the Cftr((-/-)) mice . OHL was readily detected in lung homogenates from Cftr((-/-)) mice infected with K56-2 but was only detected at levels slightly above background in a few mice infected with K56-I2 . Lung homogenates from mice infected with K56-2 had significantly higher levels of the inflammatory mediators murine macrophage inflammatory protein-2, KC/N51, interleukin-1beta and interleukin-6 than those from K56-I2-infected animals . These studies indicate that a functional CepIR quorum-sensing system contributes to the severity of B . cenocepacia infections . A zinc metalloprotease gene (zmpA) was shown to be regulated by CepR and may be one of the factors that accounts for the difference in virulence between the cepI mutant and the parent strain.

J Clin Microbiol, 2003 Dec, 41(12), 5750 - 4
Molecular comparison of isolates of Burkholderia multivorans from patients with cystic fibrosis in the United Kingdom; Turton JF et al.; Burkholderia multivorans strains from 47 cystic fibrosis (CF) patients in 28 hospitals were compared by pulsed-field gel electrophoresis (PFGE) and flagellin (fliC) PCR-restriction fragment length polymorphism (PCR-RFLP) analysis . A considerable degree of genetic variation was evident, with each patient harboring a strain with a unique PFGE profile . Four sizes of fliC amplicons were produced, and these amplicons gave 13 RFLP types with restriction enzyme MspI . B . multivorans did not appear to spread between patients, suggesting that most CF patients acquire the organism from the natural environment.

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