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 deg