Burkholderia

Int J Syst Evol Microbiol, 2001 Jan, 51(Pt 1), 183 - 5
Genotypic and chemotaxonomic evidence for the reclassification of Pseudomonas woodsii (Smith 1911) Stevens 1925 as Burkholderia andropogonis (Smith 1911) Gillis et al . 1995; Coenye T et al.; It was reported before that {Pseudomonas} woodsii and Burkholderia andropogonis are phenotypically indistinguishable and probably represent the same taxon, for which the name B . andropogonis has been proposed . In the present study, it was found that {P.} woodsii and B . andropogonis strains were indistinguishable by whole-cell protein electrophoresis and have a highly similar cellular fatty acid composition . A high DNA-DNA binding value of 95% was found between the type strains of both species . In addition, the 16S rDNA sequence of {P.} woodsii strain LMG 2362T was very similar to that of B . andropogonis LMG 2129T (99.0%) . The chemotaxonomic and genotypic data confirm that {P.} woodsii and B . andropogonis represent the same species, for which it is proposed to retain the name B . andropogonis.

J Med Microbiol, 2001 Feb, 50(2), 116 - 26
Type III secretion systems and pathogenicity islands; Winstanley C et al.; Some bacterial pathogens have evolved by acquiring pathogenicity islands (PIs), which are clusters of genes encoding virulence traits . PIs encoding the secretion of effector molecules via type III secretion (TTS) systems have been discovered in several gram-negative pathogens . TTS systems are involved in contact-dependent secretion of virulence factors and can facilitate delivery of toxins directly into target cells . The expanding list of bacteria found to contain clusters of TTS genes includes members of the genera Yersinia, Salmonella, Shigella, Escherichia, Pseudomonas, Bordetella, Burkholderia, Chlamydia and a number of plant pathogens or symbionts . This review discusses the current knowledge of the role of TTS PIs in pathogenicity, the genetic organisation and evolution of such systems,and the potential for using TTS systems as targets for novel treatments.

Zh Mikrobiol Epidemiol Immunobiol, 2000 Nov-Dec, (6), 94 - 9
{Pathogenicity of Burkholderia pseudomallei: extracellular and surface antigen functions}; Piven' NN et al.; The data of literature and the results of investigations carried out by the authors on the analysis of B . pseudomallei pathogenicity factors . They include mucoid, endotoxin, lecithinase, proteases, hemolysins, etc . In addition to fimbriae and pili, the adhesive properties of B . pseudomallei are supposed to be associated with surface capsule-like structures whose composition includes Ag8 . The characterization of exoproteases, hemolysins and lethal toxins is given . The virulence and immunogenicity of B . pseudomallei were shown to be linked with the components of surface glycoprotein Ag8 . The conclusion has been made that the analyzed pathogenicity factors are of importance for deciphering the pathogenesis of melioidosis.

Ceylon Med J, 2000 Sep, 45(3), 116 - 8
The isolation of Burkholderia cepacia in a hospital setting in Sri Lanka; Perera N et al.; INTRODUCTION: Burkholderia cepacia is widely prevalent in nature . The natural habitat of this organism is soil, water and vegetation, but the hospital environment remains the main source of infection . It causes a wide variety of infections in hospitalised patients . Since there are no reports of its prevalence in Sri Lanka, a data retrieval and analysis was undertaken to determine its incidence among patients at Sri Jayawardenepura General Hospital (SJGH) . OBJECTIVE: To determine the prevalence, morphological characteristics, biochemical profile and antibiotic susceptibility pattern of B cepacia in a Sri Lankan tertiary care hospital . METHODS: Relevant clinical data were retrieved from all isolates of B cepacia from SJGH for 12 months from October 1996 . Standard procedures were used to isolate the organism from clinical specimens . API 20E was employed for biochemical identification . Antibiotic susceptibility tests was carried out using the modified Kirby Bauer method . RESULTS: B cepacia was isolated from 17 patients . 16 of them were hospitalised and were from the medical, surgical, and intensive care units . All were in a compromised state of health . The organism was isolated from a variety of specimens which included swabs of surgical wounds, chronic ulcers, sputum, bronchial lavage, endotracheal aspirate, urine, peritoneal fluid and blood . Blood agar, MacConkey agar and cystine lactose electrolyte deficient medium supported the growth of all strains . They were motile Gram negative rods . All strains were oxidase positive . Strains gave variable results with other biochemical tests . Antibiograms too were variable . All strains were sensitive to ceftazidime, and the majority of the strains were sensitive to the other third generation cephalosporines . All strains were resistant to four or more antimicrobial agents included in the study . Of the 17 patients from whom B cepacia was isolated, only 9 seem to have had actual infection; others were probably instances of colonisation or contamination . CONCLUSIONS: The present report confirms the prevalence and importance of B cepacia as a hospital pathogen in Sri Lanka . Hospital laboratories should be equipped to isolate, identify and perform antibiotic sensitivity test on these strains . Antibiotic susceptibility testing is necessary as the patterns seem to differ among strains . The multiple drug resistant nature of the organism warrants strict infection control measures to prevent spread in a hospital setting.

J Med Microbiol, 2001 Jan, 50(1), 55 - 61
Distribution of immunoglobulin classes and IgG subclasses against a culture filtrate antigen of Burkholderia pseudomallei in melioidosis patients; Chenthamarakshan V et al.; The class and subclass distribution of antibody response to the culture filtrate antigen (CFA) of Burkholderia pseudomallei was examined in the sera of 45 septicaemic and 17 localised melioidosis cases and 40 cases clinically suspected of melioidosis and the results were compared with those from high-risk and healthy control groups . The geometric mean titre index (GMTI) values for all classes and subclasses of immunoglobulins examined were higher for sera from the proven and clinically suspected melioidosis cases than for the control groups . However, the highest response in the three patient groups was that of IgG with GMTIs ranging from 219.4 to 291.6 and the lowest was for IgM with GMTIs of 22.5, 24.3 and 28.7 . The IgA response was intermediate with GMTIs ranging from 119.2 to 170 . The GMTIs were highest for IgG in septicaemic and localised infections and for IgA and IgM in localised infections . As regards IgG subclass distribution, IgG1 and IgG2 were the predominant subclasses produced against the CFA in contrast to IgG3 and IgG4, which were produced in low amounts . None of the sera from the control groups had any significant titres of antibodies.

Am J Respir Crit Care Med, 2001 Jan, 163(1), 43 - 8
Infection with Burkholderia cepacia in cystic fibrosis: outcome following lung transplantation; Chaparro C et al.; As a result of concern over excessive mortality after lung transplantation, many transplant programs refuse to accept cystic fibrosis (CF) patients infected with Burkholderia cepacia . As a significant proportion of patients with CF in our community are infected with this organism, we have continued to provide lung transplantation as an option . A retrospective review was conducted of medical records of all patients with CF transplanted between March 1988 and September 1996 . Fifty-six transplant procedures were performed in 53 recipients with CF between March 1988 and September 1996 . Twenty-eight had B . cepacia isolated pretransplant and 25 remaining positive post-transplant . Of the 53 recipients, 19 have died (15 of 28 {54%} B . cepacia positive and 4 of 25 {16%} B . cepacia negative) . B . cepacia was responsible for or involved in 14 deaths . Nine of the deaths occurred in the first 3 mo post-transplantation . One-year survival was 67% for B . cepacia positive patients and 92% for B . cepacia negative patients . Recent modifications in antimicrobial and immunosuppressive therapy since 1995 have resulted in no deaths early post-transplant in the last five patients transplanted . We conclude that early mortality in patients with CF infected with B . cepacia is significantly higher than in those not infected with B . cepacia . Modifications in post-transplant medical therapy may improve outcome.

Environ Microbiol, 1999 Oct, 1(5), 393 - 9
Development and characterization of a lux-modified 2,4-dichlorophenol-degrading Burkholderia sp . RASC; Shaw LJ et al.; lux-marked biosensors for assessing the toxicity and bioremediation potential of polluted environments may complement traditional chemical techniques . luxCDABE genes were introduced into the chromosome of the 2,4-dichlorophenol (2,4-DCP)-mineralizing bacterium, Burkholderia sp . RASC c2, by biparental mating using the Tn4431 system . Experiments revealed that light output was constitutive and related to cell biomass concentration during exponential growth . The transposon insertion was stable and did not interrupt 2,4-DCP-degradative genes, and expression of luxCDABE did not constitute a metabolic burden to the cell . A bioluminescence response was detectable at sublethal 2,4-DCP concentrations: at < 10.26 microg ml(-1), bioluminescence was stimulated (e.g . 218% of control), but at concentrations >60 microg ml(-1) it declined to < 1% . Investigating the effect of {14C}-2,4-DCP concentration on the evolution of 14CO2 revealed that, for initial concentrations of 2.5-25 microg ml(-1), approximately equals 55% of the added 14C was mineralized after 24 h compared with <1% at 50 and 100 microg ml(-1) . Inhibition of 2,4-DCP mineralization between 25 and 50 microg ml(-1) corresponded well to the EC50 value (33.83 microg ml(-1)) obtained from bioluminescence inhibition studies . lux-marked RASC c2 may therefore be used as a functionally (i.e . 2,4-DCP degrader) and environmentally relevant biosensor of toxicity and biodegradation inhibition.

Environ Microbiol, 1999 Jun, 1(3), 199 - 212
Identification of the metabolically active members of a bacterial community in a polychlorinated biphenyl-polluted moorland soil; Nogales B et al.; The presumptive metabolically active members of a bacterial community in a moorland soil in Germany, highly polluted with polychlorinated biphenyls (PCBs), were identified by sequencing of cloned reverse transcription-polymerase chain reaction (RT-PCR) amplification products of 16S rRNA generated from total RNA extracts . Analysis of the 16S rRNA clone library revealed a considerable diversity of metabolically active bacteria in the soil, despite the acidic pH and high concentrations of PCBs . Cloned sequence types clustered within the Proteobacteria (34% alpha-, 33% beta- and 7% gamma-subclasses), the Holophaga-Acidobacterium phylum (14%), the Actinobacteria (6.5%) and the Planctomycetales (2%) . Three cloned sequence types were not affiliated to any described phylogenetic group . An unusual feature of this soil was the abundance of sequence types within the beta-subclass of the Proteobacteria, most of which were similar to the 16S rRNA gene sequences of species from only two genera, Burkholderia and Variovorax . Three other numerous 16S rRNA sequence types were similar to the sequences of Sphingomonas species, members of the Rhodopila globiformis group and Acidobacterium capsulatum . Some of the sequence types retrieved were similar to the 16S rRNA sequences of bacterial isolates able to degrade a variety of organic pollutants, including PCBs . As the PCB contamination is the major source of measurable carbon in this soil, some of the 16S rRNA sequence types detected and presumed to represent the metabolically active members of the community indicate the organisms likely to be involved, directly or indirectly, in the utilization of the PCBs as carbon and energy sources.

Phytother Res, 2001 Feb, 15(1), 39 - 43
Activity of plant flavonoids against antibiotic-resistant bacteria; Xu HX et al.; Thirty eight plant-derived flavonoids representing seven different structural groups were tested for activities against antibiotic-resistant bacteria using the disc-diffusion assay and broth dilution assay . Among the flavonoids examined, four flavonols (myricetin, datiscetin, kaempferol and quercetin) and two -flavones (flavone and luteolin) exhibited inhibitory activity against methicillin-resistant Staphylococcus aureus (MRSA) . Myricetin was also found to inhibit the growth of multidrug-resistant Burkholderia -cepacia, vancomycin-resistant enterococci (VRE) and other medically important organisms such as -Klebsiella pneumoniae and Staphylococcus epidermidis . Myricetin was bactericidal to B . cepacia . The results of the radiolabel incorporation assay showed that myricetin inhibited protein synthesis by -B . cepacia . The structure-activity relationship of these flavonoids is discussed .

Gene, 2001 Jan 10, 262(1-2), 137 - 45
Expression of 2-halobenzoate dioxygenase genes (cbdSABC) involved in the degradation of benzoate and 2-halobenzoate in Burkholderia sp . TH2; Suzuki K et al.; Burkholderia sp . TH2, isolated from soil, utilizes 2-chlorobenzoate (2CB) and benzoate (BA) as its sole source of carbon and energy . The genes for 2-halobenzoate dioxygenase (cbdABC) from Burkholderia sp . TH2 were cloned and sequenced . The predicted amino acid sequences of all the gene products are highly similar to the cbd gene products of Pseudomonas sp . 2CBS . Disruption of the promoter region of cbdA resulted in loss of growth on 2CB and BA, indicating that these genes are involved in the growth of TH2 on these substrates . Expression of the cbd genes was analyzed by transcriptional fusion assay . The cbdS gene, a possible araC/xylS-type transcriptional regulatory gene, was shown to positively regulate the expression of cbdA . In addition, the effectors of CbdS were shown to be 2CB, 2-bromobenzoate, o-toluate (2-methylbenzoate), 2-iodobenzoate, and BA . Primer extension analysis showed that the cbdA mRNA started at two positions, 14 and 15 nucleotides upstream from the cbdA start codon, ATG . A pair of direct repeats, identical to that of the Pm promoter of the TOL plasmid, was found upstream of -35 hexamer of the cbdA promoter.

FEMS Microbiol Lett, 2001 Feb 20, 195(2), 231 - 5
Screening and characterization of trehalose-oleate hydrolyzing lipase; Ishimoto R et al.; Various soil samples were collected to screen the presence of microorganisms which have ability to degrade TOE . One strain (AKU-883) with good TOE degrading activity was isolated and identified as Burkholderia cepacia and the extracellular enzyme was purified to homogeneity . The purification was achieved by ultrafiltration, Super Q anion-exchange chromatography and Superdex 200HR gel-filtration in the presence of Triton X . The enzyme was purified to 85-fold, and specific activity of 4.910 kU mg protein(-1) . The peak preparation on gel filtration showed a single band of 34 kDa on SDS-PAGE and native PAGE which indicate the monomeric nature of the enzyme . The pI of the enzyme was 6.3 . The enzyme showed the maximum activity at pH 9 and 65 degrees C, and was stable in the range of pH 5--10 and up to 60 degrees C . Almost all the activity (92%) was kept after incubation for more than 1 week at 50 degrees C (pH 7.3) . High activities remained even in water-miscible solvents such as ethanol, dimethyl formamide, diisopropyl ether, and dioxane . The N-terminal 16 amino acid residues were determined as A-N-G-Y-A-A-T-R-Y-P-I-I-L-V-G-G, which showed a consensus sequence for lipases from Burkholderia species . Thus the enzyme was concluded to be a kind of lipase.

Curr Microbiol, 2001 Apr, 42(4), 269 - 75
Phenotypic and phylogenetic characterization of Burkholderia (Pseudomonas) sp . strain LB400; Fain MG et al.; The relevant phenotypic traits and phylogenetic relationships between Burkholderia (Pseudomonas) sp . strain LB400 and B . cepacia ATCC 25416(T) were compared to determine the degree to which these two strains might be related . Strain LB400 degrades chlorinated biphenyls and has been a model system for potential use in the bioremediation of polychlorinated biphenyls, while some strains of B . cepacia are plant and human pathogens . The fatty acid methyl ester profile, sole carbon source utilization, and biochemical tests confirmed that strain LB400 was a member of the genus Burkholderia . The 16S rRNA gene sequence showed that this strain was not as closely related to B . cepacia as previously suspected or to other known pathogens of this genus, but is closely related to B . phenazinium, B . caribensis, B . graminis, and three unnamed Burkholderia spp . not known to be pathogenic.

Diagn Microbiol Infect Dis, 2001 Jan, 39(1), 1 - 7
Detection of immunoglobulins M and G using culture filtrate antigen of Burkholderia pseudomallei; Chenthamarakshan V et al.; IgM and IgG based ELISA systems were developed using the culture filtrate antigen (CFA) of Burkholderia pseudomallei . The assays were evaluated using 95 sera from 66 septicemic cases and 47 sera from 20 cases with localized melioidosis . In addition 65 sera from culture negative cases that were also serologically negative for other endemic infections clinically suspected of melioidosis were included . These were compared with sera from 260 non-melioidosis cases, 169 sera from individuals with high risk of acquiring the infection and 48 sera from healthy controls . The IgG-ELISA was 96% sensitive and 94% specific . All sera from cases with septicemic and localized infections and 61 of 63 sera from clinically suspected melioidosis cases were positive for IgG antibody . The geometric mean titre index (GMTI) values of IgG antibody in melioidosis cases were significantly higher (p < 0.0005) compared to that of healthy subjects, high risk group and subjects with non-melioidosis infections . The sensitivity and specificity of IgM ELISA was 74 and 99% respectively . The GMTI value of IgM antibody in the sera of melioidosis cases was significantly higher as compared to that of non-melioidosis disease controls (p < or = 0.001) . These results demonstrate that the detection of IgG is a better indicator of the disease in the diagnosis of melioidosis.

FEMS Immunol Med Microbiol, 2001 Feb, 30(1), 53 - 63
Study on the pathophysiology of experimental Burkholderia pseudomallei infection in mice; Gauthier YP et al.; Burkholderia pseudomallei is the etiological agent of melioidosis, a potentially fatal disease occurring in man and animals . The aim of this study was to investigate the pathophysiological course of experimental melioidosis, and to identify the target organs, in an animal model . For this purpose SWISS mice were infected intraperitoneally with the virulent strain B . pseudomallei 6068 . The bacterial load of various organs was quantified daily by bacteriological analysis and by an enzyme-linked immunosorbent assay (ELISA) based on a monoclonal antibody specific to B . pseudomallei exopolysaccharide (EPS) . Electron microscopic investigation of the spleen was performed to locate the bacteria at the cellular level . In this model of acute melioidosis, B . pseudomallei had a marked organ tropism for liver and spleen, and showed evidence of in vivo growth with a bacterial burden of 1.6x10(9) colony forming units (CFU) per gram of spleen 5 days after infection with 200 CFU . The highest bacterial loads were detected in the spleen at all time points, in a range from 2x10(6) to 2x10(9) CFU g(-1) . They were still 50-80 times greater than the load of the liver at the time of peak burden . Other investigated organs such as lungs, kidneys, and bone marrow were 10(2)-10(4)-fold less infected than the spleen, with loads ranging from 3x10(2) to 3x10(6) CFU g(-1) . The heart and the brain were sites of a delayed infection, with counts in a range from 10(3) to 10(7) times lower than bacterial counts in the spleen . The EPS-specific ELISA proved to be highly sensitive, particularly at the level of those tissues in which colony counting on agar revealed low contamination . In the blood, EPS was detected at concentrations corresponding to bacterial loads ranging from 8x10(3) to 6x10(4) CFU ml(-1) . Electron microscopic examination of the spleen revealed figures of phagocytosis, and the presence of large numbers of intact bacteria, which occurred either as single cells or densely packed into vacuoles . Sparse figures suggesting bacterial replication were also observed . In addition, some bacteria could be seen in vacuoles that seemed to have lost their membrane . These observations provide a basis for further investigations on the pathogenesis of the disease.

FEMS Microbiol Lett, 2001 Feb 5, 195(1), 91 - 6
New broad-host-range promoter probe vectors based on the plasmid RK2 replicon; Santos PM et al.; Broad-host-range plasmid RK2-based promoter probe vectors with a known nucleotide sequence were constructed . In the absence of an upstream promoter, the expression of two tested reporter genes (luc and lacZ) in Escherichia coli was virtually zero, while insertion of the Ptrc promoter resulted in strong inducer-dependent expression . The lacZ-based vectors were mobilized into Pseudomonas fluorescens ST, Pseudomonas putida KT2442, Sphingomonas spp . and Burkholderia spp . LB400, and expression analyses indicated that the properties observed in E . coli are maintained across the species barriers . In addition, the previously established knowledge of RK2 molecular biology allows easy manipulations of features such as plasmid copy number, further extending the application potential of the vectors.

Int J Antimicrob Agents, 2001 Feb, 17(2), 109 - 13
Antibiotic susceptibility of Burkholderia pseudomallei from tropical northern Australia and implications for therapy of melioidosis; Jenney AW et al.; From a prospective melioidosis study commencing in 1989 at Royal Darwin Hospital, 170 initial isolates of Burkholderia pseudomallei were available for susceptibility testing . Of these 163 (96%) were susceptible to meropenem/imipenem, ceftazidime, trimethoprim-sulphamethoxazole (SMX/TMP) and doxycycline . Seven (4%) showed primary resistance; three had low-level resistance to SMX/TMP, one to ceftriaxone and amoxycillin/clavulanate (AMOX/CA) and three to doxycycline . Of 167 patients who survived their initial presentation, seven (4%) had culture positive infections which persisted for greater than 3 months after start of therapy . All ultimately cleared carriage of B . pseudomallei though three required changing to SMX/TMP after development of doxycycline resistance . Nineteen (11%) of the initial survivors clinically relapsed and 17 of these had repeat isolates available for testing . Four of these had acquired resistance: one to doxycycline, one to AMOX/CA and ceftazidime, one to SMX/TMP and one to both SMX/TMP and doxycycline . Molecular typing using randomly amplified polymorphic DNA and pulsed-field gel electrophoresis showed all but one relapse isolate to be the same as the original strain . These data are similar to published data from Thailand . As melioidosis has a high mortality (21% in this series) these results emphasize the need for prolonged eradication therapy and regular clinical and microbiological monitoring so that the emergence of resistance can be detected early and appropriate treatment modifications made.

Microbiology, 2001 Jan, 147(Pt 1), 111 - 20
Identification of the acid phosphatase (acpA) gene homologues in pathogenic and non-pathogenic Burkholderia spp . facilitates TnphoA mutagenesis; Burtnick M et al.; Burkholderia pseudomallei and Burkholderia mallei are pathogens responsible for disease in both humans and animals . Burkholderia thailandensis, while phylogenetically similar, is considered avirulent in comparison . These three species exhibit phosphatase activity when grown on media containing chromogenic substrates such as 5-bromo-4-chloro-3-indolyl phosphate (XP) . Tn5-OT182 mutagenesis has been utilized to isolate mutants of B . pseudomallei and B . thailandensis unable to hydrolyse XP . Sequence analysis of these mutants revealed an ORF of 1734 nucleotides demonstrating a high degree of homology to the acpA gene product of Francisella tularensis . PCR primers were designed based on the B . pseudomallei acpA gene sequence and were used to amplify an acpA homologue from B . mallei . The predicted amino acid sequence of B . pseudomallei AcpA differed from those of the predicted B . thailandensis AcpA and B . mallei AcpA by 15 and 3 amino acids, respectively . Allelic exchange was used to construct DeltaacpA mutants in each of these Burkholderia spp . These mutants were shown to be devoid of phosphatase activity and have subsequently allowed for the implementation of phoA fusion transposon mutagenesis systems . Two such systems have been successfully utilized in Burkholderia spp . for the identification of several genes encoding exported proteins.

Microbiology, 2001 Jan, 147(Pt 1), 97 - 109
In vitro resistance of Burkholderia cepacia complex isolates to reactive oxygen species in relation to catalase and superoxide dismutase production; Lefebre M et al.; The Burkholderia cepacia complex comprises groups of genomovars (genotypically distinct strains with very similar phenotypes) that have emerged as important opportunistic pathogens in cystic fibrosis (CF) patients . The inflammatory response against bacteria in the airways of CF individuals is dominated by polymorphonuclear cells and involves the generation of oxidative stress, which leads to further inflammation and tissue damage . Bacterial catalase, catalase-peroxidase and superoxide dismutase activities may contribute to the survival of B . cepacia following exposure to reactive oxygen metabolites generated by host cells in response to infection . In the present study the authors investigated the production of catalase, peroxidase and SOD by isolates belonging to various genomovars of the B . cepacia complex . Production of both catalase and SOD was maximal during late stationary phase in almost all isolates examined . Native PAGE identified 13 catalase electrophoretotypes and two SOD electrophoretotypes (corresponding to an Fe-SOD class) in strains belonging to the six genomovars of the B . cepacia complex . Seven out of 11 strains displaying high-level survival after H(2)O(2) treatment in vitro had a bifunctional catalase/peroxidase, and included all the genomovar III strains examined . These isolates represent most of the epidemic isolates that are often associated with the cepacia syndrome . The majority of the isolates from all the genomovars were resistant to extracellular O(-)(2), while resistance to intracellularly generated O(-)(2)was highly variable and could not be correlated with the detected levels of SOD activity . Altogether the results suggest that resistance to toxic oxygen metabolites from extracellular sources may be a factor involved in the persistence of B . cepacia in the airways of CF individuals.

J Bacteriol, 2001 Mar, 183(5), 1511 - 6
Comparative specificities of two evolutionarily divergent hydrolases involved in microbial degradation of polychlorinated biphenyls; Seah SY et al.; 2-Hydroxy-6-oxo-6-phenylhexa-2,4-dienoate (HOPDA) hydrolase (BphD) is a key determinant in the aerobic transformation of polychlorinated biphenyls (PCBs) by Burkholderia sp . strain LB400 (S . Y . K . Seah, G . Labbe, S . Nerdinger, M . Johnson, V . Snieckus, and L . D . Eltis, J . Biol . Chem . 275:15701-15708, 2000) . To determine whether this is also true in divergent biphenyl degraders, the homologous hydrolase of Rhodococcus globerulus P6, BphD(P6), was hyperexpressed, purified to apparent homogeneity, and studied by steady-state kinetics . BphD(P6) hydrolyzed HOPDA with a k(cat)/K(m) of 1.62 (+/- 0.03) x 10(7) M(-1) s(-1) (100 mM phosphate {pH 7.5}, 25 degrees C), which is within 70% of that of BphD(LB400) . BphD(P6) was also similar to BphD(LB400) in that it catalyzed the hydrolysis of HOPDAs bearing chloro substituents on the phenyl moiety at least 25 times more specifically than those bearing chloro substituents on the dienoate moiety . However, the rhodococcal enzyme was significantly more specific for 9-Cl and 10-Cl HOPDAs, catalyzing the hydrolysis of 9-Cl, 10-Cl, and 9,10-diCl HOPDAs two- to threefold respectively, more specifically than HOPDA . Moreover, 4-Cl HOPDA competitively inhibited BphD(P6) more effectively than 3-Cl HOPDA, which is the inverse of what was observed in BphD(LB400) . These results demonstrate that BphD is a key determinant in the aerobic transformation of PCBs by divergent biphenyl degraders, but that there exists significant diversity in the specificity of these biphenyl hydrolases.

Appl Environ Microbiol, 2001 Feb, 67(2), 982 - 5
Burkholderia cepacia genomovar III Is a common plant-associated bacterium; Balandreau J et al.; A polyphasic taxonomic study involving DNA-DNA hybridization, whole-cell protein electrophoresis, and 16S ribosomal DNA sequence analysis revealed that a group of Burkholderia cepacia-like organisms isolated from the rhizosphere or tissues of maize, wheat, and lupine belong to B . cepacia genomovar III, a genomic species associated with "cepacia syndrome" in cystic fibrosis patients . The present study also revealed considerable protein electrophoretic heterogeneity within this species and demonstrated that the B . cepacia complex consists of two independent phylogenetic lineages.

Appl Environ Microbiol, 2001 Feb, 67(2), 725 - 32
Nitrogen fixation genes in an endosymbiotic Burkholderia strain; Minerdi D et al.; In this paper we report the identification and characterization of a DNA region containing putative nif genes and belonging to a Burkholderia endosymbiont of the arbuscular mycorrhizal fungus Gigaspora margarita . A genomic library of total DNA extracted from the fungal spores was also representative of the bacterial genome and was used to investigate the prokaryotic genome . Screening of the library with Azospirillum brasilense nifHDK genes as the prokaryotic probes led to the identification of a 6,413-bp region . Analysis revealed three open reading frames encoding putative proteins with a very high degree of sequence similarity with the two subunits (NifD and NifK) of the component I and with component II (NifH) of nitrogenase from different diazotrophs . The three genes were arranged in an operon similar to that shown by most archaeal and bacterial diazotrophs . PCR experiments with primers designed on the Burkholderia nifHDK genes and Southern blot analysis demonstrate that they actually belong to the genome of the G . margarita endosymbiont . They offer, therefore, the first sequence for the nif operon described for Burkholderia . Reverse transcriptase PCR experiments with primers designed on the Burkholderia nifH and nifD genes and performed on total RNA extracted from spores demonstrate that the gene expression was limited to the germination phase . A phylogenetic analysis performed on the available nifK sequences placed the endosymbiotic Burkholderia close to A . brasilense.

Curr Microbiol, 2000 Jul, 41(1), 33 - 8
Induction of stress shock proteins DnaK and GroEL by phenoxyherbicide 2,4-D in Burkholderia sp . YK-2 isolated from rice field; Cho YS et al.; The purpose of this work was to investigate the induction of stress shock proteins in Burkholderia sp . YK-2 in response to the phenoxyherbicide 2,4-dichlorophenoxyacetic acid (2,4-D) . The stress shock proteins, which contribute to the resistance of the cytotoxic effect of 2,4-D, were induced at different 2,4-D concentrations in exponentially growing cultures of Burkholderia sp . YK-2 . This response involved the induction of a 43-kDa DnaK and 41-kDa GroEL proteins, characterized by SDS-PAGE and Western blot by use of the anti-DnaK and anti-GroEL monoclonal antibodies . The total stress shock proteins were analyzed by 2-D PAGE . Survival of Burkholderia sp . YK-2 with time in the presence of different concentrations of 2,4-D was monitored, and viable counts paralleled the induction of the stress shock proteins in this strain.

PDA J Pharm Sci Technol, 1999 Jul-Aug, 53(4), 186 - 201
Application of membrane filtration for removal of diminutive bioburden organisms in pharmaceutical products and processes; Sundaram S et al.; In this report, we present results of a recent investigation in our laboratories demonstrated the effect of process conditions and/or drug product composition on the ability of 0.2 micron and 0.22 micron sterilizing grade filters to fully retain Ralstonia (formerly Burkholderia, formerly Pseudomonas) pickettii . R . pickettii is a opportunistic pathogen widely distributed in nature as well as clinical specimens and there have been several reports of nosocomial infections due to intrinsic manufacture-related R . pickettii contamination in filter-sterilized parenteral fluids . This study documents the penetration of 0.2 micron nylon 66 and 0.22 micron modified PVDF sterilizing grade filters by R . pickettii (grown and challenged) in a drug solution under conditions that simulated a pharmaceutical filling operation . Penetration was not observed for every filter disc tested, and this may be explained, in part, by the stochastic nature (i.e., governed by the rules of probability) of the retention mechanisms involved . Scanning electron microscopy revealed significant changes in the microorganism's size and morphology as a result of exposure to the drug solution; these changes are consistent with those reported for bacteria subjected to nutrient deprivation . The SEM analyses of R . pickettii challenge suspensions in the drug solution showed that the average cell length decreased from 1.25 +/- 0.27 microns to 0.84 +/- 0.17 micron between zero and 24 hours . In addition, significant changes were observed in the size (length) distributions, with approximately 35% of the cells at 24 hours being smaller than any cell observed at the start of the challenge . These data suggest that the significant reduction in bioburden size and morphology that occurred as a result of exposure to the drug solution may play a role in the reduced ability of the 0.2 micron and 0.22 micron filters tested in this study to retain these organisms . Under the same test conditions where penetration of 0.2/0.22 micron filters was observed, 0.1 micron rated membrane filters qualified with both B . diminuta and Acholeplasma laidlawii mycoplasma consistently provided sterile effluent . Bacterial penetration of 0.2 (or 0.22) micron sterilizing grade filters was not observed under identical test conditions with either R . pickettii in a standardized solution (saline lactose broth) routinely used in challenge testing filters, or with the standard test organism, B . diminuta, in the drug solution . This study thus supports the renewed emphasis on both product- and process specific validation as well as routine bioburden monitoring expressed by regulatory agencies, and the use of enhanced bacterial removal efficiency 0.1 micron rated filters to provide enhanced sterility assurance in pharmaceutical processes.

Med Microbiol Immunol (Berl), 1999 Nov, 188(2), 91 - 7
Highly resistant Burkholderia pseudomallei small colony variants isolated in vitro and in experimental melioidosis; Haussler S et al.; Burkhloderia pseudomallei is the causative agent of melioidosis, a disease in which treatment failures and relapses are common . This study reports on slow growing B . pseudomallei 'small colony variants' (SCVs), isolated either in vitro after exposure to ceftazidime, ciprofloxacin or gentamicin or from the spleen and liver in a mouse model of melioidosis after treatment with ceftazidime . Interestingly, SCVs isolated by either method or antimicrobial agent showed a significant increase in the minimal inhibitory concentrations of various unrelated classes of antimicrobial agents . B . pseudomallei SCVs did not differ from their parental strains in standard biochemical profiles, nor by pulsed field gel electrophoresis or electron microscopy . Although the SCV phenotype was stable throughout numerous passages on antibiotic-free solid media, revertants with the parental colony morphology and, most importantly, with the parental susceptibility pattern occurred . These revertants led to rapid overgrowth of SCVs in liquid media without added antibiotics . Future studies will have to determine the clinical relevance of B . pseudomallei SCVs especially in treatment failure and relapse of infection.

Int J Syst Evol Microbiol, 2000 Nov, 50 Pt 6, 2135 - 9
Pseudomonas antimicrobica Attafuah and Bradbury 1990 is a junior synonym of Burkholderia gladioli (Severini 1913) Yabuuchi et al . 1993; Coenye T et al.; Comparison of the 16S rDNA sequence of Pseudomonas antimicrobica LMG 18920T with published 16S rDNA sequences from other pseudomonads indicated that Pseudomonas antimicrobica belongs to the genus Burkholderia, with Burkholderia gladioli, Burkholderia glumae and Burkholderia plantarii as its closest neighbours . DNA-DNA hybridizations confirmed that Pseudomonas antimicrobica and Burkholderia gladioli represent the same species . Strain LMG 18920T and other Burkholderia gladioli strains were also indistinguishable by SDS-PAGE of whole-cell proteins and had similar biochemical characteristics . The whole-cell fatty acid composition, however, was different from that of other Burkholderia gladioli strains . It is concluded that Pseudomonas antimicrobica is a later synonym of Burkholderia gladioli . As Burkholderia gladioli is known to cause infections in patients with cystic fibrosis and chronic granulomatous disease, the eventual use of strain LMG 18920T as a biological control agent should be approached with caution.

Appl Microbiol Biotechnol, 2000 Dec, 54(6), 778 - 85
Cloning and characterization of EstC from Burkholderia gladioli, a novel-type esterase related to plant enzymes; Reiter B et al.; By screening a genomic library of Burkholderia gladioli (formerly Pseudomonas marginata) for clones exhibiting esterolytic activity, the gene for a novel-type esterase (EstC) showing significant homology to plant enzymes could be isolated . High homology was found to two hydroxynitrile lyases originating from Hevea brasiliensis (tropical rubber tree) and Manihot esculenta (cassava), and to two proteins from Oryza sativa (rice) that are specifically induced upon infection by Pseudomonas syringae pv . syringae . The sequenced ORF encodes for a protein of 298 amino acids . The enzyme was efficiently overexpressed in Escherichia coli, purified and characterized with respect to enzymatic capabilities . The enzyme was able to hydrolyze a variety of esterase substrates of low to medium carbonic acid chain length, but no triglycerides were hydrolyzed . Despite the high sequence homology, no hydroxynitrile lyase activity could be recognized.

Rev Argent Microbiol, 2000 Oct-Dec, 32(4), 196 - 8
{Bact-Alert system for hemocultures: evaluation of terminal subcultures}; Soloaga R et al.; Few laboratory microbiological procedures are as important as the isolation of microorganisms from blood . To evaluate the usefulness of the terminal subcultures, 5669 blood cultures giving negative results after 7 days of incubation in the Bact/Alert System (Organon Teknika) were studied . Bottles were distributed as follows: 1562 adult aerobic bottles, 119 adult anaerobic bottles, 3960 pediatric bottles and 28 FAN bottles . From 5669 blood cultures, 10 subcultures that yielded growth had not been detected by the system . These included 5 adult aerobic bottles and 5 pediatric bottles, 7 of these microorganisms were considered contaminants according to clinical data (2 Micrococcus spp, 1 staphylococci coagulase negative, 1 Burkholderia cepacia, 1 Peptoestreptococcus spp, 1 Corynebacterium spp, 1 Scedosporium spp) while the other 3 were considered true bacteremia (1 Pseudomonas aeruginosa, 1 Proteus mirabilis, 1 Streptococcus sanguis), although no one made any change in treatment on the basis of the previous isolation . Based on these results the routinary utilization of terminal subcultures is not advisable and should be used only for special cases or a second system of blood culture should be added according to clinical or epidemiological data.

J Immunol, 2001 Jan 15, 166(2), 1097 - 105
Bystander activation of CD8+ T cells contributes to the rapid production of IFN-gamma in response to bacterial pathogens; Lertmemongkolchai G et al.; The bacterium Burkholderia pseudomallei causes a life-threatening disease called melioidosis . In vivo experiments in mice have identified that a rapid IFN-gamma response is essential for host survival . To identify the cellular sources of IFN-gamma, spleen cells from uninfected mice were stimulated with B . pseudomallei in vitro and assayed by ELISA and flow cytometry . Costaining for intracellular IFN-gamma vs cell surface markers demonstrated that NK cells and, more surprisingly, CD8(+) T cells were the dominant sources of IFN-gamma . IFN-gamma(+) NK cells were detectable after 5 h and IFN-gamma(+) CD8(+) T cells within 15 h after addition of bacteria . IFN-gamma production by both cell populations was inhibited by coincubation with neutralizing mAb to IL-12 or IL-18, while a mAb to TNF had much less effect . Three-color flow cytometry showed that IFN-gamma-producing CD8(+) T cells were of the CD44(high) phenotype . The preferential activation of NK cells and CD8(+) T cells, rather than CD4(+) T cells, was also observed in response to Listeria monocytogenes or a combination of IL-12 and IL-18 both in vitro and in vivo . This rapid mechanism of CD8(+) T cell activation may be an important component of innate immunity to intracellular pathogens.

Curr Opin Hematol, 2001 Jan, 8(1), 17 - 22
Clinical aspects of chronic granulomatous disease; Johnston RB Jr; Data from a registry of 368 patients with chronic granulomatous disease (CGD) documenta shift in the most common infecting organisms away from staphylococci and enteric bacteria to Aspergillus species, although staphylococci remain a threat . A . nidulans appears to have a particular virulence in CGD . Burkholderia cepacia sepsis/pneumonia was the second most lethal infection in patients in the registry . Seventy-six percent of registry patients had the X-linked recessive (XLR) form of CGD . Chorioretinitis may be more common than previously appreciated, and boys with the XLR disease should probably have routine full eye exams . A new variant of CGD has been described that is caused by an inhibitory mutation in Rac2, which regulates activity of the neutrophil respiratory burst and actin assembly . Interferon-gamma, antibacterial prophylaxis, and, probably, antifungal prophylaxis with itraconazole reduce the rate of infection, and bone marrow transplantation can cure the disease if a histocompatible donor is available . Gene therapy can cure CGD in knockout mouse models . Having even a small percentage of phagocytes that are nicotinamide adenine dinucleotide phospate oxidase-positive can reduce the risk of serious infection, and procedures now under study appear close to achieving that goal, if not a cure.

J Pediatr Hematol Oncol, 2000 Nov-Dec, 22(6), 581 - 7
Infections in E-beta thalassemia; Wanachiwanawin W; Infection is a major complication and the leading cause of death in thalassemia, especially E-beta thalassemia . The spectrum of infections in E-beta thalassemia include mild and severe infections, therapy-related infections such as Yersinia enterocolitica infection associated with desferrioxamine (DFO) therapy, and transfusion-transmitted disease, as well as unique infections such as with pythiosis . Prospective studies in Thailand indicate that patients with E-beta thalassemia had more frequent episodes of both mild and severe infections . The former included upper respiratory tract infection, acute gastroenteritis, cutaneous abscess, and gingivitis . Severe infections occurred more commonly in patients with splenectomy and included septicemia, pneumonia, biliary tract infection, salmonellosis, and urinary tract infection . Responsible organisms were Escherichia coli (26%), Klebsiella pneumoniae (23%), Salmonella (15%), and Streptococcus pneumoniae (13%) . Other organisms included Pseudomonas, Staphylococci, Burkholderia pseudomallei (melioidosis), and Aeromonas . Patients undergoing DFO therapy are at risk for Y . enterocolitica infection which may be localized to mesenteric nodes and tonsils or occur as a generalized form such as septicemia . Recently, we have seen a unique infection so-called vascular pythiosis . Patients usually presented with clinical features of vascular occlusion of lower limbs from ascending arteritis and thrombosis . The causative organism, Pythium insidiosum, is fungus-like, in the kingdom Stramenopila, and in the class Oomycetes . The mortality rate is high and the only effective treatment has been early amputation or possibly immunotherapy . The predisposing factors of infections in thalassemia include splenectomy, iron overload, anemia, and granulocyte dysfunctions . General management of infections in thalassemia consist of prevention, i.e., immunization with pneumococcal and hepatitis vaccines, oral penicillins especially in patients with splenectomy, removal of predisposing factors such as gallstones, iron overload, and appropriate antibiotics.

J Med Microbiol, 2000 Dec, 49(12), 1075 - 8
Monoclonal antibody-based rapid identification of Burkholderia pseudomallei in blood culture fluid from patients with community-acquired septicaemia; Anuntagool N et al.; A monoclonal antibody-based latex agglutination (MAb-LA) test was employed for the rapid identification of Burkholderia pseudomallei in blood culture fluid from patients with community-acquired septicaemia . These patients were admitted to 12 hospitals in the northeastern part of Thailand which is a region known to be endemic for melioidosis . Blood samples were collected and immediately added to the blood culture bottles which were incubated in either automated (five hospitals) or manual (seven hospitals) culture systems . Of a total of 1369 culture-positive specimens, 204 specimens were culture-positive for B . pseudomallei . Of those, 194 (95%) were positive by MAb-LA and the type of blood culture system did not affect positivity rates . The performance of the MAb-LA test on these specimens was highly satisfactory compared with culture detection and confirmation by biochemical test, with 95.1% sensitivity, 99.7% specificity and 98.8% and 99.2% for positive and negative predictive values, respectively . The method described is highly reproducible, simple to perform even by inexperienced laboratory personnel and does not require expensive or elaborate equipment.

Drugs, 2000 Nov, 60(5), 1053 - 64
The treatment of respiratory pseudomonas infection in cystic fibrosis: what drug and which way?
Banerjee D, Stableforth D.
Pseudomonas aeruginosa is a non-capsulate and non-sporing gram-negative bacillus that most commonly affects the lower respiratory system in humans . Burkholderia (previously Pseudomonas) cepacia has emerged as an important respiratory pathogen in patients with cystic fibrosis (CF) . The ability of P . aeruginosa to persist and multiply in moist environments and equipment, such as humidifiers in hospital wards, bathrooms, sinks and kitchens, maybe of importance in cross-infection . P . aeruginosa infections of the lower respiratory tract can range in severity from colonisation (without an immunological response) to a severe necrotising bronchopneumonia . Infection is seen in patients with CF and other chronic lung diseases such as non-CF bronchiectasis . In patients with CF, once P . aeruginosa is established in the airways it is almost impossible to eradicate, but prior to this, aggressive treatment can delay the development of chronic infection . 30 to 40% of the present paediatric population with CF will have chronic pseudomonal infection . B . cepacia has a particular predisposition to infect patients with CF and may be distinguished from P . aeruginosa by accelerated lung disease in about one- third of patients . Overwhelming septicaemia and necrotising pneumonia are well described (cepacia syndrome); events that are rare with P . aeruginosa . With the propensity for social cross-infection, segregation policies have been accepted as means of controlling outbreaks . A number of antipseudomonal agents are available . The most commonly used are the extended-spectrum penicillins, aminoglycosides, cephalosporins, fluoroquinolones, polymixins and the monobactams . An aminoglycoside with a beta-lactam penicillin is usually considered to be the first line treatment . No trial has shown any significant clinical advantage of any particular combination regimen over another . The emergence of resistance continues to be a concern . Pipericillin, piperacillin/tazobactam and meropenem have good but equivalent antibacterial activity against P . aeruginosa . However, B . cepacia is characterised by in vitro resistance to colistin (colomycin), aminoglycosides and ciprofloxacin but better susceptibility to ceftazidime . Nebulised delivery of antipseudomonal antibiotics is thought to prevent recurrent exacerbations, reduce antibiotic usage and maintain lung function, particularly in patients with CF . Colistin, tobramycin and gentamicin are currently the most commonly prescribed nebulised antibiotics . Much effort is directed at treating chronic P . aeruginosa infection but as chronic infection is seldom if ever eradicated when first established, prevention is preferable . Early intensive treatment for P . aeruginosa infection is advocated in order to maintain pulmonary function and postpone the onset of chronic P . aeruginosa infection.

Indian J Pathol Microbiol, 1999 Oct, 42(4), 493 - 4
Burkholderia pseudomallei--abscess in an unusual site; Rao PS et al.; Meloidosis in a unusual site has been reported in a child . Complete identification of the organism has been presented.

Clin Exp Immunol, 2000 Dec, 122(3), 324 - 9
Kinetic studies of the production of nitric oxide (NO) and tumour necrosis factor-alpha (TNF-alpha) in macrophages stimulated with Burkholderia pseudomallei endotoxin; Utaisincharoen P et al.; The mechanism by which Burkholderia pseudomallei survives in macrophages is not clearly understood . In this study, we demonstrated that the mouse macrophage cell line (RAW 264.7) treated with lipopolysaccharide (LPS) from B . pseudomallei (BP-LPS) produced significantly less NO and TNF-alpha compared with those stimulated with the LPS from Escherichia coli and Salmonella typhi . The time required for the BP-LPS to trigger substantial NO and TNF-alpha release was at least 30 min, compared with < 5 min for the E . coli-LPS . A time course study of inducible nitric oxide synthase (iNOS) protein expression also indicated that the time required for macrophages stimulated with the BP-LPS to up-regulate iNOS was longer . The longer time lag for the BP-LPS to activate macrophages was probably due to the delay in up-regulation of iNOS and TNF-alpha mRNA transcription . These results indirectly suggest that the delay of the mediators' production may be due to a reduced rate of signal transduction initiated by the interaction of BP-LPS with the macrophage cell surface . The use of MoAb to phosphorylated p38 in a Western blot analysis provided data compatible with the notion that the maximum level of phosphorylated p38 from the cells activated with BP-LPS was attained at a slower rate . These results suggest that the unique structure of BP-LPS exhibits a property which may interfere with macrophage cell activation.

Infect Immun, 2001 Jan, 69(1), 34 - 44
Detection of bacterial virulence genes by subtractive hybridization: identification of capsular polysaccharide of Burkholderia pseudomallei as a major virulence determinant; Reckseidler SL et al.; Burkholderia pseudomallei, the etiologic agent of melioidosis, is responsible for a broad spectrum of illnesses in humans and animals particularly in Southeast Asia and northern Australia, where it is endemic . Burkholderia thailandensis is a nonpathogenic environmental organism closely related to B . pseudomallei . Subtractive hybridization was carried out between these two species to identify genes encoding virulence determinants in B . pseudomallei . Screening of the subtraction library revealed A-T-rich DNA sequences unique to B . pseudomallei, suggesting they may have been acquired by horizontal transfer . One of the subtraction clones, pDD1015, encoded a protein with homology to a glycosyltransferase from Pseudomonas aeruginosa . This gene was insertionally inactivated in wild-type B . pseudomallei to create SR1015 . It was determined by enzyme-linked immunosorbent assay and immunoelectron microscopy that the inactivated gene was involved in the production of a major surface polysaccharide . The 50% lethal dose (LD(50)) for wild-type B . pseudomallei is <10 CFU; the LD(50) for SR1015 was determined to be 3.5 x 10(5) CFU, similar to that of B . thailandensis (6.8 x 10(5) CFU) . DNA sequencing of the region flanking the glycosyltransferase gene revealed open reading frames similar to capsular polysaccharide genes in Haemophilus influenzae, Escherichia coli, and Neisseria meningitidis . In addition, DNA from Burkholderia mallei and Burkholderia stabilis hybridized to a glycosyltransferase fragment probe, and a capsular structure was identified on the surface of B . stabilis via immunoelectron microscopy . Thus, the combination of PCR-based subtractive hybridization, insertional inactivation, and animal virulence studies has facilitated the identification of an important virulence determinant in B . pseudomallei.

J Bacteriol, 2001 Jan, 183(1), 358 - 66
Phylogeny of the major head and tail genes of the wide-ranging T4-type bacteriophages; Tetart F et al.; We examined a number of bacteriophages with T4-type morphology that propagate in different genera of enterobacteria, Aeromonas, Burkholderia, and Vibrio . Most of these phages had a prolate icosahedral head, a contractile tail, and a genome size that was similar to that of T4 . A few of them had more elongated heads and larger genomes . All these phages are phylogenetically related, since they each had sequences homologous to the capsid gene (gene 23), tail sheath gene (gene 18), and tail tube gene (gene 19) of T4 . On the basis of the sequence comparison of their virion genes, the T4-type phages can be classified into three subgroups with increasing divergence from T4: the T-evens, pseudoT-evens, and schizoT-evens . In general, the phages that infect closely related host species have virion genes that are phylogenetically closer to each other than those of phages that infect distantly related hosts . However, some of the phages appear to be chimeras, indicating that, at least occasionally, some genetic shuffling has occurred between the different T4-type subgroups . The compilation of a number of gene 23 sequences reveals a pattern of conserved motifs separated by sequences that differ in the T4-type subgroups . Such variable patches in the gene 23 sequences may determine the size of the virion head and consequently the viral genome length . This sequence analysis provides molecular evidence that phages related to T4 are widespread in the biosphere and diverged from a common ancestor in acquiring the ability to infect different host bacteria and to occupy new ecological niches.

Clin Infect Dis, 2001 Jan, 32(1), E15 - 6 Epub 2000 Dec 11.
Travel-associated Burkholderia pseudomallei infection (Melioidosis) in a patient with cystic fibrosis: a case report; Visca P et al.; In September 1997, a 25-year-old Italian woman with cystic fibrosis (CF) spent 3 weeks in Thailand . In August 1998, her pulmonary function rapidly declined, with productive cough and intermittent fever . Chest x-ray films revealed diffuse, small, patchy opacities in the upper lobes . Burkholderia pseudomallei (BP) was isolated from specimens of the patient's sputum and was identified by means of 16S rDNA sequencing . The diagnosis of melioidosis was serologically confirmed . Continuous therapy with ceftazidime and co-trimoxazole and maintenance with co-trimoxazole, doxycycline, and chloramphenicol resulted in eradication of BP . We present the issue of whether patients with CF represent a population particularly at risk for melioidosis.

Diagn Microbiol Infect Dis, 2000 Nov, 38(3), 141 - 5
Characterization of Burkholderia pseudomallei isolated in Thailand and Malaysia; Radua S et al.; A total of 35 Burkholderia pseudomallei isolates from Thailand (16 clinical and eight soil isolates) and Malaysia (seven animal, two isolate each from clinical and soil) were investigated by their antimicrobial resistance, plasmid profiles and were typed by randomly amplified polymorphic DNA analysis . All isolates were found to be resistant to six or more of the 12 antimicrobial agents tested . Only two small plasmids of 1.8 and 2.4 megadalton were detected in two clinical isolates from Thailand . RAPD analysis with primer GEN2-60-09 resulted in the identification of 35 RAPD-types among the 35 isolates . The constructed dendrogram differentiated the 35 isolates into two main clusters and a single isolate . The wide genetic biodiversity among the 35 isolates indicate that RAPD-PCR can be a useful method to differentiate unrelated B . pseudomallei in epidemiological investigation.

Vet Pathol, 2000 Nov, 37(6), 626 - 36
Mouse model of sublethal and lethal intraperitoneal glanders (Burkholderia mallei); Fritz DL et al.; Sixty male BALB/c mice were inoculated intraperitoneally with either a sublethal or a lethal dose of Burkholderia mallei China 7 strain, then killed at multiple time points postinoculation . Histopathologic changes were qualitatively similar in both groups and consisted of pyogranulomatous inflammation . In sublethal study mice, changes were first seen at 6 hours in mediastinal lymph nodes, then in spleen, liver, peripheral lymph nodes, and bone marrow at day 3 . These changes generally reached maximal incidence and severity by day 4 but decreased by comparison in all tissues except the liver . Changes were first seen in lethal study mice also at 6 hours in mediastinal lymph nodes and in spleens . At day 1, changes were present in liver, peripheral lymph nodes, and bone marrow . The incidence and severity of these changes were maximal at day 2 . In contrast to sublethal study mice, the incidence and severity of the changes did not decrease through the remainder of the study . The most significant difference between the two groups was the rapid involvement of the spleen in the lethal study mice . Changes indicative of impaired vascular perfusion were more frequently seen in the sublethal study mice . Our findings indicate that mice are susceptible to B . mallei infection and may serve as an appropriate model for glanders infection in a resistant host such as human beings . Additionally, by immunoelectron microscopy, we showed the presence of type I O-antigenic polysaccharide (capsular) antigen surrounding B . mallei.

J Clin Microbiol, 2000 Dec, 38(12), 4305 - 9
Identification and detection of Stenotrophomonas maltophilia by rRNA-directed PCR; Whitby PW et al.; Stenotrophomonas maltophilia has recently emerged as an important nosocomial pathogen in immunocompromised patients, in transplant recipients, and in persons with cystic fibrosis (CF) . While this organism is nonpathogenic in healthy individuals, it is increasingly associated with morbidity and mortality in susceptible populations . Recent studies have indicated that for approximately 10% of CF patients with moderate lung disease, S . maltophilia can be cultured from respiratory tract secretions . Identification of S . maltophilia can be problematic, and analysis of isolates from the Burkholderia cepacia Research Laboratory and Repository showed that several isolates presumptively identified as B . cepacia by clinical microbiology laboratories were in fact S . maltophilia . To overcome the problems associated with definitive identification, we developed species-specific PCR (SS-PCR) primers, designated SM1 and SM4, directed to the 23S rRNA gene, and tested their utility to accurately identify S . maltophilia directly from sputum . The SS-PCR was developed and tested against a panel of 112 S . maltophilia isolates collected from diverse geographic locations . To test for specificity, 43 isolates from 17 different species were analyzed . PCR with the SM1-SM4 primer pair and isolated genomic DNA as a template resulted in amplification of a band from all S . maltophilia isolates and was uniformly negative for all other species tested, yielding a sensitivity and a specificity of 100% for the SS-PCR . The utility of the SS-PCR to directly identify S . maltophilia in sputum was examined . Thirteen expectorated sputum samples from CF patients were analyzed by SS-PCR . Three samples were PCR positive, in complete concordance with the conventional laboratory culture . Thus, we have developed an SS-PCR protocol that can rapidly and accurately identify S . maltophilia isolates and which can be used for the direct detection of this organism in CF patient sputum.

Braz J Infect Dis, 1998 Apr, 2(2), 43 - 61
Microbial Pathogens Associated With Cystic Fibrosis: Special Focus on Pseudomonas aeruginosa; Barth AL et al.; Cystic fibrosis (CF) is the most common inherited lethal disease among Caucasians . The disease is characterized by a high sodium sweat concentration, malabsorption, malnutrition, and chronic bronchopulmonary infection . Although CF is a multisystem disorder it is the deterioration of lung function, due mainly to bacterial colonization, that is the major determinant contributing to the high morbidity and mortality of affected patients . A relatively large variety of microbial species can be recovered from the CF sputum but it is widely accepted that Staphylococcus aureus, Haemophilus influenzae, Pseudomonas aeruginosa, and Burkholderia cepacia are the most important organisms causing infection in CF patients . Among these, P.aruginosa is the most prevalent pathogen responsible for much of the severe chronic lung infection . Antibiotic therapy aimed at clearing or reducing, the bacterial load in the CF lung, even though temporary, increases the longevity of patients and is one of the mainstays in treatment the disease.

Appl Environ Microbiol, 2000 Dec, 66(12), 5192 - 200
Zoospore homing and infection events: effects of the biocontrol bacterium Burkholderia cepacia AMMDR1 on two oomycete pathogens of pea (Pisum sativum L.); Heungens K et al.; Burkholderia cepacia AMMDR1 is a biocontrol agent that protects pea and sweet corn seeds from Pythium damping-off in field experiments . The goal of this work was to understand the effect of B . cepacia AMMDR1 on Pythium aphanidermatum and Aphanomyces euteiches zoospore homing events and on infection of pea seeds or roots . In vitro, B . cepacia AMMDR1 caused zoospore lysis, prevented cyst germination, and inhibited germ tube growth of both oomycetes . B . cepacia AMMDR1 also reduced the attractiveness of seed exudates to Pythium zoospores to nondetectable levels . However, when present at high levels on seeds, B . cepacia AMMDR1 had little net effect on zoospore attraction, probably because it also enhanced seed exudation . Seed-applied B . cepacia AMMDR1 dramatically reduced the incidence of infection by Pythium zoospores in situ compared with an antibiosis-deficient Tn5 mutant strain . This mutant strain also decreased Pythium infection incidence to some extent, but only when the pathogen inoculum potential was low . B . cepacia AMMDR1 did not affect attraction of Aphanomyces zoospores or Aphanomyces root rot incidence . These results suggest that B . cepacia AMMDR1 controls P . aphanidermatum largely through antibiosis, but competition for zoospore-attracting compounds can contribute to the effect . Differences in suppression of Aphanomyces and Pythium are discussed in relation to differences in the ecology of the two pathogens.

FEMS Microbiol Lett, 2000 Dec 1, 193(1), 179 - 85
Studies on polyhydroxyalkanoate (PHA) accumulation in a PHA synthase I-negative mutant of Burkholderia cepacia generated by homogenotization; de Andrade Rodrigues MF et al.; In the genome of Burkholderia cepacia strain IPT64, which accumulates a blend of the two homopolyesters poly(3-hydroxybutyrate), poly(3HB), and poly(3-hydroxy-4-pentenoic acid), poly(3H4PE), from sucrose or gluconate as single carbon source, the polyhydroxyalkanoate (PHA) synthase structural gene was disrupted by the insertion of a chloramphenicol-resistant gene cassette (phaC1::Cm) . The suicide vector pSUP202 harboring phaC1::Cm was transferred to B . cepacia by conjugation . The inactivated gene was integrated into the chromosome of B . cepacia by homologous recombination . This mutant and also 15 N-methyl-N'-nitrosoguanidine (NMG)-induced mutants still accumulated low amounts of PHAs and expressed low PHA synthase activity . The analysis of the mutant phaC1::Cm showed that it accumulated about 1% of PHA consisting of 68.2 mol% 3HB and 31.8 mol% 3H4PE from gluconate . The wild-type, in contrast, accumulated 49.3% of PHA consisting of 96.5 mol% 3HB and 3 . 5 mol% 3H4PE . Our results indicated that the genome of B . cepacia possesses at least two PHA synthase genes, which probably have different substrate specificities.

J Biotechnol, 2001 Nov 30, 84(2), 155 - 61
Expression of a mineral phosphate solubilizing gene from Erwinia herbicola in two rhizobacterial strains; Rodriguez H et al.; A genetic construction was carried out using the broad host range vector pKT230 and plasmid pMCG898, which encodes the Erwinia herbicola pyrroloquinoline quinone (PQQ) synthase, a gene involved in mineral phosphate solubilization (mps) . The final construction was transformed and expressed in Escherichia coli MC1061, and the recombinant plasmids were transferred to Burkholderia cepacia IS-16 and Pseudomonas sp . PSS recipient cells by conjugation . Clones containing recombinant plasmids produced higher clearing halos in plates with insoluble phosphate as the unique (P) source, in comparison with those of strains without plasmids, demonstrating the heterologous expression of the E . herbicola gene in the recipient strains . This genetic manipulation allowed the increase in mps ability of both strains, enhancing their potentialities as growth promoters of agricultural crops . These results represent the first report on the application of the recombinant DNA methodology for the obtaining of improved phosphate solubilizing ability from rhizobacterial strains for biofertilization purposes.

Infect Control Hosp Epidemiol, 2000 Nov, 21(11), 739 - 41
Outbreak of nosocomial Burkholderia cepacia infection and colonization associated with intrinsically contaminated mouthwash; Matrician L et al.; From August 1996 through June 1998, 69 ventilated, intensive care unit patients at two Arizona hospitals had nosocomial respiratory tract cultures positive for Burkholderia cepacia . Intrinsically contaminated alcohol-free mouthwash was identified by pulsed-field gel electrophoresis as the source of the outbreak.

Infect Immun, 2000 Dec, 68(12), 6554 - 60
Identification of a siderophore receptor required for ferric ornibactin uptake in Burkholderia cepacia; Sokol PA et al.; Ornibactins are linear hydroxamate siderophores produced by Burkholderia cepacia with peptide structures similar to that of pyoverdines produced by the fluorescent pseudomonads . The gene encoding the outer membrane receptor (orbA) was identified, sequenced, and demonstrated to have significant homology with hydroxamate receptors produced by other organisms . The orbA precursor was predicted to be a protein with a molecular mass of 81 kDa . An orbA mutant was constructed and demonstrated to be unable to take up (59)Fe-ornibactins or to grow in medium supplemented with ornibactins . Outer membrane protein profiles from the parent strain, K56-2, revealed an iron-regulated outer membrane protein of 78 kDa that was not detectable in the K56orbA::tp mutant . When this mutant harbored a plasmid containing the orbA gene, the 78-kDa protein was present in the outer membrane protein profiles and the mutant was able to utilize ornibactin to acquire iron . The orbA mutant was less virulent in a chronic respiratory infection model than the parent strain, indicating that ornibactin uptake and utilization are important in the pathogenesis of B . cepacia respiratory infections.

Acta Trop, 2000 Nov 2, 77(2), 229 - 37
Genotype analysis of Burkholderia pseudomallei using randomly amplified polymorphic DNA (RAPD): indicative of genetic differences amongst environmental and clinical isolates; Leelayuwat C et al.; Burkholderia pseudomallei is the causative agent of melioidosis, an infectious disease common in the tropics . Melioidosis is most prevalent in the northeastern part of Thailand . The diseases has diverse clinical manifestations ranging from mild localized to fatal septicemic forms . The bacterial genetic factors contributing to the severity of melioidosis have not been completely identified . We have developed a genotyping method based upon randomly amplified polymorphic DNA (RAPD) analysis . Eighteen deca-oligo nucleotide primers with 70% GC content, eight previously published 60%GC RAPD primers, and four random deca oligomers were tested on nine strains of B . pseudomallei isolated from five patients with localized and four with septicemic melioidosis . The RAPD patterns were analyzed by polyacrylamide gel electrophoresis using a laser based automated fragment analyzer, GS2000 . Based upon the pattern complexity, seven pairs consisting of eight primers were chosen for further analysis . Six hundred and thirty-two samples, including duplicates/triplicates, of B . pseudomallei isolated from melioidosis patients and the environment were analyzed . Two controls were included in each run of the test samples . All the samples were tested and patterns analyzed by blinded technical staff . Apparently, the method is reproducible . This is indicated by the RAPD patterns of the two controls of between run assay . Interestingly, some RAPD patterns were more prevalent in the clinical isolates than the environmental specimens and vice versa . For example, Q162KKU4-0 and Q162KKU1-0 were found 3 . 5 and 3.3 times more often in the clinical specimens (P<0.025) . Likewise, Q162KKU1-1 and Q162KKU4-1 were found 18 and 37 times more often in the environment (P<0.0000001) . In addition, there was a bias in the distribution of arabinose positive strains and particular RAPD patterns; RAPD patterns of B . pseudomallei that were found frequently in septicemic patients were less likely to be arabinose positive . The data suggest the existence of bacterial genetic differences between the clinical and environmental isolates of B . pseudomallei . Further analysis of the RAPD patterns searching for common polymorphic DNA fragments and systemic comparative genomic analysis of B . pseudomallei in accordance with the clinical data should reveal genetic factors involved in severity and bacterial pathogenesis of B . pseudomallei in melioidosis.

Acta Trop, 2000 Nov 2, 77(2), 157 - 65
Melioidosis research in China; Yang S; Research on melioidosis and its pathogen has been ongoing in China for more than two decades . It has been demonstrated that the natural foci are located predominantly in Hainan, Guangdong and Guangxi province, where there is a good correlation between soil isolation and the serum prevalence of antibodies to Burkholderia pseudomallei . The cases of melioidosis reported up to now are concentrated in the Hainan and Zhanjiang peninsula . Investigations on serotype, virulence, ecology, antibiotic susceptibility, whole cell analysis by gas chromatography, and genetics have led to a new understanding of the pathology of the disease . Immunological cross reactions between Burkholderia mallei and B . pseudomallei and the difference between melioidosis and glanders in horses is discussed.

Microb Ecol, 2000 Aug, 40(3), 189 - 199
Aquifer Protist Response and the Potential for TCE Bioremediation with Burkholderia cepacia G4 PR1; Snyder RA et al.; Bacterivorous protists have been recovered from pristine and contaminated aquifer environments, but the ecological role of these organisms in bioremediation strategies has not been well defined . Burkholderia cepacia G4 PR1 constitutively expresses a toluene ortho-monooxygenase (tom) due to a secondary transposition of a Tn5 transposable element in a trichloroethylene (TCE) degradative plasmid (TOM) . Groundwater and sediment from a potential site for a TCE bioremediation field demonstration were used in laboratory microcosms to test the survival of this organism . In nonsterile aquifer sediment slurries, the bacterium was eliminated in a logrithmic decay concomitant with an increase in bacterivorous protists . A half-life for the organism calculated from extinction coefficients increased logarithmically with increasing inoculation density above 1 x 10(6) PR1 ml(-1) . For inoculation densities below this level, the half-life of PR1 increased exponentially with decreasing inoculation density . The lowest half-lives corresponded to densities of bacteria that stimulate response of bacterivores . In a column system designed to incorporate aquifer flow, repeated addition of PR1 resulted in a buildup of bacterivore populations and reduced half-life of the bacterium . Addition of TCE and growth substrate in the eluent resulted in prolonged survival of PR1 and apparent mineralization of TCE . The results indicate significant but predictable losses due to native bacterivores would occur within and beyond a treatment zone where PR1 would be added to the aquifer, and mineralization of TCE in contaminated groundwater might be possible with repeated inoculation and addition of nutrients.

Microb Ecol, 2000 Aug, 40(3), 169 - 176
Bias Caused by Using Different Isolation Media for Assessing the Genetic Diversity of a Natural Microbial Population; Tabacchioni S et al.; The influence of isolation medium on the biodiversity of Burkholderia cepacia strains recovered from the rhizosphere of Zea mays was evaluated by comparing the genetic diversity of isolates obtained by plating serial dilutions of root macerates on the two selective media TB-T and PCAT . From each medium, 50 randomly chosen colonies were isolated . On the basis of the restriction patterns of DNA coding for 16S rRNA (16S rDNA) amplified by means of PCR (ARDRA), all strains isolated from TB-T medium were assigned to the B . cepacia species, whereas among PCAT isolates only 74% were assigned to the B . cepacia species . Genetic diversity among the PCAT and TB-T isolates was evaluated by the random amplified polymorphic DNA (RAPD) technique . The analysis of molecular variance (AMOVA) method was applied to determine the variance component for RAPD patterns . Most of the genetic diversity (90.59%) was found within the two groups of isolates, but an appreciable amount (9.41%) still separated the two groups (P < 0.001) . Mean genetic distances among PCAT isolates (10.39) and TB-T isolates (9.36) were significantly different (P < 0.0001) . The results indicate that the two different isolation media select for B . cepacia populations with a different degree of genetic diversity . Moreover, a higher degree of genetic diversity was observed among strains isolated from PCAT medium than among those isolated from TB-T medium.

J Med Microbiol, 2000 Nov, 49(11), 993 - 1001
Cloning and analysis of genomic differences unique to Burkholderia pseudomallei by comparison with B . thailandensis; Brown NF et al.; Melioidosis is an infectious disease caused by Burkholderia pseudomallei . Genomic subtractive hybridisation was performed with the closely related avirulent species B . thailandensis to identify virulence genes of B . pseudomallei . The subtractive hybridisation products were highly specific for B . pseudomallei . Sequence analysis revealed a number of putative virulence factors, as well as apparently novel sequences of unknown function . The subtracted library contained DNA regions of relatively low G + C mol% content, which were distributed throughout the B . pseudomallei genome . The distribution of subtracted sequences amongst a collection of 22 B . pseudomallei isolates was found to be variable, with the exception of three strains which almost universally lacked the subtracted sequences . These three strains also differed in that they were highly haemolytic, indicating a possible separate virotype.

Anal Biochem, 2000 Nov 15, 286(2), 187 - 92
The use of protocatechuate dioxygenase for maintaining anaerobic conditions in biochemical experiments; Patil PV et al.; The study of redox-active systems often requires the maintenance of anaerobic conditions . The glucose oxidase system has often been used to maintain anaerobic conditions, but it has some drawbacks, such as the production of H(2)O(2) and limitations on stability . Protocatechuate dioxygenase from Burkholderia cepacia and the substrate, protocatechuate, constitute an alternate effective oxygen-scrubbing system that can be used in a wide variety of biochemical experiments . We have shown its suitability for maintaining rigorous anaerobic environments in solutions of pH 6-9, at temperatures from 4 to 35 degrees C, and for periods of time up to 15 months . The enzyme system was shown to be stable under these conditions and effective for maintaining anaerobic conditions in titrations of FAD . It is also suitable for scrubbing various types of apparatus such as stopped-flow instruments for anaerobic experiments .

Biotechnol Bioeng, 2000 Dec 20, 70(6), 693 - 8
Aerobic degradation of mixtures of chlorinated aliphatics by cloned toluene-o-xylene monooxygenase and toluene o-monooxygenase in resting cells; Shim H et al.; Recombinant strains of Escherichia coli constitutively expressing toluene-o-xylene monooxygenase (ToMO) of Pseudomonas stutzeri OX1 and toluene o-monooxygenase (TOM) of Burkholderia cepacia G4 were investigated for their ability to oxidize trichloroethylene (TCE), 1,1-dichloroethylene (1,1-DCE), cis-1,2-dichloroethylene (cis-DCE), trans-1,2-dichloroethylene (trans-DCE), vinyl chloride (VC), and chloroform (CF), individually as well as in various mixtures . ToMO oxidized all of these individual compounds well, whereas TOM did not degrade VC significantly (16-fold less) and degraded cis-DCE and trans-DCE less well (3.7- and 2.4-fold, respectively) . For mixtures of these chlorinated aliphatics, ToMO was again more robust than TOM . For example, in binary mixtures including TCE, ToMO degraded all three DCE isomers and CF, but the presence of TCE inhibited VC degradation; TOM degraded both TCE/1,1-DCE and TCE/trans-DCE, but not cis-DCE for TCE/cis-DCE, and the addition of CF or VC to TCE completely inhibited degradation of both compounds and TCE . The addition of CF or trans-DCE stimulated VC degradation in the presence of TCE for ToMO, and the addition of any of the three DCE isomers stimulated VC degradation for TOM . Significant degradation of all ternary mixtures of TCE and less chlorinated ethenes, as well as a mixture of TCE, three DCEs, and VC, was achieved with ToMO (but not TOM) . In mixtures of these chlorinated compounds, degradation was found to occur simultaneously rather than sequentially, and the mineralization of many of these compounds could be confirmed through detection of chloride ions.

Appl Environ Microbiol, 2000 Nov, 66(11), 4673 - 8
Rhizosphere competitiveness of trichloroethylene-degrading, poplar-colonizing recombinant bacteria; Shim H et al.; Indigenous bacteria from poplar tree (Populus canadensis var . eugenei 'Imperial Carolina') and southern California shrub rhizospheres, as well as two tree-colonizing Rhizobium strains (ATCC 10320 and ATCC 35645), were engineered to express constitutively and stably toluene o-monooxygenase (TOM) from Burkholderia cepacia G4 by integrating the tom locus into the chromosome . The poplar and Rhizobium recombinant bacteria degraded trichloroethylene at a rate of 0.8 to 2.1 nmol/min/mg of protein and were competitive against the unengineered hosts in wheat and barley rhizospheres for 1 month (colonization occurred at a level of 1.0 x 10(5) to 23 x 10(5) CFU/cm of root) . In addition, six of these recombinants colonized poplar roots stably and competitively with populations as large as 79% +/- 12% of all rhizosphere bacteria after 28 days (0.2 x 10(5) to 31 x 10(5) CFU/cm of root) . Furthermore, five of the most competitive poplar recombinants (e.g., Pb3-1 and Pb5-1, which were identified as Pseudomonas sp . strain PsK recombinants) retained the ability to express TOM for 29 days as 100% +/- 0% of the recombinants detected in the poplar rhizosphere expressed TOM constitutively.

FEMS Microbiol Lett, 2000 Nov 1, 192(1), 67 - 72
Sequencing and characterization of a novel serine metalloprotease from Burkholderia pseudomallei; Lee MA et al.; Burkholderia pseudomallei, a Gram-negative bacterium is found in the soil and water, mainly in Southeast Asia and Northern Australia . It is responsible for melioidosis in human and animals . The bacteria produce several potential virulent factors such as extracellular protease, hemolysin, lipase and lecithinase . The isolation of virulence genes and the study of their functions will contribute to our understanding of bacterial pathogenesis . Previous studies have implicated protease as a contributing virulence factor in the pathogenesis of some bacteria . Three out of 5000 clones screened from a genomic DNA library of B . pseudomallei were found to express protease activity . The clones were found to have the same sequence . The nucleotide sequence revealed an open reading frame (designated as metalloprotease A, mprA) encoding a 500-amino acid protein, MprA, with an estimated molecular mass of 50241 Da . The predicted amino acid sequence shares homology with the subtilisin family of serine proteases.

Appl Microbiol Biotechnol, 2000 Sep, 54(3), 382 - 9
Phospholipid compositional changes of five pseudomonad archetypes grown with and without toluene; Fang J et al.; Bacterial physiological responses to toluene exposure were investigated in five reference pseudomonad strains that express different toluene degradation pathways: Pseudomonas putida mt-2, Pseudomonas putida F1, Burkholderia cepacia G4, Burkholderia pickettii PKO1, and Pseudomonas mendocina KR1 . The intact phospholipids of these archetypes, grown with and without toluene, were characterized using liquid chromatography/electrospray ionization/mass spectrometry . All strains showed significant changes in phospholipid content and composition as an adaptive response to toluene exposure, as well as considerable diversity in response mechanisms . For example, the phospholipid content of toluene-grown PKO1, F1, and KR1 were 10.9-34.7% of that found in succinate-grown strains, while the phospholipid content of mt-2 and G4 increased by 56% and 94%, respectively, when grown on toluene . In addition, PKO1, F1, and mt-2 responded to the presence of toluene by synthesizing more phosphatidylglycerol, whereas G4 and KR1 synthesized phospholipids with polyunsaturated fatty acids (C18:2) on one or both of the sn-2 positions . These changes in phospholipid composition and concentration probably reflect the sensitivity and degree of tolerance of these strains to toluene, and suggest that different mechanisms are utilized by dissimilar bacteria to maintain optimal lipid ordering in the presence of such environmental pollutants.

Appl Microbiol Biotechnol, 2000 Sep, 54(3), 354 - 60
Molecular cloning and characterization of a chitosanase from the chitosanolytic bacterium Burkholderia gladioli strain CHB101; Shimosaka M et al.; A chitosanase was purified from the culture fluid of the chitino- and chitosanolytic bacterium Burkholderia gladioli strain CHB101 . The purified enzyme (chitosanase A) had a molecular mass of 28 kDa, and catalyzed the endo-type cleavage of chitosans having a low degree of acetylation (0-30%) . The enzyme hydrolyzed glucosamine oligomers larger than a pentamer, but did not exhibit any activity toward N-acetylglucosamine oligomers and colloidal chitin . The gene coding for chitosanase A (csnA) was isolated and its nucleotide sequence determined . B . gladioli csnA has an ORF encoding a polypeptide of 355 amino acid residues . Analysis of the N-terminal amino acid sequence of the purified chitosanase A and comparison with that deduced from the csnA ORF suggests post-translational processing of a putative signal peptide and a possible substrate-binding domain . The deduced amino acid sequence corresponding to the mature protein showed 80% similarity to the sequences reported from Bacillus circulans strain MH-K1 and Bacillus ehimensis strain EAG1, which belong to family 46 glycosyl hydrolases.

J Med Microbiol, 2000 Oct, 49(10), 875 - 85
Preferential adherence of cable-piliated burkholderia cepacia to respiratory epithelia of CF knockout mice and human cystic fibrosis lung explants; Sajjan U et al.; The Burkholderia cepacia complex consists of at least five well-documented bacterial genomovars, each of which has been isolated from the sputum of different patients with cystic fibrosis (CF) . Although the world-wide prevalence of this opportunist pathogen in CF patients is low (1-3%), 'epidemic' clusters occur in geographically isolated regions . Prevalence in some of these clusters is as high as 30-40% . The majority of CF B . cepacia isolates belong to genomovar III, but the relationship between genomovar and virulence has not yet been defined . Because the initial stage of infection involves bacterial binding to host tissues, the present study investigated differences in the binding of representative isolates of all five genomovars to fixed nasal sections of UNC cftr (-/-) and (+/+) mice and to human lung explants, biopsy and autopsy tissue of CF and non-CF patients . Binding was highest for isolates of genomovar III, subgroup RAPD type 2, but only if the isolates expressed the cable pili phenotype . Antibodies to the 22-kDa adhesin of cable pili virtually abolished binding . Binding occurred only to cftr (-/-) nasal sections or to CF lung sections and was negligible in cftr (+/+) or human non-CF, histologically normal lung sections . Unlike normal epithelia, the hyperplastic epithelia of CF bronchioles were enriched in cytokeratin 13, a 55-kDa protein that has previously been shown to act as a receptor in vitro for cable-piliated B . cepacia . These findings may help to explain the high transmissibility ofCbl-positive, genomovar III strains of B . cepacia among CF patients.

Br J Neurosurg, 1998 Apr, 12(2), 170 - 2
Brain abscess as the presenting feature of melioidosis; Lath R et al.; Central nervous system involvement in melioidosis is rare and there are only a few reports of the causative organism, Burkholderia pseudomallei, causing a brain abscess . We report a patient who presented to us with a brain abscess due to this organism and emphasize the need for a high degree of suspicion for this disease in tropical countries and treatment with the appropriate antibiotics, as the mortality associated with this disease is very high.

Appl Environ Microbiol, 2000 Oct, 66(10), 4585 - 8
A rhamnolipid biosurfactant reduces cadmium toxicity during naphthalene biodegradation; Sandrin TR et al.; A model cocontaminated system was developed to determine whether a metal-complexing biosurfactant, rhamnolipid, could reduce metal toxicity to allow enhanced organic biodegradation by a Burkholderia sp . isolated from soil . Rhamnolipid eliminated cadmium toxicity when added at a 10-fold greater concentration than cadmium (890 microM), reduced toxicity when added at an equimolar concentration (89 microM), and had no effect at a 10-fold smaller concentration (8.9 microM) . The mechanism by which rhamnolipid reduces metal toxicity may involve a combination of rhamnolipid complexation of cadmium and rhamnolipid interaction with the cell surface to alter cadmium uptake.

Appl Environ Microbiol, 2000 Oct, 66(10), 4503 - 9
Detection and identification of bacterial endosymbionts in arbuscular mycorrhizal fungi belonging to the family Gigasporaceae; Bianciotto V et al.; Intracellular bacteria have been found previously in one isolate of the arbuscular mycorrhizal (AM) fungus Gigaspora margarita BEG 34 . In this study, we extended our investigation to 11 fungal isolates obtained from different geographic areas and belonging to six different species of the family Gigasporaceae . With the exception of Gigaspora rosea, isolates of all of the AM species harbored bacteria, and their DNA could be PCR amplified with universal bacterial primers . Primers specific for the endosymbiotic bacteria of BEG 34 could also amplify spore DNA from four species . These specific primers were successfully used as probes for in situ hybridization of endobacteria in G . margarita spores . Neighbor-joining analysis of the 16S ribosomal DNA sequences obtained from isolates of Scutellospora persica, Scutellospora castanea, and G . margarita revealed a single, strongly supported branch nested in the genus Burkholderia.

Biochem Biophys Res Commun, 2000 Sep 16, 276(1), 71 - 6
Enzymes leading to the nucleotide sugar precursors for exopolysaccharide synthesis in Burkholderia cepacia; Richau JA et al.; Based on the chemical composition of the exopolysaccharide produced by the cystic fibrosis bacterial isolate Burkholderia cepacia IST408, we postulated and confirmed, based on the specificity of enzymes detected in crude cell-free extracts, the pathway leading to the presumptive activated sugar precursors: UDP-D-glucose, UDP-D-galactose, UDP-D-glucuronic acid, GDP-D-mannose, and GDP-D-rhamnose . Results also suggest that regulation of the expression of the mucoid phenotype in B . cepacia does not occur at the level of synthesis of any of these enzymes .

Biotechnol Bioeng, 2000 Nov 20, 70(4), 436 - 45
Use of 16S-rRNA to investigate microbial population dynamics during biodegradation of toluene and phenol by a binary culture; Rogers JB et al.; Interspecies interactions and changes in the rate and extent of biodegradation in mixed culture-mixed substrate studies were investigated . A binary mixed culture of Pseudomonas putida F1 and Burkholderia sp . JS150 degraded toluene, phenol, and their mixture . Both toluene and phenol can serve as sole sources of carbon and energy for both P . putida F1 and strain JS150 . To investigate the population dynamics of this system, a fluorescent in-situ hybridization method was chosen because of its ability to produce quantitative data, its low standard error, and the ease of use of this method . When the binary mixed culture was grown on toluene or phenol alone, significant interactions between the species were observed . These interactions could not be explained by a pure-and-simple competition model and were substrate dependent . Strain JS150 growth was slightly inhibited when grown with P . putida F1 on phenol, and P . putida F1 grew more rapidly than expected . Conversely, when the two species were grown together on toluene alone, P . putida F1 was inhibited while strain JS150 was unaffected . During growth of the mixed culture on a combination of toluene and phenol, the interactions were similar to that observed during growth on phenol alone; P . putida F1 growth was enhanced while strain JS150 was unaffected . Because of the observed interspecies interactions, monoculture kinetic parameters were not sufficient to describe the mixed culture kinetics in any experiment . This is one of the first reports of microbial population dynamics in which molecular microbial ecology and mathematical modeling have been combined . The use of the 16S-rRNA-based method allowed for observation and understanding of interspecies interactions that were not observable with standard culture-based methods . These results suggest the need for more investigations that account for both substrate and microbial interactions when predicting the fate of organic pollutants in real systems .

Biotechnol Bioeng, 2000 Nov 20, 70(4), 428 - 35
Modeling substrate interactions during the biodegradation of mixtures of toluene and phenol by Burkholderia species JS150; Rogers JB et al.; The biodegradation kinetics of toluene, phenol, and a mixture of toluene and phenol by Burkholderia species JS150 was measured and modeled . Both of these compounds can serve as the sole source of carbon and energy for this microorganism . The single-substrate biodegradation kinetics was described well using the Monod model, with model constants of mu(max,T) = 0.39 h(-1) and K(S,T) = 0.011 mM for growth on toluene and mu(max,P) = 0.309 h(-1) and K(S,P) = 0.0054 mM for growth on phenol . Degradation of the mixture of toluene and phenol followed simultaneous utilization kinetics with toluene being the preferred substrate . Toluene was found to inhibit the rate of utilization of phenol while the presence of phenol had little effect on the rate of degradation of toluene . Of the kinetic models that were tested, one developed for microbial degradation of multiple substrates was able to describe substrate interactions and to model the mixture utilization by strain JS150 . Simple competitive, noncompetitive, or uncompetitive substrate kinetics were not sufficient to describe the observed inhibitory interactions.

J Med Assoc Thai, 2000 Aug, 83(8), 856 - 60
Comparison between the antimicrobial susceptibility of Burkholderia pseudomallei to trimethoprim-sulfamethoxazole by standard disk diffusion method and by minimal inhibitory concentration determination; Lumbiganon P et al.; Melioidosis, an infection caused by Burkholderia pseudomallei, usually occurs in immunocompromised patients and requires prolonged antibiotic therapy . Previously, oral trimethoprim-sulfamethoxazole (TM/SM), an inexpensive and effective drug has been used as a maintenance therapy . The susceptibility of B . pseudomallei to TM/SM by the standard disk diffusion method is very low . However, some patients who were treated with TM/SM as a maintenance therapy despite the in vitro resistance showed good clinical responses . There were no data comparing the susceptibility of B . pseudomallei by the standard disk diffusion method with other quantitative susceptibility tests . The objective of this study was to determine the agreement between the antimicrobial susceptibility of B . pseudomallei to TM/SM by standard disk diffusion and minimal inhibitory concentration determination (MIC) . We performed the susceptibility test of 144 strains of B . pseudomallei to TM/SM by both the standard disk diffusion and microbroth dilution MIC . The sensitivity results were 53.5 per cent and 84.0 per cent respectively . The agreement between the 2 tests was very poor (Kappa = 0.14; 95% CI = -0.01 to 0.29) . The false resistant rate by the standard disk diffusion test was 67.9 per cent . Further in vitro susceptibility and clinical study are needed to define the interpretive criteria that correlate with clinical response.

J Agric Food Chem, 2000 Sep, 48(9), 4314 - 9
Protection of tomato seed germination from the inhibitory effect of 2,4,5-trichlorophenoxyacetic acid by inoculation of soil with Burkholderia cepacia AC1100; Gangadhara KP et al.; The effect of 2,4,5-trichlorophenoxy acetic acid (2,4,5-T) on the germination and seedling vigor of different crop seeds was tested . Seeds of rice, maize, sorghum, finger millet, and horse gram were comparatively more tolerant to the chemical with no marked effect up to a concentration of 200 mg 2,4,5-T L(-)(1) as tested by the filter paper method . Tomato and brinjal (egg plant) were highly susceptible . Even at 5 and 10 mg 2,4,5-T L(-)(1), marked reduction in the germination and seedling vigor of tomato and egg plant, respectively, was observed . At 20 and 30 mg L(-)(1), the germination of tomato and egg plant seeds, respectively, were completely inhibited on filter paper, whereas the inhibitory concentrations in soil was 40 mg 2,4,5-T kg(-)(1) soil . Several abnormalities were observed in the chemically affected seedlings . Protease activity of the seeds germinating in the presence of the chemical was drastically reduced . Bioremediation of the chemically contaminated soil with Burkholderia cepacia AC1100, by inoculation of the soil 7 days before sowing the seeds, completely protected the seeds, resulting in normal germination and an improved seedling vigor.

J AOAC Int, 2000 Jul-Aug, 83(4), 963 - 6
Molecular detection of Burkholderia cepacia in toiletry, cosmetic, and pharmaceutical raw materials and finished products; Jimenez L et al.; A polymerase chain reaction (PCR) assay was developed and compared with standard methods for rapid detection of Burkholderia cepacia, a major industrial contaminant, in cosmetic and pharmaceutical raw materials and finished products . Artificially contaminated samples were incubated for 24 h in trypticase soy broth containing 4% Tween 20 and 0.5% soy lecithin . DNA was extracted from each sample using a proteinase K-tris-EDTA-Tween 20 treatment at 35 degrees C . The extracted DNA was added to Ready-To-Go PCR beads and specific DNA primers for B . cepacia . The B . cepacia DNA primers coded for a 209-base pair (bp) fragment of the 16S rRNA ribosomal gene . No DNA amplification was observed in samples that were not spiked with B . cepacia . However, all contaminated samples showed the specific 209-bp fragment for B . cepacia . There was a 100% correlation between standard methods and the PCR assay . Standard microbiological methods required 5-6 days for isolation and identification of spiked microorganisms, whereas PCR detection and identification was completed in 27 h . PCR detection of B . cepacia allows for rapid quality evaluation of cosmetic and pharmaceutical raw materials and finished products.

Infect Immun, 2000 Oct, 68(10), 5710 - 5
IS1414, an Escherichia coli insertion sequence with a heat-stable enterotoxin gene embedded in a transposase-like gene; McVeigh A et al.; Enteroaggregative Escherichia coli (EAEC) heat-stable enterotoxin 1 (EAST1) was originally discovered in EAEC but has also been associated with enterotoxigenic E . coli (ETEC) . Multiple genomic restriction fragments from each of three ETEC strains of human origin showed homology with an EAST1 gene probe . A single hybridizing fragment was detected on the plasmid of ETEC strain 27D that also encodes heat-stable enterotoxin Ib and colonization factor antigen I . We isolated and characterized this fragment, showing that it (i) carries an allele of astA nearly identical to that originally reported from EAEC 17-2 and (ii) expressed enterotoxic activity . Sequence analysis of the toxin coding region revealed that astA is completely embedded within a 1,209-bp open reading frame (ORF1), whose coding sequence is on the same strand but in the -1 reading frame in reference to the toxin gene . In vitro expression of the predicted M(r)- approximately 46,000 protein product of ORF1 was demonstrated . ORF1 is highly similar to transposase genes of IS285 from Yersinia pestis, IS1356 from Burkholderia cepacia, and ISRm3 from Rhizobium meliloti . It is bounded by 30-bp imperfect inverted repeat sequences and flanked by 8-bp direct repeats . Based on these structural features, pathognomonic of a regular insertion sequence, this element was designated IS1414 . Preliminary experiments to show IS1414 translocation were unsuccessful . Overlapping genes of the type suggested by the IS1414 core region have heretofore not been described in bacteria . It seems to offer a most efficient mechanism for intragenomic and horizontal dissemination of EAST1.

Rev Latinoam Microbiol, 1999 Jan-Mar, 41(1), 17 - 23
{The interaction between corn and Burkholderia (Pseudomonas) cepacia}; Velazquez M et al.; Burkholderia (Pseudomonas) cepacia is a plant growth promoting rhizobacteria that could improve maize productivity . In this work we studied some properties of B . cepacia strain 0057 in relation to interaction with maize . Spermosphere effect, chemostatic response, indolic compounds and siderophores production were evaluated . We observed inhibitory effect on bacterial growth by maize seeds and their soluble extracts . Root exudates had positive chemiostatic effect . We founded indolic compounds production (20.1 micrograms/ml) and siderophore presence . We think that this microorganisms could improve maize production.

Trans R Soc Trop Med Hyg, 2000 May-Jun, 94(3), 301 - 4
Melioidosis: acute and chronic disease, relapse and re-activation; Currie BJ et al.; In melioidosis-endemic regions the importance of re-activation of Burkholderia pseudomallei from latent foci remains unclear . This topic was assessed in a 10-year prospective study (1989-99) of melioidosis in the tropical north of the Northern Territory of Australia, together with other aspects of the nature of melioidosis . Incubation period from defined inoculating events was previously ascertained as 1-21 (mean 9) days . Of 252 total cases 244 (97%) were considered to be from recent acquisition of B . pseudomallei infection and 8 (3%) were considered to be re-activation from a latent focus . Acute illness occurred in 222 (88%) cases; 30 (12%) cases had chronic illness (symptomatic for > 2 months) . Of the 207 patients surviving the initial illness, 27 (13%) had a confirmed relapse (mean time from initial diagnosis of 8 months), with 5 relapsing twice . Of these 32 relapses, 15 (3 fatal) were associated with poor adherence to the eradication therapy antibiotics and 10 (none fatal) were failures of eradication with doxycycline monotherapy . Following initial intensive therapy with ceftazidime or meropenem for at least 14 days, eradication therapy with trimethoprim-sulphamethoxazole monotherapy for at least 3 months had been more successful.

Gene, 2000 Aug 22, 254(1-2), 129 - 37
Characterization and mutagenesis of fur gene from Burkholderia pseudomallei; Loprasert S et al.; A homolog of the ferric uptake regulator gene (fur) was isolated from Burkholderia pseudomallei (Bp) by a reverse genetic technique . Sequencing of a 2.2kb DNA fragment revealed an open reading frame with extensive homology to bacterial Fur proteins . The cloned gene encodes a 16kDa protein that cross-reacts with a polyclonal anti-Escherichia coli Fur serum . The transcription start site was determined by the primer extension technique . Expression analysis of fur showed no increased fur mRNA levels in response to various stresses and iron conditions . A positive selection procedure involving the isolation of manganese-resistant mutants was used to isolate mutants that produce altered Fur proteins . Sequencing of a fur mutant revealed a nucleotide change (G to A) converting a conserved amino acid arginine-69 to histidine . The fur missense mutant produced an elevated level of siderophore that could be complemented by a multicopy plasmid carrying the Bp fur . Interestingly, Fur was found to play roles as a positive regulator of FeSOD and peroxidase . The mutant showed a decreased activity of FeSOD and peroxidase, which could be important in its pathogenicity and survival in macrophages.

J Clin Microbiol, 2000 Sep, 38(9), 3165 - 73
DNA-Based diagnostic approaches for identification of Burkholderia cepacia complex, Burkholderia vietnamiensis, Burkholderia multivorans, Burkholderia stabilis, and Burkholderia cepacia genomovars I and III; Mahenthiralingam E et al.; Bacteria of the Burkholderia cepacia complex consist of five discrete genomic species, including genomovars I and III and three new species: Burkholderia multivorans (formerly genomovar II), Burkholderia stabilis (formerly genomovar IV), and Burkholderia vietnamiensis (formerly genomovar V) . Strains of all five genomovars are capable of causing opportunistic human infection, and microbiological identification of these closely related species is difficult . The 16S rRNA gene (16S rDNA) and recA gene of these bacteria were examined in order to develop rapid tests for genomovar identification . Restriction fragment length polymorphism (RFLP) analysis of PCR-amplified 16S rDNA revealed sequence polymorphisms capable of identifying B . multivorans and B . vietnamiensis but insufficient to discriminate strains of B . cepacia genomovars I and III and B . stabilis . RFLP analysis of PCR-amplified recA demonstrated sufficient nucleotide sequence variation to enable separation of strains of all five B . cepacia complex genomovars . Complete recA nucleotide sequences were obtained for 20 strains representative of the diversity of the B . cepacia complex . Construction of a recA phylogenetic tree identified six distinct clusters (recA groups): B . multivorans, B . vietnamiensis, B . stabilis, genomovar I, and the subdivision of genomovar III isolates into two recA groups, III-A and III-B . Alignment of recA sequences enabled the design of PCR primers for the specific detection of each of the six latter recA groups . The recA gene was found on the largest chromosome within the genome of B . cepacia complex strains and, in contrast to the findings of a previous study, only a single copy of the gene was present . In conclusion, analysis of the recA gene of the B . cepacia complex provides a rapid and robust nucleotide sequence-based approach to identify and classify this taxonomically complex group of opportunistic pathogens.

Med J Malaysia, 1997 Jun, 52(2), 158 - 60
In-vitro susceptibility of Burkholderia pseudomallei to cefoperazone-sulbactam combination; Koay AS et al.; Melioidosis is endemic in Malaysia . Emerging resistance with new and current antimicrobial agents has underscored the need to look further for new antimicrobial agents for the treatment of melioidosis . Hence, we evaluated the in-vitro susceptibility of fifty locally isolated strains of Burkholderia pseudomallei, the causative agent of melioidosis to cefoperazone-sulbactam combination using the method of NCCLS . All the fifty strains tested were susceptible in-vitro to cefoperazone-sulbactam . The MIC90 of the organism for cefoperazone-sulbactam was 4 mg/L . The results of our findings suggested that cefoperazone-sulbactam may be useful in the treatment of melioidosis.

Appl Environ Microbiol, 2000 Sep, 66(9), 4139 - 41
Identification and characteristics of a novel Burkholderia strain with broad-spectrum antimicrobial activity; Cain CC et al.; A Burkholderia strain isolated from soil is capable of inhibiting the growth of bacteria, plant-pathogenic fungi, pathogenic yeasts, and protozoa . Inhibition does not involve cell contact or the presence of living cells, suggesting that at least a substantial portion of the antimicrobial activity is due to the excretion of extracellular compounds.

Appl Environ Microbiol, 2000 Sep, 66(9), 4098 - 104
Toluene-degrading bacteria are chemotactic towards the environmental pollutants benzene, toluene, and trichloroethylene; Parales RE et al.; The bioremediation of polluted groundwater and toxic waste sites requires that bacteria come into close physical contact with pollutants . This can be accomplished by chemotaxis . Five motile strains of bacteria that use five different pathways to degrade toluene were tested for their ability to detect and swim towards this pollutant . Three of the five strains (Pseudomonas putida F1, Ralstonia pickettii PKO1, and Burkholderia cepacia G4) were attracted to toluene . In each case, the response was dependent on induction by growth with toluene . Pseudomonas mendocina KR1 and P . putida PaW15 did not show a convincing response . The chemotactic responses of P . putida F1 to a variety of toxic aromatic hydrocarbons and chlorinated aliphatic compounds were examined . Compounds that are growth substrates for P . putida F1, including benzene and ethylbenzene, were chemoattractants . P . putida F1 was also attracted to trichloroethylene (TCE), which is not a growth substrate but is dechlorinated and detoxified by P . putida F1 . Mutant strains of P . putida F1 that do not oxidize toluene were attracted to toluene, indicating that toluene itself and not a metabolite was the compound detected . The two-component response regulator pair TodS and TodT, which control expression of the toluene degradation genes in P . putida F1, were required for the response . This demonstration that soil bacteria can sense and swim towards the toxic compounds toluene, benzene, TCE, and related chemicals suggests that the introduction of chemotactic bacteria into selected polluted sites may accelerate bioremediation processes.

Pediatr Med Chir, 1999 Sep-Oct, 21(5 Suppl), 213 - 8
{Bacterial infections and resistance to antibiotics in cystic fibrosis}; Taccetti G et al.; Knowledge of the microbiology of pulmonary infections is critical for treatment of cystic fibrosis because sickness and mortality in this disease are mainly due to relapse occurring in the respiratory tract . The microbiology of pulmonary infections presents several singular aspects . Respiratory tract infections are caused by bacteria such as Staphylococcus aureus in the early years of life and Pseudomonas aeruginosa and Burkholderia cepacia thereafter . The patients, who are not immune compromised, are predisposed to chronic colonization and highly transmissible bacterial strains can cause cross-infections . Bacterial also develop resistance mechanisms which make them difficult to treat . Until recently the relationship between genetic defects and a predisposition to colonization was not noted, but recent studies have allowed us to form some interesting hypothesis . The present work analyzes the principal mechanisms of antibiotic resistance, with particular reference to classic cystic fibrosis pathogens, and looks at future prospects of respiratory tract infection treatment.

Mol Gen Genet, 2000 Jul, 263(6), 1031 - 7
Members of the amylovora group of Erwinia are cellulolytic and possess genes homologous to the type II secretion pathway; Riekki R et al.; A cellulase-producing clone was isolated from a genomic library of the Erwinia rhapontici (Millard) Burkholder strain NCPPB2989 . The corresponding gene, named celA, encodes an endoglucanase (EC 3.2.1.4) with the extremely low pH optimum of 3.4 and a temperature optimum between 40 and 50 degrees C . A single ORF of 999 nt was found to be responsible for the Cel activity . The corresponding protein, named CelA, showed 67% identity to the endoglucanase Y of E . chrysanthemi and 51.5% identity to the endoglucanase of Cellulomonas uda, and thus belongs to the glycosyl hydrolase family 8 . The celA gene, or its homologue, was found to be present in all E . rhapontici isolates analysed, in E . chrysanthemi, and in E . amylovora . The presence of plant cell wall-degrading enzymes in the amylovora group of Erwinia spp . had not previously been established . Furthermore, the DNA of both E . rhapontici and E . amylovora was found to exhibit homology to genes encoding the type II (GSP) secretion pathway, which is known to be responsible for extracellular targeting of cellulases and pectinases in Erwinia spp . that cause soft rotting, such as E . carotovora and E . chrysanthemi . Secretion of the CelA protein by E . rhapontici could not be verified . However, the CelA protein itself was found to include the information necessary for heterologous secretion by E . chrysanthemi.

Infect Immun, 2000 Sep, 68(9), 5377 - 84
Burkholderia pseudomallei induces cell fusion and actin-associated membrane protrusion: a possible mechanism for cell-to-cell spreading; Kespichayawattana W et al.; Burkholderia pseudomallei, a facultative intracellular bacterium, is the causative agent of a broad spectrum of diseases collectively known as melioidosis . Its ability to survive inside phagocytic and nonphagocytic cells and to induce multinucleated giant cell (MNGC) formation has been demonstrated . This study was designed to assess a possible mechanism(s) leading to this cellular change, using virulent and nonvirulent strains of B . pseudomallei to infect both phagocytic and nonphagocytic cell lines . We demonstrated that when the cells were labeled with two different cell markers (CMFDA or CMTMR), mixed, and then infected with B . pseudomallei, direct cell-to-cell fusion could be observed, leading to MNGC formation . Staining of the infected cells with rhodamine-conjugated phalloidin indicated that immediately after the infection, actin rearrangement into a comet tail appearance occurred, similar to that described earlier for other bacteria . The latter rearrangement led to the formation of bacterium-containing, actin-associated membrane protrusions which could lead to a direct cell-to-cell spreading of B . pseudomallei in the infected hosts . Results from 4', 6'-diamidine-2-phenylindole dihydrochloride (DAPI) nuclear staining, poly-ADP ribose polymerase cleavage, staining of infected cells for phosphatidylserine exposure with annexin V, and electrophoresis of the DNA extracted from these infected cells showed that B . pseudomallei could kill the host cells by inducing apoptosis in both phagocytic and nonphagocytic cells.

Int J Food Microbiol, 2000 Jul 25, 59(1-2), 67 - 72
Recovery of Burkholderia pseudomallei and B . cepacia from drinking water; Zanetti F et al.; Samples of drinking water were examined in order to evaluate the occurrence of two gram-negative bacteria: Burkholderia pseudomallei and B . cepacia . A total of 85 samples were collected from public and private buildings in the province of Bologna (Italy) . Other bacteriological indicators (heterotrophic plate count at 22 and 36 degrees C) were also examined, together with physical and chemical parameters (temperature, pH, residual chlorine, total hardness and chemical oxygen demand (COD)) . High levels of B . pseudomallei were recovered (mean value = 578 cfu/100 ml) in about 7% of samples, while B . cepacia was recovered in 3.5% (mean value = < 1) of the samples . The two microorganisms were found to correlate positively with heterotrophic plate counts at 22 and 36 degrees C, but not with the physical and chemical parameters taken into consideration.

Mol Microbiol, 2000 Jun, 36(6), 1481 - 93
Clinical and environmental isolates of Burkholderia cepacia exhibit differential cytotoxicity towards macrophages and mast cells; Melnikov A et al.; Burkholderia cepacia is an emerging opportunistic pathogen that causes fatal infections in patients suffering from cystic fibrosis (CF) and chronic granulomatous disease . Various environmental isolates of B . cepacia are, however, capable of degrading environmental pollutants, such as trichloroethylene, 2,4,5-trichlorophenoxyacetic acid (2,4,5-T), etc., and are also highly effective in controlling plant diseases caused by nematodes and fungi . Such strains have therefore been proposed for environmental release to clean up toxic dump sites or as biopesticides . Various efforts to distinguish between clinical and environmental isolates of B . cepacia with regard to their virulence characteristics have produced ambiguous results, suggesting that newer methods are needed to test for the presence or absence of pathogenic potential in B . cepacia strains proposed for environmental release . We now report that several clinical strains of B . cepacia secrete cytotoxic factors that allow macrophage and mast cell death in the presence of external ATP . Several environmental strains had reduced activity in this regard . We also demonstrate that, while all the strains secrete enzymes that have nucleoside diphosphate kinase (Ndk), adenylate kinase (Ak) and 5'-nucleotidase activity, the level of secretion of the 5'-nucleotidase (and/or ATPase/phosphatase) appears to be lower in the environmental strains than in the clinical strains . The secretion of these enzymes is specifically activated in the presence of eukaryotic proteins such as alpha2-macroglobulin . As macrophage-or mast cell surface-associated P2Z receptors promote their cell death in the presence of mM concentrations of ATP, and as the secreted ATP-using enzymes generate various phosphorylated or non-phosphorylated adenine nucleotides that may even be better agonists than ATP in activating the P2Z receptors or may act through the activation of additional purinergic receptors, such enzymes may play an important role in allowing B . cepacia to evade host defence.

Br J Clin Pharmacol, 2000 Aug, 50(2), 184 - 91
Pharmacokinetic-pharmacodynamic evaluation of ceftazidime continuous infusion vs intermittent bolus injection in septicaemic melioidosis; Angus BJ et al.; AIMS: Experimental studies have suggested that constant intravenous infusion would be preferable to conventional intermittent bolus administration of beta-lactam antibiotics for serious Gram-negative infections . Severe melioidosis (Burkholderia pseudomallei infection) carries a mortality over 40% despite treatment with high dose ceftazidime . The aim of this study was to measure the pharmacokinetic and pharmacodynamic effects of continuous infusion of ceftazidime vs intermittent bolus dosing in septicaemic melioidosis . METHODS: Patients with suspected septicaemic melioidosis were randomised to receive ceftazidime 40 mg kg(-1) 8 hourly by bolus injection or 4 mg kg(-1) h(-1) by constant infusion following a 12 mg kg(-1) priming dose and pharmacokinetic and pharmacodynamic parameters were compared . RESULTS: Of the 34 patients studied 16 (59%) died . Twenty patients had cultures positive for B . pseudomallei of whom 12 (60%) died . The median MIC90 of B . pseudomallei was 2 mg l(-1), giving a minimum target concentration (4*MIC) of 8 mg l(-1) . The median (range) estimated total apparent volume of distribution, systemic clearance and terminal elimination half-lives of ceftazidime were 0.468 (0.241-0 . 573) l kg(-1), 0.058 (0.005-0.159) l kg(-1) h(-1) and 7.74 (1.95-44.71) h, respectively . Clearance of ceftazidime and creatinine clearance were correlated closely (r = 0.71; P < 0.001) and there was no evidence of significant nonrenal clearance . CONCLUSIONS: Simulations based on these data and the ceftazidime sensitivity of the B . pseudomallei isolates indicated that administration by constant infusion would allow significant dose reduction and cost saving . With conventional 8 h intermittent dosing to patients with normal renal function, plasma ceftazidime concentrations could fall below the target concentration but this would be unlikely with a constant infusion . Correction for renal failure, which is common in patients with meliodosis is Clearance = k(*) creatinine clearance where k = 0.72 . Calculation of a loading dose gives median (range) values of loading dose, DL of 18.7 mg kg(-1) (9.5-23) and infusion rate I = 3.5 mg k(-1) h(-1) (0.4-13) (which equals 84 mg kg(-1) day(-1)) . A nomogram for adjustment in renal failure is given.

Southeast Asian J Trop Med Public Health, 1999 Dec, 30(4), 760 - 3
Genotypic and phenotypic relationship in Burkholderia pseudomallei indicates colonization with closely related isolates; Radu S et al.; Seven isolates of Burkholderia pseudomallei from cases of melioidosis in human (2 isolates) and animal (2 isolates), cat (one isolate) and from soil samples (2 isolates) were examined for in vitro sensitivity to 14 antimicrobial agents and for presence of plasmid DNA . Randomly amplified polymorphic DNA (RAPD) analysis was used to type the isolates, using two arbitrary primers . All isolates were sensitive to chloramphenicol, kanamycin, carbenicillin, rifampicin, enrofloxacin, tetracycline and sulfamethoxazole-trimethoprim . No plasmid was detected in all the isolates tested . RADP fingerprinting demonstrated genomic relationship between isolates, which provides an effective method to study the epidemiology of the isolates examined.

Southeast Asian J Trop Med Public Health, 1999 Dec, 30(4), 756 - 9
Epidemiology of arabinose assimilation in burkholderia pseudomallei isolated from patients and soil in Thailand; Trakulsomboon S et al.; Burkholderia pseudomallei is an environmental saprophyte that has been isolated widely from soil in Southeast Asia and the relationship between environmental contamination and clinical melioidosis has been established . It has been shown that the arabinose assimilation property of B . pseudonrallei is probably one of the determinants indicating virulence of this organism . Therefore, the distribution of arabinose assimilation biotypes of B . pseudomallei collected from four geographic regions of Thailand was studied in order to determine an association between arabinose assimilation of B . pseudomallei and the uneven distribution of melioidosis found among these four areas . A total of 830 isolates of B . pseudomallei (412 patient isolates and 418 soil isolates) collected from the patients and soil in four regions of Thailand in 1997 were tested for an ability to grow on a minimal agar medium supplemented with L-arabinose . All patient isolates except one could not utilise arabinose (Ara-) . For 418 soil isolates, 232 (55.5%) isolates were identified as Ara type . They comprised 180 (62.5%), 36 (46.8%), 6 (35.3%) and 10 (27.8%) isolates derived from northeastern, southern, northern and central regions respectively . The ratios of Ara- to Ara, were 1.7, 0.9 . 0.5 and 0.4 among isolates collected from northeastern, southern, northern and central regions respectively . The prevalence of Ara- in soil isolates in northeast is significantly higher than those in other regions . This observation suggests that in addition to the presence of B . pseudomallei in soil which is one of the factors contributing to a burden of melioidosis in northeastern Thailand, the distribution of more virulent biotype (Ara-) soil isolates is a factor contributing to a high prevalence of melioidosis in northeastern Thailand as well.

Zh Mikrobiol Epidemiol Immunobiol, 2000 May-Jun, (3), 73 - 5
{The role of capsule formation in Burkholderia mallei for its persistence in vivo}; Popov SF et al.; When studied in vivo (in guinea pigs) with the use of electron microscopy, B . mallei (strains C-5, 10230) were found to form a capsule . In the subacute course of infection, the encapsulated forms of B . mallei parasitized mainly in the cells of the system of mononuclear phagocytes in the liver, the spleen and the lungs . The capsule formed by B . mallei was shown to be one of the factors facilitating its persistence in the body.

J Vector Ecol, 2000 Jun, 25(1), 90 - 3
Isolations of enteric pathogens from synanthropic flies trapped in downtown Kuala Lumpur; Sulaiman S et al.; Four species of synanthropic flies were trapped in downtown Kuala Lumpur: Chrysomya megacephala, Chrysomya rufifacies, Musca domestica, and Musca sorbens . Burkholderia pseudomallei, the organism causing melioidosis, was the dominant bacteria isolated from Chrysomya megacephala . Klebsiella oxytoca, commonly associated with nosocomial infections, was commonly isolated from Chrysomya megacephala, Musca domestica, and Musca sorbens . Aeromonas hydrophila, the bacteria causing gastroenteritis, was predominantly isolated from Chrysomya megacephala and also from Musca domestica and Musca sorbens . A total of 18 bacterial species was isolated from the synanthropic flies trapped . Burkholderia pseudomallei had been reported for the first time.

Biochem Biophys Res Commun, 2000 Aug 11, 274(3), 626 - 30
Kinetics of biodegradation of p-nitrophenol by different bacteria; Bhushan B et al.; Three bacterial species, i.e., Ralstonia sp . SJ98, Arthrobacter protophormiae RKJ100, and Burkholderia cepacia RKJ200, have been examined for their efficiency and kinetics behavior toward PNP degradation . All the three bacteria utilized PNP as the sole source of carbon, nitrogen, and energy . The rates of radiolabeled {U-(14)C}PNP degradation by all the bacteria were higher in the nitrogen-free medium compared to the medium with nitrogen . The apparent K(m) values of PNP degradation by SJ98, RKJ100, and RKJ200 were 0.32, 0.28, and 0.23 mM, respectively, as determined from the Michaelis-Menten curves . The maximum rates of PNP degradation (V(max)) according to Lineweaver-Burk's plots were 11.76, 7.81, and 3.84 micromol PNP degraded/min/mg dry biomass, respectively . The interpretation drawn from the Lineweaver-Burk's plots showed that the PNP degradation by SJ98 was stimulated by 4-nitrocatechol and 1, 2,4-benzenetriol . Benzoquinone and hydroquinone inhibited PNP degradation by RKJ100 noncompetitively and competitively, respectively, whereas in the case of RKJ200, benzoquinone and hydroquinone inhibited PNP degradation in an uncompetitive manner . beta-Ketoadipate did not affect the rate of PNP degradation in any case .

Appl Environ Microbiol, 2000 Aug, 66(8), 3399 - 407
Comparison of 2,4-dichlorophenoxyacetic acid degradation and plasmid transfer in soil resulting from bioaugmentation with two different pJP4 donors; Newby DT et al.; A pilot field study was conducted to assess the impact of bioaugmentation with two plasmid pJP4-bearing microorganisms: the natural host, Ralstonia eutropha JMP134, and a laboratory-generated strain amenable to donor counterselection, Escherichia coli D11 . The R . eutropha strain contained chromosomal genes necessary for mineralization of 2,4-dichlorophenoxyacetic acid (2,4-D), while the E . coli strain did not . The soil system was contaminated with 2,4-D alone or was cocontaminated with 2,4-D and Cd . Plasmid transfer to indigenous populations, plasmid persistence in soil, and degradation of 2,4-D were monitored over a 63-day period in the bioreactors . To assess the impact of contaminant reexposure, aliquots of bioreactor soil were reamended with additional 2,4-D . Both introduced donors remained culturable and transferred plasmid pJP4 to indigenous recipients, although to different extents . Isolated transconjugants were members of the Burkholderia and Ralstonia genera, suggesting multiple, if not successive, plasmid transfers . Upon a second exposure to 2,4-D, enhanced degradation was observed for all treatments, suggesting microbial adaptation to 2,4-D . Upon reexposure, degradation was most rapid for the E . coli D11-inoculated treatments . Cd did not significantly impact 2,4-D degradation or transconjugant formation . This study demonstrated that the choice of donor microorganism might be a key factor to consider for bioaugmentation efforts . In addition, the establishment of an array of stable indigenous plasmid hosts at sites with potential for reexposure or long-term contamination may be particularly useful.

Appl Environ Microbiol, 2000 Aug, 66(8), 3180 - 6
Identification of the dimerization domain of dehalogenase IVa of Burkholderia cepacia MBA4; Tsang JS et al.; Haloacid dehalogenases are enzymes that catalyze the hydrolytic removal of halogens from haloalkanoic acids . Dehalogenase IVa (DehIVa) from Burkholderia cepacia MBA4 and dehalogenase CI (DehCI) from Pseudomonas sp . strain CBS3 exhibit 68% identity . Despite their similarity DehIVa is a dimeric enzyme while DehCI is a monomer . In this work, we describe the identification of the domain that confers the dimerization function of DehIVa . Recombinant DNA molecules were constructed by fusion of the respective dehalogenase genes hdlIVa and dehCI . When amino acids 73 to 89 of DehCI were replaced by amino acids 74 to 90 of DehIVa, the recombinant molecule migrated like that of DehIVa in a nondenaturing activity-stained gel . Similarly, when residues 73 to 89 of DehIVa were replaced by the corresponding residues of DehCI, the chimera migrated as a monomer . These 17 amino acid changes were able to determine the aggregation states of the molecules . The retention of the catalytic function in these chimeras indicated that the overall folding of these proteins was not affected . Site-directed mutagenesis on hdlIVa however indicated that amino acids Phe58, Thr65, Leu78, and Phe92 of DehIVa are also important for the aggregation state of the protein . This indicates that the 17 residues are not sufficient for the dimerization of the protein.

MMWR Morb Mortal Wkly Rep, 2000 Jun 23, 49(24), 532 - 5
Laboratory-acquired human glanders--Maryland, May 2000; Utility of commercial systems for identification of Burkholderia cepacia complex from cystic fibrosis sputum culture; Clinical Microbiology Laboratory, Children's Hospital of Philadelphia, Philadelphia, Pennsylvania 19104, USAPerformances of several commercial test systems were reviewed to determine their relative levels of accuracy in identifying Burkholderia cepacia complex isolates recovered from cystic fibrosis sputum culture . Positive predictive values ranged from 71 to 98%; negative predictive values ranged from 50 to 82% . All systems misidentified B . cepacia complex . The species most frequently misidentified as B . cepacia was Burkholderia gladioli . These data support the results of previous studies that recommend confirmatory testing, including the use of DNA-based methods, for sputum culture isolates presumptively identified as B . cepacia.

J Clin Microbiol, 2000 Aug, 38(8), 2962 - 5
Identification of members of the Burkholderia cepacia complex by species-specific PCR; Whitby PW et al.; Definitive identification of the species in the Burkholderia cepacia complex by routine clinical microbiology methods is difficult . Phenotypic tests to identify B . multivorans and B . vietnamiensis have been established; more recent work indicates B . stabilis may also be identified by growth characteristics and biochemical tests . However, attempts to identify genomovars I and III have, thus far, proved unsuccessful . Previously, we demonstrated the utility of two primer pairs, directed to the rRNA operon, to specifically identify the B . cepacia complex in a PCR . One of these primer pairs, G1-G2, only amplified a DNA fragment from genomovars I and III and B . stabilis in a PCR with genomic DNA isolated from prototypical strains representing the five genomovars . Sequence analysis of the rRNA operon for all the genomovars indicated that this primer pair targeted a region shared by these isolates . Further analysis revealed a region of heterogeneity between genomovar III and B . stabilis internal to the amplified product of G1-G2 . Primers designed to target this region were tested with prototypical strains following an initial amplification with the G1-G2 primer pair . New primers specific for the prototypical genomovar III and B . stabilis were designated SPR3 and SPR4, respectively . Analysis of 93 isolates representing 18 genomovar I, 13 B . multivorans, 36 genomovar III, 11 B . stabilis, and 15 B . vietnamiensis isolates was performed . DNA from all isolates of genomovars I and III and B . stabilis was amplified by G1-G2 . Genomovar III isolates yielded a product with SPR3/G1 while B . stabilis amplified with SPR4-G1 . Genomovar I isolates were amplified by either SPR3-G1 or SPR4-G1, but not both . B . multivorans yielded a product with SPR3-G1 but not G1-G2, and B . vietnamiensis isolates were negative in all PCRs . Thus using an algorithm with G1-G2, SPR3-G1, and SPR4-G1 primers in a PCR analysis, genomovar III isolates can be separated from B . stabilis and the identity of B . multivorans and B . vietnamiensis can be confirmed.

J Med Assoc Thai, 1997 Oct, 80(10), 671 - 4
Diabetic ketoacidosis and melioidosis in a child; Panamonta O et al.; A case of 5 year old diabetic girl with melioidosis was reported . She presented with the symptoms and signs of intraabdominal infection, septicemia and diabetic ketoacidosis . Abdominal ultrasonography showed multiple splenic and liver abscesses, melioidosis was suspected . Hemoculture and pus culture yielded Burkholderia pseudomallei which was susceptible to ceftazidime and cotrimoxazole . Correction of fluid and electrolyte combined with insulin therapy and proper antibiotics resulted in a good outcome in this patient.

Chemosphere, 2000 Oct, 41(8), 1257 - 61
Bacterial response to photocatalytic degradation of 6-chlorovanillin; Yeber MC et al.; The oxidation of a 186 ppm 6-chlorovanillin solution was performed using impregnated TiO2 glass rings in a 1 l photochemical reactor . Fifty per cent degradation was obtained after 60 min with recirculation of the solution . Then, oxidised samples were submitted under aerobic conditions to bacterial degradation in the Pseudomonas paucimobilis (S37) and Burkholderia cepacia (PZK) . Both selected aerobic bacteria degrade more efficiently the photocatalysed samples, being PZK strain better than S37 . A first-order kinetic was observed in both systems photocatalytic and bacterial degradation.

Chest, 2000 Jul, 118(1), 85 - 91
Pregnancy in cystic fibrosis . Fetal and maternal outcome; Gilljam M et al.; OBJECTIVE: To assess the effect of pregnancy on pulmonary function and survival in women with cystic fibrosis (CF) and to assess the fetal outcome . DESIGN: Cohort study . The data analyzed were collected from the Toronto CF database, chart review, and patient questionnaire . SETTING: Tertiary-care center . PATIENTS: All women with CF who, at the time of diagnosis or pregnancy, attended the Toronto Cystic Fibrosis Clinics between 1961 and 1998 . RESULTS: From 1963 to 1998, there were 92 pregnancies in 54 women . There were 11 miscarriages and 7 therapeutic abortions . Forty-nine women gave birth to 74 children . The mean follow-up time was 11 +/- 8 years . One patient was lost to follow-up shortly after delivery, and one was lost after 12 years . The overall mortality rate was 19% (9 of 48 patients) . Absence of Burkholderia cepacia (p < 0.001), pancreatic sufficiency (p = 0.01), and prepregnancy FEV(1) > 50% predicted (p = 0.03) were associated with better survival rates . When adjusted for the same parameters, pregnancy did not affect survival compared to the entire adult female CF population . The decline in FEV(1) was comparable to that in the total CF population . Three women had diabetes mellitus, and seven developed gestational diabetes . There were six preterm infants and one neonatal death . CF was diagnosed in two children . CONCLUSIONS: The maternal and fetal outcome is good for most women with CF . Risk factors for mortality are similar to those for the nonpregnant CF population . Pregnancies should be planned so that there is opportunity for counseling and optimization of the medical condition . Good communication between the CF team and the obstetrician is important.

FEMS Immunol Med Microbiol, 2000 Aug, 28(4), 307 - 12
Sensitivity of the Burkholderia cepacia complex and Pseudomonas aeruginosa to transducing bacteriophages; Nzula S et al.; Burkholderia cepacia is now recognised as a life-threatening pathogen among several groups of immunocompromised patients . In this context, the proposed large-scale use of these bacteria in agriculture has increased the need for a better understanding of the genetics of the species forming the B . cepacia complex . Until now, little information has been available on the bacteriophages of the B . cepacia complex . Transducing phages, named NS1 and NS2, were derived from the lysogenic B . cepacia strains ATCC 29424 and ATCC 17616 . The frequency of transduction per phage particle ranged from 1.0x10(-8) to 7.0x10(-6) depending on the phage and recipient strain used . The host range of NS1 and NS2 differed but in each case included environmental and clinical isolates, and strains belonging to several species and genomovars of the B . cepacia complex . The host range of both phages also included Pseudomonas aeruginosa . Some B . cepacia complex isolates were sensitive to the well-characterised P . aeruginosa transducing phages, B3, F116L and G101 . The lytic activity of NS1 and NS2 was inhibited by B . cepacia lipopolysaccharide suggesting that this moiety is a binding site for both phages . The molecular size of the NS1 and NS2 genomes was approximately 48 kb.

Biochem Biophys Res Commun, 2000 Jul 14, 273(3), 1088 - 94
Structural study of the exopolysaccharide produced by a clinical isolate of Burkholderia cepacia; Cescutti P et al.; The primary structure of the exopolysaccharide produced by a clinical isolate of the bacterium Burkholderia cepacia was studied by means of methylation analysis, selective degradation, NMR spectroscopy, and electrospray mass spectrometry . The resulting data showed that the parent repeating unit of the exopolysaccharide is a highly branched heptasaccharide with the following structure: Two acetyl groups are present per repeating unit, as noncarbohydrate substituents .

Lett Appl Microbiol, 2000 Jul, 31(1), 20 - 4
Susceptibility of multiresistant strains of Burkholderia cepacia to honey; Cooper RA et al.; Twenty strains of Burkholderia cepacia, isolated principally from the sputum of cystic fibrosis patients, were tested for their susceptibility to eight antibiotics with a modified Kirby-Bauer Disc diffusion technique . All strains exhibited multiple but not identical patterns of antibiotic resistance . The sensitivity of all strains to honey was assessed with an agar dilution method . All strains exhibited susceptibility to concentrations of honey below 6% (v/v) . This suggests that honey may have a potential role in the clinical management of B . cepacia infections.

J Infect Dis, 2000 Jul, 182(1), 206 - 13 Epub 2000 Jul 06.
Soluble granzymes are released during human endotoxemia and in patients with severe infection due to gram-negative bacteria; Lauw FN et al.; Extracellular release of granzymes is considered to reflect the involvement of cytotoxic T lymphocytes and NK cells in various disease states . To obtain insight into granzyme release during bacterial infection, granzyme levels were measured during experimental human endotoxemia and in patients with melioidosis, a severe infection due to gram-negative bacteria . Plasma concentrations of granzyme A (GrA) and GrB increased transiently after endotoxin administration, peaking after 2-6 h . In patients with bacteremic melioidosis, GrA and GrB levels were elevated on admission and remained high during the 72-h study period . In whole blood stimulated with heat-killed Burkholderia pseudomallei, neutralization of tumor necrosis factor, interleukin-12, or interleukin-18 inhibited granzyme secretion, which was independent of interferon-gamma . Stimulation with endotoxin and other gram-negative and gram-positive bacteria also strongly induced the secretion of granzymes, suggesting that granzyme release is a general immune response during bacterial infection . The interaction between the cytokine network and granzymes may play an important immunoregulatory role during bacterial infections.

Appl Environ Microbiol, 2000 Jul, 66(7), 2928 - 33
Influence of chlorine substituents on rates of oxidation of chlorinated biphenyls by the biphenyl dioxygenase of Burkholderia sp . strain LB400; Arnett CM et al.; Biphenyl dioxygenase from Burkholderia (Pseudomonas) sp . strain LB400 catalyzes the first reaction of a pathway for the degradation of biphenyl and a broad range of chlorinated biphenyls (CBs) . The effect of chlorine substituents on catalysis was determined by measuring the specific activity of the enzyme with biphenyl and 18 congeners . The catalytic oxygenase component was purified and incubated with individual CBs in the presence of electron transport proteins and cofactors that were required for enzyme activity . The rate of depletion of biphenyl from the assay mixture and the rate of formation of cis-biphenyl 2,3-dihydrodiol, the oxidation product, were almost equal, indicating that the assay accurately measured enzyme-specific activity . Four classes of CBs were defined based on their oxidation rates . Class I contained 3-CB and 2,5-CB, which gave rates that were approximately twice that of biphenyl . Class II contained 2,5,3',4'-CB, 2,3,2',5'-CB, 2,3,4,5-CB, 2,3,2',3'-CB, 2,4, 5,2',5'-CB, 2,5,3'-CB, 2,5,4'-CB, 2-CB, and 3,4,5-CB, which gave rates that ranged from 97 to 35% of the biphenyl rate . Class III contained only 2,3,4,2',5'-CB, which gave a rate that was 4% of the biphenyl rate . Class IV contained 2,4,4'-CB, 2,4,2',4'-CB, 3,4,5, 2'-CB, 3,4,5,3'-CB, 3,5,3',5'-CB, and 3,4,5,2',5'-CB, which showed no detectable depletion . Rates were not significantly correlated with the aqueous solubilities of the CBs or the number of chlorine substituents on the rings . Oxidation products were detected for all class I, II, and III congeners and were identified as chlorinated cis-dihydrodiols for classes I and II . The specificity of biphenyl dioxygenase for the CBs examined in this study was determined by the relative positions of the chlorine substituents on the aromatic rings rather than the number of chlorine substituents on the rings.

Appl Environ Microbiol, 2000 Jul, 66(7), 2703 - 10
Effect of model sorptive phases on phenanthrene biodegradation: molecular analysis of enrichments and isolates suggests selection based on bioavailability; Friedrich M et al.; Reduced bioavailability of nonpolar contaminants due to sorption to natural organic matter is an important factor controlling biodegradation of pollutants in the environment . We established enrichment cultures in which solid organic phases were used to reduce phenanthrene bioavailability to different degrees (R . J . Grosser, M . Friedrich, D . M . Ward, and W . P . Inskeep, Appl . Environ . Microbiol . 66:2695-2702, 2000) . Bacteria enriched and isolated from contaminated soils under these conditions were analyzed by denaturing gradient gel electrophoresis (DGGE) and sequencing of PCR-amplified 16S ribosomal DNA segments . Compared to DGGE patterns obtained with enrichment cultures containing sand or no sorptive solid phase, different DGGE patterns were obtained with enrichment cultures containing phenanthrene sorbed to beads of Amberlite IRC-50 (AMB), a weak cation-exchange resin, and especially Biobead SM7 (SM7), a polyacrylic resin that sorbed phenanthrene more strongly . SM7 enrichments selected for mycobacterial phenanthrene mineralizers, whereas AMB enrichments selected for a Burkholderia sp . that degrades phenanthrene . Identical mycobacterial and Burkholderia 16S rRNA sequence segments were found in SM7 and AMB enrichment cultures inoculated with contaminated soil from two geographically distant sites . Other closely related Burkholderia sp . populations, some of which utilized phenanthrene, were detected in sand and control enrichment cultures . Our results are consistent with the hypothesis that different phenanthrene-utilizing bacteria inhabiting the same soils may be adapted to different phenanthrene bioavailabilities.

Microb Ecol, 2000 May, 39(4), 322 - 329
Occurrence and Multiple Antibiotic Resistance Profiles of Non-fermentative Gram-Negative Microflora in Five Brands of Non-carbonated French Bottled Spring Water; Mary P et al.; Five brands of French bottled mineral water were analyzed by heterotrophic plate counts (HPC) and for the presence of multiple antibiotic resistant bacteria . HPC at 22 degrees C were around 10(4) colony forming units ml(-1) on R2A medium . Enumeration on PCA/10, MH, and especially PCA and King B media was less efficient . At 37 degrees C, HPC were two to three orders of magnitude less than at 22 degrees C . Moreover, phenotypic diversity (7 to 15 phenotypes) was optimal on R2A incubated at 22 degrees C . All isolates were identified as non-fermentative Gram-negative rods and 75% were non-identifiable with the API 20NE system . Stenotrophomonas maltophilia and fluorescent Pseudomonas were isolated on VIA and CFC selective agar media, respectively . Burkholderia cepacia strains were not isolated on BCSA medium . The species S . maltophilia was found in 33%, 28%, and 11% of sample from springs A, D, and E, respectively . Independent of brand, isolates from HPC media were less efficient to achieve confluent growth in 18 h on MH at 30 or 37 degrees C (0 to 40%) than isolates from selective media (28 to 63%) . Seventy percent of the total isolates from dominant microflora (1-5 x 10(3) CFU ml(-1) on HPC media) were resistant against two or four antibiotics . The antibiotics concerned were principally aztreonam, ampicillin, and nalidixic acid . The remaining dominant bacteria showed a 6-9 multiple antibiotic resistant (MAR) pattern . All isolates were susceptible to newer antimicrobial agents . Owing to their low nutrient and temperature requirements, these isolates are unlikely to cause concern to public heath . Fifty percent of strains isolated from selective media (non-dominant microflora, 4-40 CFU l(-1)) showed a 10-18 MAR pattern and 33%, identified as S . maltophilia, a 20-27 MAR pattern . However, minocycline was effective against all isolates . Owing to its low concentration, colonization of human intestine by MAR S . maltophilia is unlikely.

Zh Mikrobiol Epidemiol Immunobiol, 1999 Nov-Dec, (6), 24 - 7
{The enzyme-antienzyme interactions of a number of Burkholderia pseudomallei hydrolases with monoclonal immunoglobulins}; Tikhonov NG et al.; Phosphatase, phospholipase C and a proteolytic complex, including casein- and hemoglobin-hydrolases, have been isolated from cell-free extracts of B . pseudomallei cultivation medium . A set of monoclonal antibodies (McAb) to the antigenic complexes of this infective agent has been obtained . Conditions for the study of the interaction of the enzymes and McAb have been worked out, and most of the 14 immunoglobulins under study have been shown to neutralize 2-3 hydrolases each . Three types of McAb, selectively inhibiting only the proteolytic complex, only casein-hydrolase and only phospholipase C, have been identified.

Plasmid, 2000 Jul, 44(1), 44 - 53
The novel insertion sequences IS1417, IS1418, and IS1419 from Burkholderia glumae and their strain distribution; Hasebe A et al.; Three insertion sequences, IS1417, IS1418, and IS1419, were isolated from Burkholderia glumae (formerly Pseudomonas glumae), a gram-negative rice pathogenic bacterium, on the basis of their abilities to activate the expression of the neo gene of the entrap vector pSHI1063 . The 1335-bp IS1417 element with 17-bp imperfect terminal inverted repeats was found to be flanked by 5-bp direct repeats of the vector sequence . IS1418 is 865 bp in length and carries 15-bp inverted repeats with a target duplication of 3 bp . The 1215-bp IS1419 sequence is bounded by the 36-bp terminal inverted repeats of the element and 7-bp direct repeats of the vector sequence . IS1417 and IS1418 belong to the IS2 subgroup of the IS3 family and the IS427 subgroup of the IS5 family, respectively, whereas IS1419 does not appear to be a member of any known IS family . Southern blot analysis of DNAs from B . glumae field isolates indicated that those IS elements are widely distributed, but the host range of the three IS elements appears to be limited to B . glumae and some other related species such as B . plantarii . The polymorphisms exhibited in B . glumae isolates suggest that those elements are useful for molecular epidemiological studies of B . glumae infections .

Biodegradation, 1999, 10(5), 363 - 71
Degradation of anaerobic reductive dechlorination products of Aroclor 1242 by four aerobic bacteria; Maltseva OV et al.; We studied the aerobic degradation of eight PCB congeners which comprise from 70 to 85% of the anaerobic dechlorination products from Aroclor 1242, including 2-, 4-, 2,4-, 2,6-, 2,2'-, 2,4'-, 2,2', 4-, and 2,4,4'-chlorobiphenyl (CB), and the biodegradation of their mixtures designed to simulate anaerobic dechlorination profiles M and C . Strains Comamonas testosteroni VP44 and Rhodococcus erythreus NY05 preferentially oxidized a para-substituted ring, while Rhodococcus sp . RHA1, similar to well known strain Burkholderia sp . LB400, preferably attacked an ortho-chlorinated ring . Strains with ortho-directed attack extensively degraded 2,4'- and 2,4,4'-CB into 4-chlorobenzoate, while bacteria with para-directed attack transformed these congeners mostly into potentially problematic meta-cleavage products . The strains that preferentially oxidized an ortho-substituted ring readily degraded seven of the eight congeners supplied individually; only 2,6-CB was poorly degraded . Degradation of 2,2'- and 2,4,4'-CB was reduced when present in mixtures M and C . Higher efficiencies of degradation of the individual congeners and defined PCB mixtures M and C and greater production of chlorobenzoates were observed with bacteria that preferentially attack an ortho-substituted ring . PCB congeners 2,4'-, 2,2',4-, and 2,4,4'-CB can be used to easily identify bacteria with ortho-directed attack which are advantageous for use in the aerobic stage of the two-phase (anaerobic/aerobic) PCB bioremediation scheme.

Lancet, 2000 May 27, 355(9218), 1885 - 6
Differential binding of mannose-binding lectin to respiratory pathogens in cystic fibrosis; Davies J et al.; We have found that mannose-binding lectin binds to Burkholderia cepacia, an important pathogen in patients with cystic fibrosis, and leads to complement activation, but that this is not the case for Pseudomonas aeruginosa, the more common colonising organism in this disease . We suggest that patients with cystic fibrosis with mannose-binding-lectin deficiency will be at particular risk of B cepacia colonisation.

J Biol Chem, 2000 Sep 1, 275(35), 26885 - 91
Role of the lipase-specific foldase of Burkholderia glumae as a steric chaperone; El Khattabi M et al.; Most lipases of Gram-negative bacteria require a lipase-specific foldase (Lif) in order to fold in the periplasm into their active, protease-resistant conformation prior to their secretion . The periplasmic domain of the Lif (amino acids 44-353) of Burkholderia glumae was purified as a His-tagged protein, and its function in the folding of lipase was studied in vitro . Refolding of the denatured lipase into its active conformation was dependent on the presence of the Lif . Circular dichroism revealed that the lipase refolded in the absence of Lif into a form with a native-like conformation, which was more stable against heat-induced denaturation than the native form, but was enzymatically inactive . This form of the protein could be activated by adding Lif after several hours, which demonstrates that the function of this chaperone is to help lipase to overcome an energetic barrier in the productive folding pathway rather than to prevent it from entering a non-productive pathway . The Lif was shown to interact with the native lipase in protease-protection experiments as well as by affinity chromatography, consistent with a role of the Lif late in the folding process . These results demonstrate that the Lif functions in a way analogous to the propeptides of many bacterial proteases and indicate that the amino acid sequence of the lipase does not contain all the information required for the protein to adopt its three-dimensional structure.

Chest, 2000 Jun, 117(6), 1661 - 5
Misidentification of Burkholderia cepacia in US cystic fibrosis treatment centers: an analysis of 1,051 recent sputum isolates; McMenamin JD et al.; BACKGROUND: Burkholderia cepacia remains a significant pathogen in persons with cystic fibrosis (CF) . The medical and psychosocial consequences of pulmonary colonization with this bacterium are enormous . However, B cepacia may be frequently misidentified from CF sputum culture . Study objectives: To determine the rate of misidentification of B cepacia recently recovered from CF sputum culture of persons receiving care in US treatment centers . DESIGN: Bacterial isolates cultured from CF sputum and putatively identified as B cepacia or other related nonlactose-fermenting Gram-negative species were referred from participating treatment centers . Isolates underwent polyphasic analyses employing phenotypic (selective media and biochemical testing) and genotypic (polymerase chain reaction) assays to determine species identification . Taxonomic evaluations were performed by using sodium dodecyl sulfate-polyacrylamide gel electrophoresis of whole-cell proteins and amplified-fragment length polymorphism analysis . MEASUREMENTS AND RESULTS: A total of 1,051 isolates recovered from 608 patients were received from 115 treatment centers in 91 US cities . Among the isolates identified as B cepacia by referring laboratories, 11% could not be confirmed as B cepacia by polyphasic analyses . In addition, 36% of isolates not specifically identified by the referring laboratory or identified as a species other than B cepacia were, in fact, found to be members of the B cepacia complex . CONCLUSIONS: Rates of misidentification of B cepacia remain unacceptably high among US treatment centers . These data suggest the need for increased awareness of this problem among CF centers and their affiliated laboratories, better adherence to recommended protocols for evaluation of CF sputum, and greater use of reference laboratories equipped to provide advanced analyses.

Infect Immun, 2000 Jul, 68(7), 3888 - 93
The CXC chemokines gamma interferon (IFN-gamma)-inducible protein 10 and monokine induced by IFN-gamma are released during severe melioidosis; Lauw FN et al.; Gamma interferon (IFN-gamma)-inducible protein 10 (IP-10) and monokine induced by IFN-gamma (Mig) are related CXC chemokines which bind to the CXCR3 receptor and specifically target activated T lymphocytes and natural killer (NK) cells . The production of IP-10 and Mig by various cell types in vitro is strongly dependent on IFN-gamma . To determine whether IP-10 and Mig are released during bacterial infection in humans, we measured plasma levels of IP-10 and Mig in patients with melioidosis, a severe gram-negative infection caused by Burkholderia pseudomallei . IP-10 and Mig were markedly elevated in patients with melioidosis on admission, particularly in blood culture-positive patients, and remained elevated during the 72-h study period . Levels of IP-10 and Mig showed a positive correlation with IFN-gamma concentrations and also correlated with clinical outcome . In whole blood stimulated with heat-killed B . pseudomallei, neutralization of IFN-gamma and tumor necrosis factor alpha (TNF-alpha) partly attenuated IP-10 and Mig release, while anti-interleukin-12 (IL-12) and anti-IL-18 had a synergistic effect . Stimulation with other bacteria or endotoxin also induced strong secretion of IP-10 and Mig . These data suggest that IP-10 and Mig are part of the innate immune response to bacterial infection . IP-10 and Mig may contribute to host defense in Th1-mediated host defense during infections by attracting CXCR3(+) Th1 cells to the site of inflammation.

Int J Antimicrob Agents, 2000 Jul, 15(2), 125 - 9
Comparison of methods for assessing synergic antibiotic interactions; Mackay ML et al.; Twenty-five combined microtitre chequerboard/time kill curves were performed on ten isolates from patients with relapsing infection to assess the potential for combination therapy . The isolates were Burkholderia cepacia, Staphylococcus aureus and Klebsiella pneumoniae . No antagonism (FIC index or FBC index >4) was observed with any combination . Synergy by time kill curve (present in 21 combinations) was more often seen at 24 h than 2 or 5 h (P<0.001) . On comparing the mean of the FIC and FBC indices, there were significant differences in only four chequerboards (P<0.05) . The same checkerboard was repeated on 3 separate days to test reproducibility . There were no significant differences (P>0.05) . All combinations showing synergism by FBC index were synergic by FIC index . Synergy by FIC index predicted synergy by FBC index in 67% . All combinations showing synergism by FIC index were synergic by time kill at 24 h but there was poor correlation between synergy at 2 or 5 h and synergy by FIC index or FBC index . In conclusion combining time kill and chequerboard tests gives reproducible results and good correlation between FIC and FBC indices . FIC indices showing synergy were also predictive of synergy in time kill studies . For bactericidal combinations unlikely to be antagonistic, calculation of FIC index may be a good indicator of synergic bactericidal activity.

Zh Mikrobiol Epidemiol Immunobiol, 1999 May-Jun, (3), 52 - 5
{The outlook for the development of live vaccines for the prevention of melioidosis}; Iliukhin VI et al.; The effectiveness of immunization with Burkholderia pseudomallei attenuated strains (Pur and Ts), heterologous vaccines and the recombinant culture of Francisella tularensis RM2 carrying a plasmid with fragments of B . pseudomallei chromosome was studied in four species of experimental animals, essentially differing in their sensitivity to melioidosis . The most immunogenic B . pseudomallei mutants, introduced subcutaneously, created a statistically significant level of protection in animals, moderately sensitive to melioidosis, but proved to be ineffective in highly sensitive animal models when tested under the same conditions . In aerogenic infection the effectiveness of the experimental vaccines under study in all species of the animals was on the same level . The study showed good prospects of using tularemia vaccine for inducing heterologous immunity to melioidosis, as well as the possibility of its use as the basis of a bivalent gene-engineering vaccine.

Medicine (Baltimore), 2000 May, 79(3), 155 - 69
Chronic granulomatous disease . Report on a national registry of 368 patients; Winkelstein JA et al.; A registry of United States residents with chronic granulomatous disease (CGD) was established in 1993 in order to estimate the minimum incidence of this uncommon primary immunodeficiency disease and characterize its epidemiologic and clinical features . To date, 368 patients have been registered; 259 have the X-linked recessive form of CGD, 81 have 1 of the autosomal recessive forms, and in 28 the mode of inheritance is unknown . The minimum estimate of birth rate is between 1/200,000 and 1/250,000 live births for the period 1980-1989 . Pneumonia was the most prevalent infection (79% of patients; Aspergillus most prevalent cause), followed by suppurative adenitis (53% of patients; Staphylococcus most prevalent cause), subcutaneous abscess (42% of patients; Staphylococcus most prevalent cause), liver abscess (27% of patients; Staphylococcus most prevalent cause), osteomyelitis (25% of patients; Serratia most prevalent cause), and sepsis (18% of patients; Salmonella most prevalent cause) . Fifteen percent of patients had gastric outlet obstruction, 10% urinary tract obstruction, and 17% colitis/enteritis . Ten percent of X-linked recessive kindreds and 3% of autosomal recessive kindreds had family members with lupus . Eighteen percent of patients either were decreased when registered or died after being registered . The most common causes of death were pneumonia and/or sepsis due to Aspergillus (23 patients) or Burkholderia cepacia (12 patients) . Patients with the X-linked recessive form of the disease appear to have a more serious clinical phenotype than patients with the autosomal recessive forms of the disease, based on the fact that they are diagnosed significantly earlier (mean, 3.01 years of age versus 7.81 years of age, respectively), have a significantly higher prevalence of perirectal abscess (17% versus 7%), suppurative adenitis (59% versus 32%), bacteremia/fungemia (21% versus 10%), gastric obstruction (19% versus 5%), and urinary tract obstruction (11% versus 3%), and a higher mortality (21.2% versus 8.6%).

Mol Microbiol, 2000 Jun, 36(5), 1085 - 100
The NRAMP proteins of Salmonella typhimurium and Escherichia coli are selective manganese transporters involved in the response to reactive oxygen; Kehres DG et al.; NRAMPs (natural resistance-associated macrophage proteins) have been characterized in mammals as divalent transition metal transporters involved in iron metabolism and host resistance to certain pathogens . The mechanism of pathogen resistance is proposed to involve sequestration of Fe2+ and Mn2+, cofactors of both prokaryotic and eukaryotic catalases and superoxide dismutases, not only to protect the macrophage against its own generation of reactive oxygen species, but to deny the cations to the pathogen for synthesis of its protective enzymes . NRAMP homologues are also present in bacteria . We report the cloning and characterization of the single NRAMP genes in Escherichia coli and Salmonella enterica ssp . typhimurium, and the cloning of two distinct NRAMP genes from Pseudomonas aeruginosa and an internal fragment of an NRAMP gene in Burkholderia cepacia . The genes are designated mntH because the two enterobacterial NRAMPs encode H+-stimulated, highly selective manganese(II) transport systems, accounting for all Mn2+ uptake in each species under the conditions tested . For S . typhimurium MntH, the Km for 54Mn2+ ( approximately 0.1 microM) was pH independent, but maximal uptake increased as pH decreased . Monovalent cations, osmotic strength, Mg2+ and Ca2+ did not inhibit 54Mn2+ uptake . Ni2+, Cu2+ and Zn2+ inhibited uptake with Kis greater than 100 microM, Co2+ with a Ki of 20 microM and Fe2+ with a Ki that decreased from 100 microM at pH 7 . 6 to 10 microM at pH 5.5 . Fe3+ and Pb2+ inhibited weakly, exhibiting Kis of 50 microM, while Cd2+ was a potent inhibitor with a Ki of about 1 microM . E . coli MntH had a similar inhibition profile, except that Kis were three- to 10-fold higher . Both S . typhimurium and E . coli MntH also transport 55Fe2+ however, the Kms are equivalent to the Kis for Fe2+ inhibition of Mn2+ uptake, and are thus too high to be physiologically relevant . In both S . typhimurium and E . coli, mntH:lacZ constructs were strongly induced by hydrogen peroxide, weakly induced by EDTA and unresponsive to paraquat, consistent with the presence of Fur and OxyR binding sites in the promoters . Strains overexpressing mntH were more susceptible to growth inhibition by Mn2+ and Cd2+ than wild type, and strains lacking a functional mntH gene were more susceptible to killing by hydrogen peroxide . In S . typhimurium strain SL1344, mntH mutants showed no defect in invasion of or survival in cultured HeLa or RAW264.7 macrophage cells; however, expression of mntH:lacZ was induced severalfold by 3 h after invasion of the macrophages . S . typhimurium mntH mutants showed only a slight attenuation of virulence in BALB/c mice . Thus, the NRAMP Mn2+ transporter MntH and Mn2+ play a role in bacterial response to reactive oxygen species and possibly have a role in pathogenesis.

J Infect, 2000 Mar, 40(2), 164 - 70
Antibody response to Burkholderia cepacia in patients with cystic fibrosis colonized with Burkholderia cepacia and Pseudomonas aeruginosa; Hendry J et al.; INTRODUCTION: This study was designed to determine the relationship between formation of serum antibodies to lipopolysaccharide (LPS) core antigen of Burkholderia cepacia and pulmonary colonization with B . cepacia and Pseudomonas aeruginosa in patients with cystic fibrosis (CF), and to define if an enhanced host humoral immune response to B . cepacia was related to a poor clinical outcome . METHODS: Serum IgG to B . cepacia LPS core antigen was measured in adult cystic fibrosis patients colonized with B . cepacia and P . aeruginosa, and serial titres were measured in 13 B . cepacia and 41 P . aeruginosa colonized patients followed prospectively over 18 months . RESULTS: The median B . cepacia antibody titre was significantly greater in the patients colonized with B . cepacia compared to those colonized with P . aeruginosa, a group which grew B . cepacia intermittently from their sputum . and nine healthy controls . The median antibody titre at recruitment into the study was significantly greater in patients who later went into exacerbations compared with those who remained clinically stable . but there was no difference between B . cepacia antibody titres in patients who died and those who survived the study duration . DISCUSSION: The degree of overlap of serum IgG levels to B . cepacia LPS core antigen in cystic fibrosis patients colonized with B . cepacia and P . aeruginosa does not allow this antibody to be used in a clinical context to define infection status . The magnitude of the humoral response to B . cepacia may influence occurrence of pulmonary exacerbations, but a more exuberant humoral immune response to B . cepacia core LPS is not the mechanism by which pulmonary deterioration occurs.

J Antimicrob Chemother, 2000 Jun, 45(6), 813 - 8
Comparison of efficacy of ciprofloxacin and doxycycline against experimental melioidosis and glanders; Russell P et al.; Melioidosis and glanders are caused by the closely related species Burkholderia pseudomallei and Burkholderia mallei, respectively . Whereas melioidosis is a significant cause of morbidity in south-east Asia, glanders is extremely rare . The efficacies of ciprofloxacin and doxycycline were assessed against a strain of B . pseudomallei and a strain of B . mallei which were susceptible to both antimicrobials in vitro . Porton outbred mice and Syrian hamsters were given 40 mg/kg of either doxycycline or ciprofloxacin twice daily by sc injection according to one of three regimens: dosing starting 48 h before challenge and continuing for 5 days postchallenge; 5 days' therapy starting immediately after challenge; 5 days' therapy starting 24 h after challenge . Mice were challenged ip with B . pseudomallei 4845 and hamsters were challenged ip with B . mallei 23344 . Antimicrobial efficacy was determined by the shift in the median lethal dose (MLD) . Ciprofloxacin prophylaxis and immediate therapy both raised the MLD of B . pseudomallei to 4 x 10(6) cfu from 19 cfu in untreated animals, but therapeutic ciprofloxacin only raised the MLD to 180 cfu . The results for doxycycline were similar . Ciprofloxacin prophylaxis raised the MLD of B . mallei 23344 to 4.6 x 10(5) cfu compared with 4 cfu in untreated controls . Immediate therapy raised the MLD to 7.0 x 10(4) cfu and therapy raised the MLD to 1.6 x 10(3) cfu . All regimens of doxycycline protected hamsters against challenges of up to 2 x 10(7) cfu . Despite using a susceptible strain of B . pseudomallei, neither antimicrobial was effective when used therapeutically . The timely administration of either antimicrobial, however, was effective in preventing symptomatic infection . Doxycycline was the superior of the two antimicrobials against experimental glanders although relapse did occur in treated animals approximately 4-5 weeks after challenge.

Microbiol Immunol, 2000, 44(4), 307 - 17
Burkholderia uboniae sp . nov., L-arabinose-assimilating but different from Burkholderia thailandensis and Burkholderia vietnamiensis; Yabuuchi E et al.; A polar multitrichous gram-negative motile rod, EY 3383, originally identified as Burkholderia thailandensis, revealed a DNA-DNA reassociation rate of 36.7%, under stringent conditions, with the type strain of B . thailandensis, despite the 16S rDNA homology value between two type strains being as high as 97.9% . The strain was clearly differentiated from the type strain of B . thailandensis by physiological, bio-chemical, and nutritional characteristics, without significant difference in cellular fatty acid and lipid composition . Based on the results of 16S rDNA sequence analysis, DNA-DNA hybridization and phenotypic characterization, Burkholderia uboniae sp . nov . is herein proposed . The type strain is NCTC 13147=EY 3383, isolated on 8 December 1989 from surface soil along the roadside in Ubon Ratchathani, Thailand . Major respiratory quinone is ubiquinone-8(Q8) . G+C content of DNA is 69.71%.

Microb Ecol, 2000 Feb, 39(2), 137 - 144
A Burkholderia Strain Living Inside the Arbuscular Mycorrhizal Fungus Gigaspora margarita Possesses the vacB Gene, Which Is Involved in Host Cell Colonization by Bacteria; Ruiz-Lozano JM et al.; The arbuscular mycorrhizal (AM) fungus Gigaspora margarita harbors a resident population of endosymbiontic Burkholderia in its cytoplasm . Nothing is known about the acquisition of such bacteria and about the molecular bases which allow colonization of the fungus . We wondered whether the intracellular Burkholderia strain possesses genetic determinants involved in colonization of a eukaryotic cell . Using degenerated oligonucleotide primers for vacB, a gene involved in host cell colonization by pathogenic bacteria, an 842 bp DNA fragment was cloned, sequenced, and identified as a part of the vacB gene in Burkholderia sp . The insert was used as a probe to screen a fungal library that, because of the presence of intracellular Burkholderia cells, was also representative of the bacterial genome . The complete nucleotide sequence of vacB and flanking genes was determined . The bacterial origin of this genomic region was established by PCR, using specific vacB primers on DNA from Gigasporaceae that did or did not contain cytoplasmic Burkholderia, as well as on DNA from other bacteria, including free-living Burkholderia . We hypothesize that the vacB gene is part of a new genetic region acquired by a rhizospheric Burkholderia strain, which became able to establish a symbiotic interaction with the AM fungus G . margarita.

Curr Microbiol, 2000 Jun, 40(6), 362 - 6
Isolation of a gene from Burkholderia cepacia IS-16 encoding a protein that facilitates phosphatase activity; Rodriguez H et al.; A genomic library from Burkholderia cepacia IS-16 was constructed in Escherichia coli by partial Sau3AI digestion of the chromosomal DNA, with the plasmid vector Bluescript SK . This library was screened for clones able to grow as green stained colonies on selective medium developed for detecting phosphatase-positive colonies . Three green-stained clones (pFS1, pFS2, and pFS3) carried recombinant plasmids harboring DNA inserts of 5.0, 8.0, and 0.9 kb, respectively . DNA hybridization experiments demonstrated the presence of overlapping DNA fragments in the three clones and that these three clones were all derived from Burkholderia cepacia IS-16 genomic DNA . DNA sequence analysis, together with polyacrylamide gels of proteins encoded by E . coli containing pFS3, suggested that the isolated 0 . 9-kb DNA fragment encodes the functional portion of a phosphate transport protein.

Int J Syst Evol Microbiol, 2000 Jan, 50 Pt 1, 235 - 46
Acidovorax anthurii sp . nov., a new phytopathogenic bacterium which causes bacterial leaf-spot of anthurium; Gardan L et al.; The bacterial leaf-spot of anthurium emerged during the 1980s, in the French West Indies and Trinidad . This new bacterial disease is presently wide spread and constitutes a serious limiting factor for commercial anthurium production . Twenty-nine strains isolated from leaf-spots of naturally infected anthurium were characterized and compared with reference strains belonging to the Comamonadaceae family, the genera Ralstonia and Burkholderia, and representative fluorescent pseudomonads . From artificial inoculations 25 out of 29 strains were pathogenic on anthurium . Biochemical and physiological tests, fatty acid analysis, DNA-DNA hybridization, 16S rRNA gene sequence analysis, DNA-16S RNA hybridization were performed . The 25 pathogenic strains on anthurium were clustered in one phenon closely related to phytopathogenic strains of the genus Acidovorax . Anthurium strains were 79-99% (deltaTm range 0.2-1.6) related to the strain CFBP 3232 and constituted a discrete DNA homology group indicating that they belong to the same species . DNA-rRNA hybridization, 16S rRNA sequence and fatty acid analysis confirmed that this new species belongs to the beta-subclass of Proteobacteria and to rRNA superfamily III, to the family of Comamonadaceae and to the genus Acidovorax . The name Acidovorax anthurii is proposed for this new phytopathogenic bacterium . The type strain has been deposited in the Collection Francaise des Bacteries Phytopathogenes as CFBP 3232T.

J Infect Dis, 2000 May, 181(5), 1682 - 92 Epub 2000 May 15.
Immunization with a Pseudomonas aeruginosa elastase peptide reduces severity of experimental lung infections due to P . aeruginosa Or Burkholderia cepacia; Sokol PA et al.; Pseudomonas aeruginosa and Burkholderia cepacia produce metalloproteases that effect lung injury . Two epitopes (peptides 15 and 42) previously identified on P . aeruginosa elastase induce the production of antibodies that neutralize protease activity . The effects of immunization with synthetic peptides based on these epitopes on experimental lung infections due to P . aeruginosa or B . cepacia were examined . Rats were immunized with peptides conjugated to keyhole limpet hemocyanin or tetanus toxoid before infection . Immunization with peptide 15 (pep15) resulted in a decrease in total cells and polymorphonuclear leukocytes in bronchoalveolar lavage (BAL) fluid and a 50%-70% decrease in lung histopathologic changes, compared with findings in controls . Immunization with peptide 42 decreased cells in BAL fluid but did not decrease lung pathologic changes . Immunization with pep15 alone was just as effective in protecting against lung injury as immunization with a combination of both peptides . These studies suggest that immunization with pep15 can reduce the severity of lung infections due to P . aeruginosa or B . cepacia.

J Biol Chem, 2000 May 26, 275(21), 15701 - 8
Identification of a serine hydrolase as a key determinant in the microbial degradation of polychlorinated biphenyls; Seah SY et al.; The ability of 2-hydroxy-6-oxo-6-phenylhexa-2,4-dienoate (HOPDA) hydrolase (BphD) of Burkholderia cepacia LB400 to hydrolyze polychlorinated biphenyl (PCB) metabolites was assessed by determining its specificity for monochlorinated HOPDAs . The relative specificities of BphD for HOPDAs bearing chlorine substituents on the phenyl moiety were 0.28, 0.38, and 1.1 for 8-Cl, 9-Cl, and 10-Cl HOPDA, respectively, versus HOPDA (100 mm phosphate, pH 7.5, 25 degrees C) . In contrast, HOPDAs bearing chlorine substituents on the dienoate moiety were poor substrates for BphD, which hydrolyzed 3-Cl, 4-Cl, and 5-Cl HOPDA at relative maximal rates of 2.1 x 10(-3), 1.4 x 10(-4), and 0.36, respectively, versus HOPDA . The enzymatic transformation of 3-, 5-, 8-, 9-, and 10-Cl HOPDAs yielded stoichiometric quantities of the corresponding benzoate, indicating that BphD catalyzes the hydrolysis of these HOPDAs in the same manner as unchlorinated HOPDA . HOPDAs also underwent a nonenzymatic transformation to products that included acetophenone . In the case of 4-Cl HOPDA, this transformation proceeded via the formation of 4-OH HOPDA (t(12) = 2.8 h; 100 mm phosphate, pH 7.5, 25 degrees C) . 3-Cl HOPDA (t(12) = 504 h) was almost 3 times more stable than 4-OH HOPDA . Finally, 3-Cl, 4-Cl and 4-OH HOPDAs competitively inhibited the BphD-catalyzed hydrolysis of HOPDA (K(ic) values of 0.57 +/- 0 . 04, 3.6 +/- 0.2, and 0.95 +/- 0.04 microm, respectively) . These results explain the accumulation of HOPDAs and chloroacetophenones in the microbial degradation of certain PCB congeners . More significantly, they indicate that in the degradation of PCB mixtures, BphD would be inhibited, thereby slowing the mineralization of all congeners . BphD is thus a key determinant in the aerobic microbial degradation of PCBs.

Am J Trop Med Hyg, 2000 Feb, 62(2), 297 - 300
Diagnostic and prognostic value of an immunofluorescent assay for melioidosis; Vadivelu J et al.; Melioidosis caused by Burkholderia pseudomallei is endemic in southeast Asia . The clinical manifestations range from wound infections to acute septicemia . In some cases, recurrence can also occur following complete recovery . Case fatality rates are high and a major factor is the delay in the culture and identification of the bacterium . An immunofluorescent assay (IFAT) using whole-cell antigen for the detection of total antibodies to B . pseudomallei was tested with 650 sera . Using a cut-off value of 1:80, 66 sera from culture-confirmed cases were positive with titers > or = 320 . In another 523 sera from patients in which no other etiology could be found, 149 (23.4%) were positive . To monitor disease activity, persistence of antibody levels was investigated on 61 serial sera samples collected from 14 other confirmed cases on follow-up visits while on oral maintenance therapy . The IFAT demonstrated a reduction in titers in cases of localized infections, suggesting that either the infection was being resolved or arrested while septicemic patients maintained high IFAT titers on follow-up, suggesting the possibility of continuous sequestration of antigen from an intracellular source.

Am J Trop Med Hyg, 2000 Feb, 62(2), 232 - 9
Pseudomonas (Burkholderia) pseudomallei in Thailand, 1964-1967: geographic distribution of the organism, attempts to identify cases of active infection, and presence of antibody in representative sera; Finkelstein RA et al.; The purpose of this study, initiated in 1964 and concluded in 1967, was to define the distribution of Pseudomonas (now Burkholderia) pseudomallei in Thailand, to evaluate its importance as an etiologic agent, and to survey the presence of antibody in people that might indicate prior infection and/or contact with the microorganism.

Curr Opin Pulm Med, 1998 Nov, 4(6), 337 - 41
Burkholderia cepacia epidemiology and pathogenesis: implications for infection control; LiPuma JJ; Much has been learned during the past 2 years about the microbiology and taxonomy of Burkholderia cepacia . Several distinct species have been identified in what is now referred to as the B . cepacia complex . Preliminary studies indicate that certain of these species are more likely to colonize and cause severe pulmonary infection in persons with cystic fibrosis . Ongoing investigations will expand these findings and have the potential to modify current infection control policies . The commercial use of B . cepacia as an antifungal biopesticide and in bioremediation has attracted increasing attention from industry recently and raises concerns within the cystic fibrosis community . Consensus regarding the potential threat of such uses to cystic fibrosis patients is being sought by governmental agencies and agricultural and biomedical researchers.

Appl Microbiol Biotechnol, 2000 Apr, 53(4), 453 - 60
Polyhydroxyalkanoate accumulation in Burkholderia sp.: a molecular approach to elucidate the genes involved in the formation of two homopolymers consisting of short-chain-length 3-hydroxyalkanoic acids; Rodrigues MF et al.; Burkholderia sp . accumulates polyhydrox-yalkanoates (PHAs) containing 3-hydroxybutyrate and 3-hydroxy-4-pentenoic acid when grown on mineral media under limited phosphate or nitrogen, and using sucrose or gluconate as a carbon and energy source . Solvent fractionation and NMR spectroscopic characterization of these polyesters revealed the simultaneous accumulation of two homopolyesters rather than a co-polyester with random sequence distribution of the monomers {Valentin HE, Berger PA, Gruys KJ, Rodrigues MFA, Steinbuchel A, Tran M, Asrar J (1999) Macromolecules 32: 7389-7395} . To understand the genetic requirements for such unusual polyester accumulation, we probed total genomic DNA from Burkholderia sp . by Southern hybridization experiments using phaC-specific probes . These experiments indicated the presence of more than one PHA synthase gene within the genome of Burkholderia sp . However, when total genomic DNA from Burkholderia sp . was used to complement a PHA-negative mutant of Ralstonia eutropha for PHA accumulation, only one PHA synthase gene was obtained resembling the R . eutropha type of PHA synthases, based on amino acid sequence similarity . In addition to the PHA synthase gene, based on high sequence homology, genes encoding a beta-ketothiolase and acetoacetyl-CoA reductase were identified in a gene cluster with the PHA synthase gene . The arrangement of the three genes is quite similar to the R . eutropha poly-beta-hydroxybutyrate biosynthesis operon.

Dakar Med, 1998, 43(2), 144 - 6
{Burkholderia cepacia isolation and characterization from hospital infections}; Cisse MF et al.; The evolution of reanimation and functional exploration techniques has led to and explosion of nosocomial infections . They are prevailing in Intensive Care and Neonatal Units . This study deals with B . cepacia strains isolated in 1996 in a pediatric hospital of the Dakar University Hospital Center, following the installation of tracheo-bronchial exhausters which are used for obstruction removal among children . The 44 B . cepacia strains examined come from 42 blood cultures done among 29 boys and 13 girls aged between 5 days and 7 years, and from 2 exhausters . After identification by API20 NE (bio Merieux), a standard antibiogram, a 3 characters biotyping (O.N.P.G., esculin, nitrate reductase) and a study of the polymorphism of the DNA enzymatic restriction profile obtained by an pulsed field electrophoresis are performed on the isolates . The contamination come from the exhausters . All the strains produce an orange-colored yellow pigment . Only an O.N.P.G . (+), nitrate reductase (+) biotype was identified . The antibiotic susceptibility profile is almost pathognomonic for the 44 tested strains: sensitivity (100%) to ceftriaxone, to ceftazidime, to aztreonam: to contrimoxazole (96%) and to chloramphenicol (91%) . Search for widen spectrum beta-lactamses and antibiotics resistance plasmids was negative . However, those strains that are multiples resistant, discharge others 8.1 . isofocal point beta-lactamases . The R.F.L.P . study demonstrated a unique profile . The B . cepacia transmission is the result of the installation of medical reanimation equipment that are not well taken care of . The nosocomial infections ascertained so far are ordinary bacteremias . Strain's phenotypical and genotypical identification shows the presence of only one clone . To overcome there nosocomial infections, hygienic measures have to be reinforced.

Arch Microbiol, 2000 Feb, 173(2), 86 - 90
Properties of the trihydroxytoluene oxygenase from Burkholderia cepacia R34: an extradiol dioxygenase from the 2,4-dinitrotoluene pathway; Johnson GR et al.; Burkholderia cepacia R34 mineralizes 2,4-dinitrotoluene via an oxidative pathway . The initial steps in the degradative pathway lead to formation of 2,4,5-trihydroxytoluene, which serves as the substrate for the ring cleavage dioxygenase . The trihydroxylated substrate differs from the usual substituted catechols found in pathways for aromatic compound degradation . To determine whether the characteristics of the trihydroxytoluene oxygenase reflect the unusual ring cleavage substrate of the 2,4-dinitrotoluene pathway, the gene encoding trihydroxytoluene oxygenase (dntD) was cloned and sequenced, and ring cleavage activity determined from recombinant bacteria carrying the cloned gene . The findings were compared to the trihydroxytoluene oxygenase from Burkholderia sp . strain DNT and to other previously described ring cleavage dioxygenases . The comparison revealed that only 60% identity was shared by the two trihydroxytoluene oxygenases, but the amino acid residues involved in cofactor binding, catalysis, and protein folding were conserved in the DntD sequence . The enzyme catalyzed meta-fission of trihydroxytoluene as well as the substrate analogues 1,2,4-benzenetriol, catechol, 3-methylcatechol, 4-methylcatechol, 3-chlorocatechol, 4-chlorocatechol and 2,3-dihydroxybiphenyl . However, results from enzyme assays indicated a strong preference for trihydroxytoluene, implying that it was the native substrate for the enzyme . The apparent enzyme specificity, its similarity to the trihydroxytoluene oxygenase from Burkholderia sp . strain DNT, and the distant genetic relationship to other ring cleavage enzymes suggest that dntD evolved expressly to carry out trihydroxytoluene transformation.

Syst Appl Microbiol, 1999 Dec, 22(4), 534 - 45
Serological and molecular size characterization of flagellins of Pseudomonas syringae pathovars and related bacteria
Malandrin L, Samson R.
Flagella from a total of 118 strains representing mostly pathovars of the phytopathogenic group Pseudomonas syringae, but also P . chlororaphis, P . cichorii, P . corrugata, P . fluorescens, P . fuscovaginae, P . stutzeri, P . viridiflava, as well as related phytopathogenic genera (Burkholderia cepacia and Ralstonia solanacearum) were characterized by immuno-fluorescent staining, SDS-PAGE, and immunoblotting . Eighty-six strains of the P . syringae group pathovars, P . cichorii and P . viridiflava were shown to possess flagella of serotypes H1 or H2, composed of a unique flagellin, whose molecular size varied between 31 and 31.5 kDa . Similarities between the P . syringae flagellin and a 31 kDa surface protein involved in pathogenicity are pointed out . The distribution of H1 and H2 antigens in the nine recently described genomospecies of P . syringae-P . viridiflava group suggested that flagellin would represent a phylogenetic marker within the arginin-dihydrolase-negative fluorescent pseudomonads . The characterization of flagellin was proposed as an identification tool at a level situated between genus and species.

J Appl Microbiol, 2000 May, 88(5), 764 - 72
Degradation of 4-nitrocatechol by Burkholderia cepacia: a plasmid-encoded novel pathway; Chauhan A et al.; Pseudomonas cepacia RKJ200 (now described as Burkholderia cepacia) has been shown to utilize p-nitrophenol (PNP) as sole carbon and energy source . The present work demonstrates that RKJ200 utilizes 4-nitrocatechol (NC) as the sole source of carbon, nitrogen and energy, and is degraded with concomitant release of nitrite ions . Several lines of evidence, including thin layer chromatography, gas chromatography, 1H-nuclear magnetic resonance, gas chromatography-mass spectrometry, spectral analyses and quantification of intermediates by high performance liquid chromatography, have shown that NC is degraded via 1,2, 4-benzenetriol (BT) and hydroquinone (HQ) formation . Studies carried out on a PNP- derivative and a PNP+ transconjugant also demonstrate that the genes for the NC degradative pathway reside on the plasmid present in RKJ200; the same plasmid had earlier been shown to encode genes for PNP degradation, which is also degraded via HQ formation . It is likely, therefore, that the same sets of genes encode the further metabolism of HQ in NC and PNP degradation.

Br J Clin Pharmacol, 2000 May, 49(5), 445 - 52
Pharmacokinetic-pharmacodynamic evaluation of ceftazidime continuous infusion vs intermittent bolus injection in septicaemic melioidosis; Angus BJ et al.; AIMS: Experimental studies have suggested that constant intravenous infusion would be preferable to conventional intermittent bolus administration of beta-lactam antibiotics for serious Gram-negative infections . Severe melioidosis (Burkholderia pseudomallei infection) carries a mortality of 40% despite treatment with high dose ceftazidime . The aim of this study was to measure the pharmacokinetic and pharmacodynamic effects of continuous infusion of ceftazidime vs intermittent bolus dosing in septicaemic melioidosis . METHODS: Patients with suspected septicaemic melioidosis were randomised to receive ceftazidime 40 mg kg-1 8 hourly by bolus injection or 4 mg kg-1 h-1 by constant infusion following a 12 mg kg-1 priming dose to perform estimation of pharmacokinetic and pharmacodynamic parameters . RESULTS: Of the 34 patients studied 16 (59%) died . Twenty patients had cultures positive for B . pseudomallei of whom 12 (60%) died . The median MIC90 of B . pseudomallei was 2 mg l-1, giving a target concentration CT, of 8 mg l-1 . The median (range) estimated total apparent volume of distribution, systemic clearance and terminal elimination half-lives of ceftazidime were 0.468 (0.241-0.573) l kg-1, 0.058 (0.005-0.159) l kg-1 h-1 and 7.74 (1.95-44.71) h, respectively . Clearance of ceftazidime and creatinine clearance were correlated closely (r = 0 . 71; P < 0.001) and there was no evidence of significant nonrenal clearance . CONCLUSIONS: Simulations based on these data and the ceftazidime sensitivity of the B . pseudomallei isolates indicated that administration by constant infusion would allow significant dose reduction and cost saving . With conventional 8 h intermittent dosing to patients with normal renal function, plasma ceftazidime concentrations could fall below the target concentration but this would be unlikely with a constant infusion . Correction for renal failure which is common in these patients is Clearance = k * creatinine clearance where k = 0.072 . Calculation of a loading dose gives median (range) values of loading dose, DL of 3.7 mg kg-1 (1 . 9-4.6) and infusion rate I = 0.46 mg kg h-1 (0.04-1.3) (which equals 14.8 mg kg-1 day-1) . A nomogram for adjustment in renal failure is given.

Clin Lab, 2000, 46(3-4), 119 - 30
A review on melioidosis with special respect on molecular and immunological diagnostic techniques; Zysk G et al.; Melioidosis is an 'emerging' tropical disease which causes diagnostic problems in endemic and especially in nonendemic areas e.g . Germany when imported . In the last decade, many efforts have been made to develop new molecular and immunological techniques for diagnostic use . However, an actual comprehensive review does not exist . Apart from classical microbiological procedures (microscopy, culture and biochemical identification) efforts have been made to identify Burkholderia pseudomallei using specific antibodies and PCR . The direct antigen detection can be done within a few hours leading to diagnosis days before cultural proof . ELISA and immunoblot techniques were examined for their ability to replace indirect hemagglutination and immunofluorescence test in serology . The diagnostic value of serological procedures for early detection of melioidosis is limited, however . The rapid and reliable diagnosis of melioidosis is required for an adequate onset of therapy . But no evaluated test kit based on the detection of specific antibodies, specific antigens, or on the amplification of species-specific DNA sequences is commercially available up to now . Even PCR testing--primers can easily be ordered by many gene technology companies--can not be recommended as B . pseudomallei DNA for positive controls is not available . Therefore, only microscopy and biochemical identification systems like the API 20NE can be used in the routine laboratory up to now . At this point, it has to be stressed that B . pseudomallei is a level 3 agent in many countries e.g . Germany and that only laboratories with high containment are allowed to handle it . In all cases the help of an experienced reference laboratory is adviced.

J Clin Microbiol, 2000 May, 38(5), 1876 - 84
Distinguishing species of the Burkholderia cepacia complex and Burkholderia gladioli by automated ribotyping; Brisse S et al.; Several species belonging to the genus Burkholderia are clinically relevant, opportunistic pathogens that inhabit major environmental reservoirs . Consequently, the availability of means for adequate identification and epidemiological characterization of individual environmental or clinical isolates is mandatory . In the present communication we describe the use of the Riboprinter microbial characterization system (Qualicon, Warwick, United Kingdom) for automated ribotyping of 104 strains of Burkholderia species from diverse sources, including several publicly accessible collections . The main outcome of this analysis was that all strains were typeable and that strains of Burkholderia gladioli and of each species of the B . cepacia complex, including B . multivorans, B . stabilis, and B . vietnamiensis, were effectively discriminated . Furthermore, different ribotypes were discerned within each species . Ribotyping results were in general agreement with strain classification based on restriction fragment analysis of 16S ribosomal amplicons, but the resolution of ribotyping was much higher . This enabled automated molecular typing below the species level . Cluster analysis of the patterns obtained by ribotyping (riboprints) showed that within B . gladioli, B . multivorans, and B . cepacia genomovar VI, the different riboprints identified always clustered together . Riboprints of B . cepacia genomovars I and III, B . stabilis, and B . vietnamiensis did not show distinct clustering but rather exhibited the formation of loose assemblages within which several smaller, genomovar-specific clusters were delineated . Therefore, ribotyping proved useful for genomovar identification . Analysis of serial isolates from individual patients demonstrated that infection with a single ribotype had occurred, despite minor genetic differences that were detected by pulsed-field gel electrophoresis of DNA macrorestriction fragments . The automated approach allows very rapid and reliable identification and epidemiological characterization of strains and generates an easily manageable database suited for expansion with information on additional bacterial isolates.

J Clin Microbiol, 2000 May, 38(5), 1763 - 6
Distribution of genes encoding putative transmissibility factors among epidemic and nonepidemic strains of Burkholderia cepacia from cystic fibrosis patients in the United Kingdom; Clode FE et al.; In the last 15 years, Burkholderia cepacia has emerged as a significant pathogen in cystic fibrosis (CF) patients, mainly due to the severity of infection observed in a subset of patients and the fear of transmission of the organism to noncolonized patients . Although patients who deteriorate rapidly cannot be predicted by microbiological characteristics, three genetic markers have been described for strains that spread between patients . These are the cblA gene, encoding giant cable pili; a hybrid of two insertion sequences, IS1356 and IS402; and a 1.4-kb open reading frame known as the B . cepacia epidemic strain marker (BCESM) . The latter two are of unknown function . An epidemic strain lineage was previously identified among CF patients in the United Kingdom that apparently had spread from North America and that was characterized by a specific random amplified polymorphic DNA (RAPD) pattern . We searched for the described genetic markers using specific PCR assays with 117 patient isolates of B . cepacia from 40 United Kingdom hospitals . Isolates were grouped according to genomovar and epidemic strain lineage RAPD pattern with a 10-base primer, P272 . A total of 41 isolates from patients in 12 hospitals were classified as the epidemic strain, and 40 of these were distributed in genomovars IIIa (11 isolates), IIIb (1 isolate), and IIIc (28 isolates) . All isolates of the epidemic strain were positive for the cblA gene and BCESM, but two lacked the insertion sequence hybrid . None of the 76 sporadic isolates contained cblA or the insertion sequence hybrid, but 11 of them were positive for BCESM . Nonepidemic isolates were distributed among genomovars I or IV (9), II (49), IIIa (11), IIIb (3), and IIIc (4) . There were three clusters of cross-infection (one involving two patients and two involving three patients) with isolates of genomovar II . We conclude that in the United Kingdom, a single clonal lineage has spread between and within some hospitals providing care for CF patients . The presence of the cblA gene is the most specific marker for the epidemic strain . We recommend that all isolates of B . cepacia from CF patients should be screened by PCR to influence segregation and infection control strategies.

Appl Environ Microbiol, 2000 May, 66(5), 2139 - 47
Aerobic degradation of dinitrotoluenes and pathway for bacterial degradation of 2,6-dinitrotoluene; Nishino SF et al.; An oxidative pathway for the mineralization of 2,4-dinitrotoluene (2, 4-DNT) by Burkholderia sp . strain DNT has been reported previously . We report here the isolation of additional strains with the ability to mineralize 2,4-DNT by the same pathway and the isolation and characterization of bacterial strains that mineralize 2, 6-dinitrotoluene (2,6-DNT) by a different pathway . Burkholderia cepacia strain JS850 and Hydrogenophaga palleronii strain JS863 grew on 2,6-DNT as the sole source of carbon and nitrogen . The initial steps in the pathway for degradation of 2,6-DNT were determined by simultaneous induction, enzyme assays, and identification of metabolites through mass spectroscopy and nuclear magnetic resonance . 2,6-DNT was converted to 3-methyl-4-nitrocatechol by a dioxygenation reaction accompanied by the release of nitrite . 3-Methyl-4-nitrocatechol was the substrate for extradiol ring cleavage yielding 2-hydroxy-5-nitro-6-oxohepta-2,4-dienoic acid, which was converted to 2-hydroxy-5-nitropenta-2,4-dienoic acid . 2, 4-DNT-degrading strains also converted 2,6-DNT to 3-methyl-4-nitrocatechol but did not metabolize the 3-methyl-4-nitrocatechol . Although 2,6-DNT prevented the degradation of 2,4-DNT by 2,4-DNT-degrading strains, the effect was not the result of inhibition of 2,4-DNT dioxygenase by 2,6-DNT or of 4-methyl-5-nitrocatechol monooxygenase by 3-methyl-4-nitrocatechol.

Appl Environ Microbiol, 2000 May, 66(5), 1814 - 7
Quantification of phnAc and nahAc in contaminated new zealand soils by competitive PCR; Laurie AD et al.; Unculturable polycyclic aromatic hydrocarbon (PAH)-degrading bacteria are a significant reservoir of the microbial potential to catabolize low-molecular-weight PAHs . The population of these bacteria is larger than the population of nah-like bacteria that are the dominant organisms in culture-based studies . We used the recently described phn genes of Burkholderia sp . strain RP007, which feature only rarely in culture-based studies, as an alternative genotype for naphthalene and phenanthrene degradation and compared this genotype with the genotypically distinct but ubiquitous nah-like class in different soils . Competitive PCR quantification of phnAc and nahAc, which encode the iron sulfur protein large (alpha) subunits of PAH dioxygenases in nah-like and phn catabolic operons, revealed that the phn genotype can have a greater ecological significance than the nah-like genotype.

Mod Pathol, 2000 Apr, 13(4), 369 - 72
Histopathologic features of Burkholderia cepacia pneumonia in patients without cystic fibrosis; Belchis DA et al.; We present the histopathologic features of fatal Burkholderia cepacia pneumonia in three adults (one man {age 44 years} and two women {aged 40 and 43 years}) . In all patients, the pulmonary infiltrates initially were localized (right middle lobe, left upper lobe, and right middle lobe) but rapidly progressed . Two open-lung biopsies and one pneumonectomy specimen showed necrotizing granulomatous inflammation merging with areas of more conventional necrotizing bronchopneumonia In one patient, a mediastinal lymph node also showed stellate necrotizing granulomas . Vasculitis was absent . B . cepacia was cultured from the open-lung biopsies and bronchial wash specimens in two patients and from postmortem cultures of lung, subcarinal lymph nodes, and blood in the third . The histopathology in these patients resembles that of melioidosis, which is caused by a related organism, Burkholderia pseudomallei . B . cepacia needs to be considered in the differential diagnosis of necrotizing granulomatous inflammation . In addition, given the rarity with which B . cepacia is identified as a cause of pneumonia in the immunocompetent host, isolation of B . cepacia should trigger a workup for underlying immunodeficiency or lead to an investigation to exclude the possibility of a nosocomial infection.

J Clin Pathol, 2000 Feb, 53(2), 159 - 60
Interaction of insulin with Burkholderia pseudomallei may be caused by a preservative; Simpson AJ et al.; AIM: To re-examine the previously reported in vitro interaction of insulin with Burkholderia pseudomallei, in the light of a suggestion that the interaction may have resulted from the presence of the preservative m-cresol in commercial preparations . METHODS: Broth culture studies of B pseudomallei were performed with and without the addition of m-cresol and various preparations of insulin . RESULTS: Growth of B pseudomallei was inhibited by m-cresol at the concentrations found in pharmaceutical insulin preparations, and by the insulin preparation Humulin R, but not by pure insulin . CONCLUSIONS: The results of previous experiments may have been confounded by the presence of the preservative m-cresol.

Am J Respir Crit Care Med, 2000 Apr, 161(4 Pt 1), 1206 - 12
Multiple combination bactericidal antibiotic testing for patients with cystic fibrosis infected with Burkholderia cepacia; Aaron SD et al.; Most Burkholderia cepacia strains are resistant to many, or all, of the antibacterial agents commonly used in cystic fibrosis (CF), and selection of appropriate antibiotics for treatment of pulmonary exacerbations is therefore difficult . We developed a technique for rapid in vitro testing of multiple antibiotic combinations for B . cepacia isolates . For each of 119 multi-drug-resistant isolates of B . cepacia, our multiple combination bactericidal test (MCBT) studied the bactericidal activity of 10 to 15 antimicrobial agents using 225 +/- 97 single, double, and triple antibiotic combinations . Of the 119 isolates, 50% were resistant to all single antibiotics tested, 8% were resistant to all two-drug antibiotic combinations, but all were inhibited by at least one bactericidal triple-drug combination . When used alone, meropenem, ceftazidime and high-dose tobramycin (200 microg/ml) were bactericidal against only 47, 15, and 14% of in vitro isolates, respectively . Using a double antibiotic combination improved bactericidal activity; meropenem-minocycline, meropenem-amikacin, and meropenem-ceftazidime combinations were bactericidal against 76, 73, and 73% of isolates, respectively . However, 47% of isolates demonstrated antagonism (growth of an organism when a second antibiotic was added to a bactericidal single antibiotic) . Triple antibiotic combinations that contained tobramycin, meropenem, and an additional antibiotic were most effective, and were bactericidal against 81 to 93% of isolates . We conclude that triple-antibiotic combinations are more likely than double and single antibiotic combinations to be bactericidal against B . cepacia in vitro . MCBT testing is a useful technique to help clinicians decide on appropriate nonantagonistic combination antibiotic therapy for patients with CF infected with B . cepacia.

Int J Syst Evol Microbiol, 2000 Mar, 50 Pt 2, 887 - 99
Description of Pandoraea gen . nov . with Pandoraea apista sp . nov., Pandoraea pulmonicola sp . nov., Pandoraea pnomenusa sp . nov., Pandoraea sputorum sp . nov . and Pandoraea norimbergensis comb . nov; Coenye T et al.; A polyphasic taxonomic study was performed on a group of isolates tentatively identified as Burkholderia cepacia, Ralstonia pickettii or Ralstonia paucula (formerly known as CDC group IVc-2) . The isolates were mainly cultured from sputum of cystic fibrosis patients or from soil . SDS-PAGE of whole-cell proteins and AFLP fingerprinting distinguished at least five different species, and this was confirmed by DNA-DNA hybridizations . 16S rDNA sequence analysis of representative strains indicated that these organisms belong to the beta-subclass of the Proteobacteria, with the genera Burkholderia and Ralstonia as closest neighbours . Based on genotypic and phenotypic characteristics, the organisms were classified in a novel genus, Pandoraea . The DNA base composition of the members of the new genus is between 61.2 and 64.3 mol% . This novel genus includes four new species, Pandoraea apista (the type species) (type strain is LMG 16407T), Pandoraea pulmonicola (type strain is LMG 18106T), Pandoraea pnomenusa (type strain is LMG 18087T) and Pandoraea sputorum (type strain is LMG 18819T), and Pandoraea norimbergensis (Wittke et al . 1997) comb . nov . (type strain is LMG 18379T) . The available clinical data indicate that at least some of these organisms may cause chronic infection in, and can be transmitted amongst, cystic fibrosis patients.

Int J Syst Evol Microbiol, 2000 Mar, 50 Pt 2, 743 - 9
Burkholderia kururiensis sp . nov., a trichloroethylene (TCE)-degrading bacterium isolated from an aquifer polluted with TCE; Zhang H et al.; A trichloroethylene (TCE)-degrading bacterium was isolated from an aquifer sample collected at a TCE-polluted site in Japan by enriching with phenol as sole carbon source . The isolate, designated strain KP23T, was a Gram-negative, oval-shaped micro-organism . A phylogenetic study based on 16S rRNA gene sequences indicated that strain KP23T should be placed in the genus Burkholderia . Cellular fatty acids of the strain were mainly composed of C16:0, cyclopropanic acid C17:0 and cyclopropanic acid C19:0 . Strain KP23T also contained notable amounts of C13:1 and C17:1 . The G + C content of total DNA was 64.8 mol% . Strain KP23T oxidized various sugars and sugar alcohols as sole carbon source such as galactose, glucose, mannose, maltose, glycerol, inositol and mannitol . Comparisons of its phenotypic and genotypic characteristics with other known species belonging to the genus Burkholderia suggested that strain KP23T represents a new species in the genus . The name Burkholderia kururiensis is proposed for this species, with strain KP23T as the type strain (= JCM 10599T).

J Chromatogr A, 2000 Mar 17, 873(1), 53 - 61
Application of solid-phase microextraction-gas chromatography-mass spectrometry to characterize intermediates in a joint solar-microbial process for total mineralization of Aroclor 1254; Rhofir C et al.; A combined solid-phase microextraction-GC-MS analytical technique was used to monitor the formation of metabolites in the biodegradation of biphenyl, which were originally obtained from the solar photodechlorination of Aroclor 1254 by Pseudomonas pseudoalcaligenes KF707 and Burkholderia sp LB400 . In both cases, the following metabolites were detected: 2-hydroxybiphenyl (2-OH-BP), 2,3-dihydroxybiphenyl (2,3-di-OH-BP), and benzoic acid, which was detected as its benzoate derivative 1-methylethylbenzoate . A time course study for the formation and disappearance of these metabolites was used to construct a degradation pathway, which in both cases, involved the formation of 2-OH-BP and 2,3-di-OH-BP.

FEMS Microbiol Lett, 2000 Apr 15, 185(2), 243 - 6
Exopolysaccharide production by mucoid and non-mucoid strains of Burkholderia cepacia; Cerantola S et al.; Thirteen strains of Burkholderia cepacia from various origins with mucoid and non-mucoid phenotypes were assayed for exopolysaccharide (EPS) production . The EPS were characterized by glycosyl composition analysis and examination of the products resulting from lithium-ethylenediamine and Smith degradations . The results showed that all strains, including the non-mucoid strains, were able to produce EPS exhibiting the same structural features, i.e . presence of one rhamnosyl, three galactosyl, one mannosyl, one glucosyl and one glucuronosyl residues, suggesting that this EPS is representative of the B . cepacia species.

Ann Acad Med Singapore, 2000 Jan, 29(1), 108 - 9
Endobronchial mass in a patient with Burkholderia pseudomallei infection; Khoo KL et al.; INTRODUCTION: Burkholderia pseudomallei infection, the great mimicker of infectious diseases, has protean clinical manifestations . CLINICAL PICTURE: A 37-year-old man who presented with community-acquired pneumonia affecting the right upper lobe had unremitting fever . Bronchoscopy showed an endobronchial mass in the right upper lobe bronchus . TREATMENT: Intravenous ceftriaxone and oral erythromycin, with empiric antituberculous treatment added later . This was subsequently switched to intravenous ceftazidime and oral doxycycline after the diagnosis was made . OUTCOME: There was resolution of the endobronchial mass . CONCLUSION: This case illustrates a unique and unreported presentation of melioidosis.

J Clin Microbiol, 2000 Apr, 38(4), 1670 - 1
Evaluation of a new commercially available immunoglobulin M and immunoglobulin G immunochromatographic test for diagnosis of melioidosis infection; Cuzzubbo AJ et al.; An immunochromatographic test for the rapid determination of immunoglobulin M (IgM) and IgG antibodies to Burkholderia pseudomallei was evaluated by using sera from bacteriologically confirmed melioidosis patients and high-risk and clinically suspected patients, along with disease control groups . The sensitivities were 100 and 93% for the IgG and IgM tests, respectively, while the specificity was 95% for both assays . The test was rapid and simple to perform, with results obtained in 10 min.

J Clin Microbiol, 2000 Apr, 38(4), 1651 - 5
Molecular typing and exopolysaccharide biosynthesis of Burkholderia cepacia isolates from a Portuguese cystic fibrosis center; Richau JA et al.; This work describes the first epidemiological survey of Burkholderia cepacia involved in pulmonary infections among the Portuguese population with cystic fibrosis (CF) who attended the major CF treatment Center in Lisbon at Sta . Maria Hospital from 1995 to the end of 1997 . The characterization of the genomic relatedness of the isolates was based on the analysis of their ribopatterns (with EcoRI) followed by construction of a ribotype-based phylogenetic tree . This study was complemented with macrorestriction fragment analysis by pulsed-field gel electrophoresis . After optimization of the solid growth medium, we found that exopolysaccharide (EPS) production by B . cepacia CF isolates is not as rare a phenomenon as was thought before; indeed, 70% of the isolates examined were EPS producers.

Eur J Clin Microbiol Infect Dis, 2000 Feb, 19(2), 121 - 3
Survival and growth of Burkholderia cepacia within the free-living amoeba Acanthamoeba polyphaga; Landers P et al.; The aim of this study was to investigate whether Burkholderia cepacia is capable of survival and growth within the free-living amoeba Acanthamoeba polyphaga using a differential immunofluorescence assay of bacterial-amoebal cocultures and viable counts of bacteria determined after amoebal lysis . The numbers of intra-amoebal bacteria and the numbers of infected amoebae increased over time; although, when heat-killed bacteria were used, no intracellular bacteria were observed . These findings should be taken into account in future studies of environmental reservoirs of Burkholderia cepacia.

Appl Environ Microbiol, 2000 Apr, 66(4), 1737 - 40
Involvement of two plasmids in fenitrothion degradation by Burkholderia sp . strain NF100; Hayatsu M et al.; A bacterium capable of utilizing fenitrothion (O,O-dimethyl O-4-nitro-m-tolyl phosphorothioate) as a sole carbon source was isolated from fenitrothion-treated soil . This bacterium was characterized taxonomically as being a member of the genus Burkholderia and was designated strain NF100 . NF100 first hydrolyzed an organophosphate bond of fenitrothion, forming 3-methyl-4-nitrophenol, which was further metabolized to methylhydroquinone . The ability to degrade fenitrothion was found to be encoded on two plasmids, pNF1 and pNF2.

FEMS Microbiol Lett, 1998 Dec 15, 169(2), 283 - 7
Characterization of three capsular polysacharides produced by Burkholderia pseudomallei; Kawahara K et al.; Three kinds of capsular polysaccharide (CP) were found to be produced by Burkholderia pseudomallei . When the bacterium was grown with the medium without glycerol, CP-1a and CP-1b were produced . CP-1a was mainly 1.4-linked glucan and CP-1b was identified as a polymer composed of galactose and 3-deoxy-D-manno-octulosonic acid, whose chemical structure was recently reported by other laboratories . When the bacterium was grown with the medium containing 5" glycerol . CP-2 was synthesized . CP-2 contained galactose, rhamnose, mannose, glucose and a uronic acid in a ratio of approximately 3:1:0.3:1:1 . Methylation analysis of the purified polysaccharides demonstrated that the two acidic polysaccharides . CP-1b and CP-2 shared no common structure, indicating that CP-2 was an acidic capsular polysaccharide whose chemical characters were not reported previously.

Biosci Biotechnol Biochem, 2000 Feb, 64(2), 363 - 8
Synthesis of regioselectively protected forms of cytidine based on enzyme-catalyzed deacetylation as the key step; Kuboki A et al.; N4-Acetylcytidine (77%) and 2',3'-O, N4-triacetylcytidine (95%) were obtained from the hydrolysis of a common precursor, the peracetylated form of cytidine with Aspergillus niger lipase (Amano A) and Burkholderia cepacia esterase (SC esterase S), respectively, under very mild conditions . The experimental procedure for the conversion of triacetylcytidine to a corresponding phosphoramidite (82%), an intermediate for sugar nucleotide synthesis, is also elaborated.

J Appl Microbiol, 2000 Jan, 88(1), 154 - 60
Use of oligotrophic bacteria for the biological monitoring of heavy metals; Tada Y et al.; Oligotrophic bacteria exhibited active growth even in nutritionally deficient medium made with nutrient broth that had been diluted with distilled water, 1 : 10 000 . The oligotrophic bacteria, Sphingomonas paucimobilis KPS01 and Burkholderia cepacia KPC01 and KPC02 were found to be highly susceptible to heavy metals and to be potentially useful as sensors for the assessment of toxicity . The susceptibility of the bacteria to metals was measured by incubating the bacteria with metals of varying concentrations in the nutritionally deficient medium at 30 degrees C for 24 h . Bacteria were considered susceptible when the growth inhibition rate (EC50 was more than 50% of the control . The EC50 value of Ag+, Pb2+ and Cd2+ was 10(5)mmol l(-1) and Zn2+, Cr3+, Cr6+, Cu2+ and Hg2+ was 10(-4) mmol l(-1) in S . paucimobilis KPS01 . Other strains also showed similar results . No difference in the EC50 was found using either the chloride or sulphate forms of these metals . The optimum incubation time was 24 h and a longer incubation time did not necessarily lead to more inhibition . The EC50 value rose in proportion to the concentration of nutrition in media . Environmental samples were tested and 14 out of 88 samples inhibited the growth of S . paucimobilis KPS01.

Infect Immun, 2000 Apr, 68(4), 2374 - 8
Xanthine oxidase contributes to host defense against Burkholderia cepacia in the p47(phox-/-) mouse model of chronic granulomatous disease; Segal BH et al.; Chronic granulomatous disease (CGD) is an inherited disorder of the NADPH oxidase in which phagocytes are defective in generating superoxide and downstream microbicidal reactive oxidants, leading to recurrent life-threatening bacterial and fungal infections . Xanthine oxidase (XO) is another enzyme known to produce superoxide in many tissues . Using the p47(phox-/-) mouse model of CGD, we evaluated the residual antibacterial activity of XO . Clearance of Burkholderia cepacia, a major pathogen in CGD, was reduced in p47(phox-/-) mice compared to that in wild-type mice and was further inhibited in p47(phox-/-) mice by pretreatment with the specific XO inhibitor allopurinol . Hepatic B . cepacia burden was similar in the two genotypes, but allopurinol significantly reduced net hepatic killing and killing efficiency only in p47(phox-/-) mice . Clearance and killing of intravenous Escherichia coli was intact in p47(phox-/-) mice and was unaffected by pretreatment with allopurinol . In CGD, XO may contribute to host defense against a subset of reactive oxidant-sensitive pathogens.

Infect Immun, 2000 Apr, 68(4), 2034 - 42
Cytokine gene expression in innately susceptible BALB/c mice and relatively resistant C57BL/6 mice during infection with virulent Burkholderia pseudomallei; Ulett GC et al.; Production of cytokines including gamma interferon (IFN-gamma) and tumor necrosis factor alpha (TNF-alpha) is an important early-stage host response following infection with intracellular pathogens . Development of immunity to these pathogens is determined to a large extent by the timing and relative level of expression of the cytokines . Numerous studies have shown that early cytokine responses involving interleukin-12 (IL-12) and IFN-gamma are important for resistance to intracellular pathogens, whereas responses involving IL-4 and IL-10 increase host susceptibility . These often-indistinct early cytokine responses influence the differentiation of naive CD4(+) T helper cells, which later develop into what have commonly been termed Th1- and Th2-type cells . The characterization of CD4(+) T-helper-cell responses as Th1 or Th2 type is based largely on the cytokine profiles during the specific phase and has been used in recent years to account for the innate resistance and susceptibility of different inbred strains of mice to several intracellular pathogens . Studies investigating cytokine production in terms of CD4(+) T-helper-cell polarization in Burkholderia pseudomallei infection have not been undertaken . In this study, we used semiquantitative reverse transcription-PCR to assess induction of cytokine mRNA in liver and spleen of B . pseudomallei-susceptible BALB/c and relatively resistant C57BL/6 mice following infection with virulent B . pseudomallei . The levels of mRNA for IFN-gamma, TNF-alpha, IL-1beta, IL-6, IL-10, and IL-12 increased in both BALB/c and C57BL/6 mice 24 to 36 h after infection . A comparison of BALB/c and C57BL/6 responses revealed the relative levels of expression of mRNA for several of these cytokines, including IFN-gamma, were greater in BALB/c mice, suggesting a role for endotoxic shock and cytokine-mediated immunopathology in the development of acute melioidosis . Early induction of mRNA for the cytokines classically associated with development of Th1- and Th2-type responses was absent or minimal, and induction levels in both strains of mice were similar . During the specific phase, cytokine mRNA profiles occurred as a combination of Th1- and Th2-type patterns . Collectively, these results demonstrate that cytokine mRNA responses in BALB/c and C57BL/6 mice following infection with virulent B . pseudomallei do not develop as polarized Th1- or Th2-type profiles . Considering the role of TNF-alpha and IFN-gamma in the processes of endotoxic shock, these results also indicate that selected cytokines, while important for resistance to B . pseudomallei infection, are also potential contributors to immunopathology and the development of acute fulminating disease.

Infect Immun, 2000 Apr, 68(4), 1787 - 95
Cable-piliated Burkholderia cepacia binds to cytokeratin 13 of epithelial cells; Sajjan US et al.; Although the Burkholderia cepacia complex consists of several genomovars, one highly transmissible strain of B . cepacia has been isolated from the sputa of cystic fibrosis (CF) patients throughout the United Kingdom and Canada . This strain expresses surface cable (Cbl) pili and is thought to be the major strain associated with the fatal "cepacia syndrome." In the present report we characterize the specific 55-kDa buccal epithelial cell (BEC) protein that binds cable pilus-positive B . cepacia . N-terminal sequences of CNBr-generated internal peptides identified the protein as cytokeratin 13 (CK13) . Western blots of BEC extracts probed with a specific monoclonal antibody to CK13 confirmed the identification . Mixed epidermal cytokeratins (which contain CK13), cytokeratin extract from BEC (which consists essentially of CK13 and CK4), and a polyclonal antibody to mixed cytokeratins inhibited B . cepacia binding to CK13 blots and to normal human bronchial epithelial (NHBE) cells . Preabsorption of the antikeratin antibody with the BEC cytokeratin fraction reversed the inhibitory effect of the antibody . A cytokeratin mixture lacking CK13 was ineffective as an inhibitor of binding . Colocalization of CK13 and B . cepacia by confocal microscopy demonstrated that intact nonpermeabilized NHBE cells express small amounts of surface CK13 and bind Cbl-positive B . cepacia in the same location . Binding to intact NHBE cells was dependent on bacterial concentration and was saturable, whereas a Cbl-negative isolate exhibited negligible binding . These findings raise the possibility that surface-accessible CK13 in respiratory epithelia may be a biologically relevant target for the binding of cable piliated B . cepacia.

J Infect Dis, 2000 Mar, 181(3), 1014 - 9
Differential antibiotic-induced endotoxin release in severe melioidosis; Simpson AJ et al.; Severe melioidosis is a life-threatening, systemic bacterial infection caused by Burkholderia pseudomallei . A prospective, randomized treatment trial was conducted in northeast Thailand to compare ceftazidime (a penicillin-binding protein {PBP}-3-specific agent that causes release of large amounts of endotoxin in vitro) and imipenem (a PBP-2-specific agent that kills B . pseudomallei more rapidly but releases low amounts of endotoxin) in severe melioidosis over a 6-h time course after the first dose of antibiotic . Despite similar clinical, microbiological, endotoxin, and cytokine measures at study entry, ceftazidime-treated patients (n=34) had significantly greater systemic endotoxin (P<.001) than patients treated with imipenem (n=34) after the first dose of antibiotic . No overall difference in mortality was observed (35% in both groups {95% confidence interval, 20%-50%}) . Differential antibiotic-induced endotoxin release is demonstrable in severe melioidosis . These differences in endotoxin release did not appear to have a significant impact on survival in this group of patients.

Scand J Infect Dis, 2000, 32(1), 92 - 3
Melioidosis presenting as urinary tract infection in a previously healthy tourist; Carlson P et al.; Melioidosis is a tropical disease caused by Burkholderia pseudomallei, which is common in southeast Asia and Australia, but which is rarely diagnosed in Scandinavia . An increasing number of cases are being reported among tourists to infected areas . We report the first Finnish case of melioidosis, which presented as urinary tract infection in a previously healthy male tourist.

FEMS Microbiol Lett, 2000 Mar 15, 184(2), 273 - 8
Production of N-acyl-L-homoserine lactones by P . aeruginosa isolates from chronic lung infections associated with cystic fibrosis; Geisenberger O et al.; The N-acyl-L-homoserine lactones (AHLs) produced by sequential Pseudomonas aeruginosa isolates from chronically infected patients with cystic fibrosis were analyzed by thin-layer chromatography . It is demonstrated that both the amounts and the types of molecules synthesized by isolates from patients who were monitored over periods of up to 11 years do not change significantly during chronic colonization . However, in the case of a patient who became co-infected with an AHL-producing Burkholderia cepacia strain a dramatic reduction in the amounts of AHLs produced by the co-residing P . aeruginosa isolates was observed.

Microbiol Immunol, 2000, 44(1), 41 - 50
The dsbA-dsbB disulfide bond formation system of Burkholderia cepacia is involved in the production of protease and alkaline phosphatase, motility, metal resistance, and multi-drug resistance; Hayashi S et al.; In a previous study, we isolated a dsbB mutant of Burkholderia cepacia KF1 and showed that phenotypes of protease production and motility are dependent on DsbB, a membrane-bound disulfide bond oxidoreductase . We have now isolated a dsbA mutant by transposon mutagenesis, cloned the dsbA gene encoding a periplasmic disulfide bond oxidoreductase, and characterized the function of the DsbA-DsbB disulfide bond formation system in B . cepacia . The complementing DNA fragment had an open reading frame for a 212-amino acid polypeptide with a potential redox-active site sequence of Cys-Pro-His-Cys that is homologous to Escherichia coli DsbA . The dsbA mutant, as well as the previously isolated dsbB mutant, was defective in the production of extracellular protease and alkaline phosphatase, as well as in motility . In addition, mutation in the DsbA-DsbB system resulted in an increase in sensitivity to Cd2+ and Zn2+ as well as a variety of antibiotics including beta-lactams, kanamycin, erythromycin, novobiocin, ofloxacin and sodium dodecyl sulfate . These results suggested that the DsbA-DsbB system might be involved in the formation of a metal efflux system as well as a multi-drug resistance system.

Appl Microbiol Biotechnol, 2000 Feb, 53(2), 185 - 95
Novel regioselective hydroxylations of pyridine carboxylic acids at position C2 and pyrazine carboxylic acids at position C3; Tinschert A et al.; We have previously described the isolation of the new bacterial species, Ralstonia/Burkholderia sp . strain DSM 6920, which grows with 6-methylnicotinate and regioselectively hydroxylates this substrate in the C2 position by the action of 6-methylnicotinate-2-oxidoreductase to yield 2-hydroxy-6-methylnicotinate (Tinschert et al . 1997) . In the present study we show that this enzymatic activity can be used for the preparation of a series of hydroxylated heterocyclic carboxylic acid derivatives . The following products were obtained from the unhydroxylated educts by biotransformation using resting cells: 2-hydroxynicotinic acid, 2-hydroxy-6-methylnicotinic acid, 2-hydroxy-6-chloronicotinic acid, 2-hydroxy-5,6-dichloronicotinic acid, 3-hydroxypyrazine-2-carboxylic acid, 3-hydroxy-5-methylpyrazine-2-carboxylic acid and 3-hydroxy-5-chloropyrazine-2-carboxylic acid . Thus the respective educts were all regioselectively mono-hydroxylated at the carbon atom between the ring-nitrogen and the ring-carbon atom carrying the carboxyl group . In contrast to its relatively broad biotransformation abilities, the strain shows a limited heterocyclic nutritional spectrum . It could grow only with three of the seven transformed educts: 6-methylnicotinate, 2-hydroxy-6-methylnicotinate and 5-methylpyrazine-2-carboxylate . 2-Hydroxynicotinate, 2-hydroxy-6-chloronicotinate, 2-hydroxy-5,6-dichloronicotinate, 3-hydroxypyrazine-2-carboxylate and 3-hydroxy-5-chloropyrazine-2-carboxylate were not degraded by the strain . Therefore, unlike 6-methylnicotinate-2-oxidoreductase, which has a broad substrate spectrum, the second enzyme of the 6-methylnicotinate pathway seems to have a much more limited substrate range . Among 28 aromatic heterocyclic compounds tested as the sole source of carbon and energy, only pyridine-2,5-dicarboxylate was found as a further growth substrate, and this was degraded by a pathway which did not involve 6-methylnicotinate-2-oxidoreductase . To the best of our knowledge the microbial production of 2-hydroxy-6-chloronicotinic acid, 2-hydroxy-5,6-dichloronicotinic acid and 3-hydroxy-5-methylpyrazine-2-carboxylic acid have not been reported before . Strain DSM 6920 is so far the only known strain which allows the microbial production of both these compounds and 3-hydroxypyrazine-2-carboxylic acid and 3-hydroxy-5-chloroypyrazine-2-carboxylic acid.

Antonie Van Leeuwenhoek, 2000 Jan, 77(1), 1 - 5
Role of cytosine deaminase and beta-alanine-pyruvate transaminase in pyrimidine base catabolism by Burkholderia cepacia; West TP; A determination of the possible role of the salvage enzyme cytosine deaminase or beta-alanine-pyruvate transaminase in the catabolism of the pyrimidine bases uracil and thymine by the opportunistic pathogen Burkholderia cepacia ATCC 25416 was undertaken . It was of interest to learn whether these enzymes were influenced by cell growth on pyrimidine bases and their respective catabolic products to the same degree as the pyrimidine reductive catabolic enzymes were . It was found that cytosine deaminase activity was influenced very little by cell growth on the pyrimidines tested . Using glucose as the carbon source, only B . cepacia growth on 5-methylcytosine as a nitrogen source increased deaminase activity by about three-fold relative to (NH4)2SO4-grown cells . In contrast, the activity of beta-alanine-pyruvate transaminase was observed to be at least double in glucose-grown ATCC 25416 cells when pyrimidine bases and catabolic products served as nitrogen sources instead of (NH4)2SO4 . Transaminase activity in the B . cepacia glucose-grown cells was maximal after the strain was grown on either uracil or 5-methylcytosine as a nitrogen source compared to (NH4)2SO4-grown cells . A possible role for beta-alanine-pyruvate transaminase in pyrimidine base catabolism by B . cepacia would seem to be suggested from the similarity in how its enzyme activity responded to cell growth on pyrimidine bases and catabolic products when compared to the response of the three reductive catabolic enzymes.

J Clin Microbiol, 2000 Mar, 38(3), 1042 - 7
Identification and population structure of Burkholderia stabilis sp . nov . (formerly Burkholderia cepacia genomovar IV); Vandamme P et al.; The Burkholderia cepacia complex currently comprises five genomic species, i.e., B . cepacia genomovar I, B . multivorans (formerly known as B . cepacia genomovar II), B . cepacia genomovar III, B . cepacia genomovar IV, and B . vietnamiensis (also known as B . cepacia genomovar V) . In the absence of straightforward diagnostic tests for the identification of B . cepacia genomovars I, III, and IV, the last two genomic species were not formally classified as novel Burkholderia species (genomovar I contains the type strain and therefore retains the name B . cepacia) . In the present study, we describe differential biochemical tests and a recA gene-based PCR assay for the routine identification of strains currently known as B . cepacia genomovar IV and propose formal classification of this organism as Burkholderia stabilis sp . nov . B . stabilis can indeed be differentiated from all other B . cepacia complex strains by the absence of beta-galactosidase activity, from strains of B . cepacia genomovars I and III and B . vietnamiensis by the inability to oxidize sucrose, and from B . multivorans by the lack of growth at 42 degrees C . In addition, analysis with the recA gene-derived primers BCRG41 (5'-ACCGGCGAGCAGGCGCTT-3') and BCRG42 (5'-ACGCCATCGGGCATGGCA-3') specifically allows the detection of B . stabilis strains in a conventional PCR assay . Examination of a set of 21 B . stabilis strains by means of random amplified polymorphic DNA analysis and pulsed-field gel electrophoresis typing suggested that the genome of this organism is highly conserved, which is in sharp contrast to the generally accepted genomic diversity, variability, and plasticity among B . cepacia strains.

Epidemiol Infect, 1999 Dec, 123(3), 437 - 43
Acute melioidosis outbreak in Western Australia; Inglis TJ et al.; A cluster of acute melioidosis cases occurred in a remote, coastal community in tropical Western Australia . Molecular typing of Burkholderia pseudomallei isolates from culture-confirmed cases and suspected environmental sources by pulsed-field gel electrophoresis (PFGE) of XbaI chromosomal DNA digests showed that a single PFGE type was responsible for five cases of acute infection in a community of around 300 during a 5 week period . This temporal and geographical clustering of acute melioidosis cases provided a unique opportunity to investigate the environmental factors contributing to this disease . B . pseudomallei isolated from a domestic tap at the home of an asymptomatic seroconverter was indistinguishable by PFGE . Possible contributing environmental factors included an unusually acid communal water supply, unrecordable chlorine levels during the probable exposure period, a nearby earth tremor, and gusting winds during the installation of new water and electricity supplies . The possible role of the potable water supply as a source of B . pseudomallei was investigated further.

J Vet Diagn Invest, 2000 Jan, 12(1), 46 - 50
Procedurally similar competitive immunoassay systems for the serodiagnosis of Babesia equi, Babesia caballi, Trypanosoma equiperdum, and Burkholderia mallei infection in horses; Katz J et al.; Procedurally similar competitive enzyme-linked immunoassay (cELISA) methods were developed for the serodiagnosis of Babesia equi and Babesia caballi (piroplasmosis), Trypanosoma equiperdum (dourine), and Burkholderia mallei (glanders) infections in horses . Apparent test specificities for the B . equi, B . caballi, T . equiperdum, and B . mallei cELISAs were 99.2%, 99.5%, 98.9%, and 98.9%, respectively . Concordances and kappa values between the complement fixation (CF) and the cELISA procedures for the serodiagnosis of B . equi, B . caballi, T . equiperdum, and B . mallei infections in experimentally exposed horses were 76% and 0.55, 89% and 0.78, 97% and 0.95, and 70% and 0.44, respectively . The cELISA method may be a technically more reproducible, objective, and convenient approach for piroplasmosis, dourine, and glanders serodiagnosis in qualifying animals for international movement and disease eradication programs than the CF systems currently in use . Use of the cELISA method also obviated the problems associated with testing hemolyzed or anticomplementary sera.

FEMS Microbiol Lett, 2000 Mar 1, 184(1), 95 - 101
Response of soybean rhizosphere communities to human hygiene water addition as determined by community level physiological profiling (CLPP) and terminal restriction fragment length polymorphism (TRFLP) analysis; Kerkhof L et al.; In this report, we describe an experiment conducted at Kennedy Space Center in the biomass production chamber (BPC) using soybean plants for purification and processing of human hygiene water . Specifically, we tested whether it was possible to detect changes in the root-associated bacterial assemblage of the plants and ultimately to identify the specific microorganism(s) which differed when plants were exposed to hygiene water and other hydroponic media . Plants were grown in hydroponics media corresponding to four different treatments: control (Hoagland's solution), artificial gray water (Hoagland's+surfactant), filtered gray water collected from human subjects on site, and unfiltered gray water . Differences in rhizosphere microbial populations in all experimental treatments were observed when compared to the control treatment using both community level physiological profiles (BIOLOG) and molecular fingerprinting of 16S rRNA genes by terminal restriction fragment length polymorphism analysis (TRFLP) . Furthermore, screening of a clonal library of 16S rRNA genes by TRFLP yielded nearly full length SSU genes associated with the various treatments . Most 16S rRNA genes were affiliated with the Klebsiella, Pseudomonas, Variovorax, Burkholderia, Bordetella and Isosphaera groups . This molecular approach demonstrated the ability to rapidly detect and identify microorganisms unique to experimental treatments and provides a means to fingerprint microbial communities in the biosystems being developed at NASA for optimizing advanced life support operations.

Infect Immun, 2000 Mar, 68(3), 1681 - 6
Interaction between Burkholderia pseudomallei and Acanthamoeba species results in coiling phagocytosis, endamebic bacterial survival, and escape; Inglis TJ et al.; Burkholderia pseudomallei causes melioidosis, a potentially fatal disease whose clinical outcomes include rapid-onset septicemia and relapsing and delayed-onset infections . Like other facultative intracellular bacterial pathogens, B . pseudomallei is capable of survival in human phagocytic cells, but unlike mycobacteria, Listeria monocytogenes, and Salmonella serovar Typhimurium, the species has not been reported to survive as an endosymbiont in free-living amebae . We investigated the consequences of exposing Acanthamoeba astronyxis, A . castellani, and A . polyphaga to B . pseudomallei NCTC 10276 in a series of coculture experiments . Bacterial endocytosis was observed in all three Acanthamoeba species . A more extensive range of cellular interactions including bacterial adhesion, incorporation into amebic vacuoles, and separation was observed with A . astronyxis in timed coculture experiments . Amebic trophozoites containing motile intravacuolar bacilli were found throughout 72 h of coculture . Confocal microscopy was used to confirm the intracellular location of endamebic B . pseudomallei cells . Transmission electron microscopy of coculture preparations revealed clusters of intact bacilli in membrane-lined vesicles inside the trophozoite cytoplasm; 5 x 10(2) CFU of bacteria per ml were recovered from lysed amebic trophozoites after 60 min of coculture . Demonstration of an interaction between B . pseudomallei and free-living acanthamebae in vitro raises the possibility that a similar interaction in vivo might affect environmental survival of B . pseudomallei and subsequent human exposure . Endamebic passage of B . pseudomallei warrants further investigation as a potential in vitro model of intracellular B . pseudomallei infection.

Pediatr Pulmonol, 2000 Mar, 29(3), 210 - 2
In vitro activity of minocycline against respiratory pathogens from patients with cystic fibrosis; Kurlandsky LE et al.; Our objective was to determine the in vitro activity of minocycline against isolates of Burkholderia cepacia (BC), Stenotrophomonas maltophilia (SM), and Pseudomonas aeruginosa (PA) cultured from the respiratory tract of patients with cystic fibrosis (CF) . Cultures of BC, SM, and PA were isolated in a hospital bacteriology laboratory from the sputum or oropharyngeal cultures obtained from patients attending a Cystic Fibrosis Center, and were prospectively tested for in vitro sensitivity to minocycline by Kirby-Bauer disk diffusion . From January 1994 to July 1995, 116 cultures from 61 patients had at least one of the three pathogens; 9/61 (15%) patients had an isolate of BC, and 7/9 (78%) had an initial isolate sensitive to minocycline, of which 3 were sensitive only to minocycline; 2 cultures were resistant to all antibiotics . Four of 7 patients with BC were treated with minocycline; 3 patients developed resistant isolates 3-13 months after therapy . Five of 61 patients (8%) had an isolate of SM: 4/5 (80%) of these isolates were sensitive to minocycline, of which 1 was sensitive only to minocycline . Fifty-five of 61 patients (90%) had at least one PA isolate, with 112 morphotypes recovered from 90 cultures: 40/112 morphotypes (36%) were sensitive to minocycline, 65 (58%) were resistant, and 7 (6%) were intermediate in sensitivity . We conclude that the marked in vitro activity of minocycline against BC and SM isolated from patients with CF suggests that minocycline may have an adjunct role in the antimicrobial therapy of multidrug resistant, respiratory pathogens in CF .

FEMS Microbiol Lett, 2000 Feb 15, 183(2), 295 - 9
Bacterial triterpenoids of the hopane series as biomarkers for the chemotaxonomy of Burkholderia, Pseudomonas and Ralstonia spp; Cvejic JH et al.; Hopanoid fingerprints allowed to differentiate bacteria formerly connected to the genus Pseudomonas . Whereas all strains related to Pseudomonas and Ralstonia were devoid of any detectable hopanoid, these pentacyclic triterpenoids were found in the Burkholderia species and in related soil isolates, which contained as main hopanoid a bacteriohopanetetrol carbapseudopentose ether, accompanied by significant amounts of its novel Delta(6) unsaturated homologue . Unsaturated hopanoids represent an extremely rare feature in soil bacteria and the only known indication for a catabolism of this pentacyclic carbon skeleton in bacteria.

Acta Trop, 2000 Feb 5, 74(2-3), 247 - 51
Comparison of three PCR primer sets for diagnosis of septicemic melioidosis; Kunakorn M et al.; Several sets of PCR primers have recently been developed for detection of Burkholderia pseudomallei . In this report, the performance of 16S rRNA gene primers (16S), rRNA spacer gene primers (spacer), and 'LPS' primers (LPS) were compared . All primer sets were tested by PCR amplification of the same DNA samples extracted from blood specimens of 46 patients from northeastern Thailand, of which 29 had melioidosis based on blood culture as a gold standard . The sensitivities were 41, 35.7, and 31% while the specificities were 47, 59, and 100% for the 16S, spacer, and LPS primers, respectively . The positive predictive values were 60, 59, and 100%, while negative predictive values were 35, 34, and 46%, for these primers . The low sensitivity of PCR was suspected to be because of small numbers of bacteria in the samples . In addition, one primer set could not detect all B . pseudomallei strains . To make PCR for melioidosis more practical, bacterial concentration steps must be added . Lastly, mixed infection of patients in endemic areas may be the cause of controversial false positive PCR results, and should be further investigated.

Acta Trop, 2000 Feb 5, 74(2-3), 229 - 34
Proinflammatory cytokine mRNA responses in experimental Burkholderia pseudomallei infection in mice; Ulett GC et al.; Melioidosis is a potentially fatal disease of both human and animals caused by the bacterium Burkholderia pseudomallei . Disease is endemic in tropical and subtropical regions of Southeast Asia and Northern Australia . The pathogenesis of melioidosis is poorly understood . In particular, the host responses that occur following infection, and the specific host-pathogen interactions that result in the development of either acute or chronic infection are unclear . Using an established murine model, we investigated early proinflammatory cytokine responses believed to be critical in the development of acute and chronic B . pseudomallei infection . Semi-quantitative reverse transcription polymerase chain reaction (RT-PCR) was used to assess levels of mRNA for tumor necrosis factor-alpha (TNF-alpha), interleukin 1beta (IL-1beta) and interleukin 6 (IL-6) in the liver of mice following infection . We demonstrate that the level of mRNA for these cytokines increase moderately in chronic infection in C57BL/6 mice . However, in acute infection in BALB/c mice, mRNA responses for these cytokines were shown to be comparatively greater . These results demonstrate that early proinflammatory cytokine responses are important in the immunopathogenesis of melioidosis.

Acta Trop, 2000 Feb 5, 74(2-3), 221 - 8
Shedding of lipopolysaccharide and 200-kDa surface antigen during the in vitro growth of virulent Ara- and avirulent Ara+ Burkholderia pseudomallei; Anuntagool N et al.; Non-virulent Ara+ B . pseudomallei environmental isolates differ from virulent Ara- clinical isolates by their ability to assimilate L-arabinose and the absence of a 200 kDa antigen on their surface . The latter, present only on the Ara- isolates from either clinical or environmental origin, was recently demonstrated by its immunoreactivity with monoclonal antibody (MAb) 5F8 . We recently demonstrated that lipopolysaccharide (LPS) from both biotypes were indistinguishable from one another with regard to SDS-PAGE profiles and immunoreactivities with immune sera . In this study, the shedding of LPS and 200-kDa antigen into the culture medium during the in vitro growth of Ara- was compared with that of its Ara+ counterpart, using MAb-based sandwich ELISAs . The results showed that the LPS shedding profiles from the two biotypes were similar to one another . This was in contrast to the situation with the 5F8-reactive antigen . The culture fluid of all Ara- isolates and none of the Ara+ isolates were found to react strongly with the MAb 5F8 during the early log phase of growth . However, during the late stationary phase, a trace amount of the 5F8-reactive material could also be detected in the culture fluid of the Ara+ isolates.

Acta Trop, 2000 Feb 5, 74(2-3), 215 - 20
Protease production by Burkholderia pseudomallei and virulence in mice; Gauthier YP et al.; The aim of this study was to assess protease production and virulence of various Burkholderia pseudomallei strains . Protease activity was evaluated in filtrates from cultures grown for 50 h in TSB Dialysate by azocasein hydrolysis, and expressed as absorbancy at 405 nm . Virulence was assessed in 8 weeks old SWISS mice, by intraperitoneal injection of 6-6 x 10(5) CFU, and the LD50 was calculated after 30 days by the method of Reed and Muench . The lethal activity was studied for five strains of B . pseudomallei and the type strains of Burkholderia pseudomallei, Burkholderia mallei, and Burkholderia cepacia . The three type strains appeared to be low protease producers (A405 = 0.11, 0.09 and 0.00, respectively) and avirulent . The two more virulent B . pseudomallei strains exhibited significantly different LD50, 3.5 x 10(2) (IPP 6068 VIR) versus 2.1 x 10(5) CFU/mouse (40/97), and protease activities (A405 = 0.046 and 0.79, respectively) . Moreover, the avirulent parent of IPP 6068 (AG), was a better protease producer than the 6068 VIR strain, A405 = 0.26 versus 0.046 . These results suggest that there is no correlation between virulence and level of exoproteolytic activity, when B . pseudomallei is injected to mice via the intraperitoneal route.

Acta Trop, 2000 Feb 5, 74(2-3), 201 - 10
Pathogenesis of and immunity to melioidosis; Brett PJ et al.; While Burkholderia pseudomallei, the causative agent of melioidosis, is becoming increasingly recognized as a significant cause of morbidity and mortality in regions to which it is endemic, no licensed vaccine preparation currently exists for immunization against the disease . Therefore, one of the primary goals of our research has been to identify and characterize antigens expressed by B . pseudomallei isolates for the intended purpose of developing a vaccine construct that can be used to actively immunize specific high risk populations against the disease . By utilizing a combination of biochemical, immunological and molecular approaches, our studies now indicate that some of the most promising candidates for this task include flagellin proteins and the endotoxin derived O-polysaccharide (PS) antigens expressed by the organism . In this review, we have attempted to summarize the current status of B . pseudomallei research while endeavoring to provide a rationale for our approach towards the development of a melioidosis vaccine.

Acta Trop, 2000 Feb 5, 74(2-3), 193 - 9
Use of multiplex PCR patterns as genetic markers for Burkholderia pseudomallei; Wongratanacheewin S et al.; A simple PCR-based typing method was developed to differentiate between strains of Burkholderia pseudomallei . Two pairs of primers, based on sequences from two specific DNA probes, were used to amplify the bacterial DNA by multiplex PCR . We evaluated the PCR method for epidemiological typing of B . pseudomallei and compared this with restriction fragment length polymorphism (RFLP) and random amplified polymorphic DNA (RAPD) methods . In RFLP, the DNA of B . pseudomallei was digested with HindIII and the pKKU-S23L was used as a probe while 5' GTTTCGCTCC 3' primer was used in RAPD . DNA was obtained from 37 B . pseudomallei environmental and clinical isolates from humans or animals . These isolates were also classified by their ability to assimilate L-arabinose . A total of 21 type patterns were identified by multiplex PCR . Among human and animal isolates, multiplex PCR revealed ten types, all of which were arabinose negative (Ara-), whereas six of the 11 types of environmental isolates were Ara- . There are two environmental patterns that also were found in clinical isolates . The RFLP technique showed 12 different types in the 37 isolates, and three of these contained both Ara+ and Ara- isolates . The RAPD technique revealed 33 different types in the 37 isolates . Multiplex PCR, therefore, is the genetic marker that best correlates with the ability of the organism to assimilate L-arabinose . Moreover, two types (M4, M15) correlated with disseminated septicemic melioidosis in the northeast Thailand . If a greater number of isolates are tested, the multiplex PCR technique may prove to be useful for rapid epidemiological typing of B . pseudomallei.

Acta Trop, 2000 Feb 5, 74(2-3), 187 - 91
Genome fingerprinting by pulsed-field gel electrophoresis of isolates of Burkholderia pseudomallei from patients with melioidosis in Thailand; Koonpaew S et al.; A total of 95 isolates of Burkholderia pseudomallei from 53 sporadic cases in Thailand were examined by pulsed-field gel electrophoresis . Digestion of genomic DNA of all isolates by NcoI generated a macrorestriction pattern similar to that of B . pseudomallei which cannot assimilate L-arabinose . Analysis using restriction enzymes SpeI and AvrII demonstrated greater sensitivity than NcoI digestion in the differentiation of B . pseudomallei and could be used for epidemiological groupings . Four cluster groups were evident among 37 isolates tested and the majority of isolates within each cluster displayed more than 65% similarity . Furthermore, multiple isolates from 18 and 35 patients with single and recurrent episodes of melioidosis, respectively, were examined . All patients with a single episode yielded genetically identical isolates and four of 35 patients with recurrent episodes were infected with strains of different genotypic patterns from the primary isolate(s) . Hence, most repeated episodes of infection in melioidosis are as a result of the original infecting strains.

Acta Trop, 2000 Feb 5, 74(2-3), 181 - 5
Molecular phylogeny of Burkholderia pseudomallei; Pitt TL et al.; In terms of population structure, the species Burkholderia pseudomallei contains both clonal and non-clonal elements . By indexing variation in rRNA loci using the restriction endonuclease BamHI, we found that two ribotypes (types 1 and 3) are predominant in nature . Ribotype 3 is prevalent in Asian countries while ribotype 1 is more widespread . Some disease association was suggested for 4 ribotypes and strains of ribotype 4 were markedly associated with a fatal outcome . DNA macrorestriction (XbaI) profiles resolved by pulsed-field gel electrophoresis revealed great heterogeneity within the prevalent ribotypes and these profiles appeared to be reliable strain markers . Arabinose environmental strains were characterised by BamHI ribotypes that were markedly distinct form clinical and environmental isolates of the arabinose negative phenotype.

Acta Trop, 2000 Feb 5, 74(2-3), 169 - 79
Multiple replicons constitute the 6.5-megabase genome of Burkholderia pseudomallei; Songsivilai S et al.; Burkholderia pseudomallei is a causative agent of melioidosis, a fatal tropical infectious disease endemic in Southeast Asia and Northern Australia . In order to determine the size and characteristics of the bacterial genome, the B . pseudomallei genome and genes were analyzed by pulsed field gel electrophoresis of the undigested, intact megabase DNA, and by computational analysis of nucleotide sequences of B . pseudomallei genes which have been sequenced by several investigators and already deposited in a public database . The results showed that the B . pseudomallei genome consists of two large replicons, and that both contain ribosomal RNA gene sequences, indicating the presence of two chromosomes . The classical arabinose-negative B . pseudomallei isolate K96243 has chromosomes of approximately 3563 +/- 73 and 2974 +/- 40 kilobase-pairs in size, giving a total genome size of about 6.5 million base-pairs . The arabinose-positive nonvirulent biotype of B . pseudomallei also has two replicons which are smaller than those of the arabinose-negative biotype . Analysis of the publicly-available nucleotide sequences showed that the average B . pseudomallei gene is approximately 1031 base-pairs in size, with an average G + C content of 65.7% . The genome is gene-rich and about 89% of the coding capacity is used as coding sequences . It can therefore be estimated that the entire B . pseudomallei genome encodes about 5600 genes.

Acta Trop, 2000 Feb 5, 74(2-3), 159 - 68
Ecology of Burkholderia pseudomallei and the interactions between environmental Burkholderia spp . and human-animal hosts; Dance DA; Early workers thought that melioidosis was a zoonosis with a reservoir in rodents, but we now know that Burkholderia pseudomallei is a widely distributed environmental saprophyte . In northeast Thailand, two thirds of paddy fields yield the organism, and 80% of children have antibodies by the time they are 4 years old . However, interpretation of these results has been complicated by the recent recognition of avirulent, antigenically cross-reacting environmental organisms for which the name B . thailandensis has been proposed . We still know very little about the climatic, physical, chemical and biological factors which control the proliferation and survival of Burkholderia spp . in the environment, although epidemiological studies show space-time clustering of melioidosis . It is assumed that most human and animal melioidosis arises through exposure to contaminated soil or muddy water, although only 6% of human cases have a clear history of inoculation, and a further 0.5% of cases follow near-drowning . Laboratory animals have also been infected by ingestion, inhalation and insect bites, but evidence of infection acquired naturally by these routes remains anecdotal . Sporadic cases have resulted from iatrogenic inoculation, laboratory accidents, and person-to-person or animal-to-person spread . Whether exposure to B . pseudomallei will result in disease probably depends on the balance between the virulence of the strain, the immune status of the host (e.g . diabetes mellitus) and the size of the inoculum.

Acta Trop, 2000 Feb 5, 74(2-3), 153 - 8
Animal melioidosis in Australia; Choy JL et al.; Melioidosis was first diagnosed in Australia in sheep in 1949 . While it has been considered endemic in tropical Australia, there have been animal outbreaks in southwest Western Australia and southern Queensland . Infection occurs in many species, with both latency and a wide range of clinical manifestations . Some species may develop melioidosis only if immunocompromised . Sheep and goats are particularly susceptible, resulting in the requirement for pasteurisation of tropical commercial goat's milk . Nine out of 43 (21%) goats had aortic lesions at autopsy and seven died from aortic aneurysm rupture . Transplacental transmission in goats has also been documented . Asymptomatic organ abscesses are common in pigs but bovine melioidosis is very rare . Camels moved north and an alpaca brought to Darwin have died from melioidosis . It also occurs in wildlife, including birds, crocodiles and kangaroos . Zoonotic transmission to humans is extremely unusual, but there are many similar epidemiological and clinical features of melioidosis in animals and humans . There have been three possible zoonotic cases in Australia . Molecular typing has found identical Burkholderia pseudomallei organisms from animals, humans and soil . The study of melioidosis in animals, especially the use of molecular genetic techniques for organism identification and typing, will continue to unravel aspects of the disease that remain unclear in humans.

Acta Trop, 2000 Feb 5, 74(2-3), 145 - 51
Neurological melioidosis; Currie BJ et al.; Neurological abnormalities have long been recognised in animals with melioidosis, including laboratory rodents and sheep in the first Australian outbreak in 1949 . Autopsies in animals have shown microabscesses and lymphocytic infiltration to be present on occasion in the same animal, but Burkholderia pseudomallei is usually able to be grown from central nervous system (CNS) tissue . In humans CNS melioidosis is unusual, but both macroscopic brain abscesses and encephalitis occur . There has been a recently recognised syndrome of meningoencephalitis with varying involvement of brainstem, cerebellum and spinal cord . The prospective melioidosis study at Royal Darwin Hospital has documented 12 cases of CNS melioidosis over 9 years out of a total of 232 cases of melioidosis (5%) . Prominent features on presentation were unilateral limb weakness (6), predominant cerebellar signs (2), mixed cerebellar and brainstem features with peripheral weakness (2) and flaccid paraparesis (2) . Eight patients had unilateral VIIth nerve palsy and six bulbar palsy, with five requiring prolonged ventilation . Brain CT scans are usually normal initially, but MRI shows dramatic changes . Three patients died and only three made a full recovery . In two patients with predominant mononuclear CSF pleocytosis, B . pseudomallei was cultured from CSF and autopsy in one of these showed necrotising encephalitis with microabscesses . Although it has been postulated that a neurotropic exotoxin may account for melioidosis encephalomyelitis, the recent findings and comparison with the animal data suggest that direct organism spread within the CNS may be primarily responsible . Preliminary molecular typing of isolates shows no evidence of a specific strain of B . pseudomallei responsible for CNS melioidosis end further studies are required to determine if the apparent higher rate of CNS disease in Australia is due to true regional differences or is from increased ascertainment.

Acta Trop, 2000 Feb 5, 74(2-3), 121 - 7
The epidemiology of melioidosis in Australia and Papua New Guinea; Currie BJ et al.; Melioidosis was first described in Australia in an outbreak in sheep in 1949 in north Queensland (22 degrees S) . Human melioidosis was first described from Townsville (19 degrees S) in 1950 . Melioidosis is hyperendemic in the Top End of the Northern Territory (NT) and as in parts of northeastern Thailand it is the commonest cause of fatal community-acquired septicemic pneumonia . In the 9 years since 1989 the prospective NT melioidosis study at Royal Darwin Hospital (12 degrees S) has documented 206 culture confirmed cases of melioidosis, with an average annual incidence of 16.5/100,000 . Melioidosis is also seen in the north of Western Australia and north Queensland, including the Torres Strait Islands, but is uncommon in adjacent Papua New Guinea . Serological studies suggest that infection is rare in the Port Moresby region, but there is emerging evidence of melioidosis from Western Province . The NT study has documented inoculating events in 52 (25%) of cases, with an incubation period of 1-21 days (mean 9 days); 84% of cases had acute disease from presumed recent acquisition and 13% had chronic disease (sick, > 2 months) . In 4% there was evidence of possible reactivation from a latent focus; 28 of 153 (18%) males had prostatic abscesses . The overall mortality was 21% (43 cases), with a mortality rate in septicemic cases (95) of 39% and in non-septicemic cases (103) of 4% . Pneumonia was the commonest presentation in both groups and, in addition, eight patients (two deaths) presented with melioidosis encephalomyelitis . Melioidosis clusters in temperate Australia are attributed to animals imported from the north . Molecular typing of Burkholderia pseudomallei isolates from temperate southwest Western Australia showed clonality over 25 years . In this outbreak and in studies from the NT, some soil isolates are molecularly identical to epidemiologically related animal and human isolates . Molecular typing has implicated the water supply in two clonal outbreaks in remote aboriginal communities in northern Australia . Further prospective collaborative studies are required to evaluate whether there are truly regional differences in clinical features of melioidosis and to better understand how B . pseudomallei is acquired from the environment.

Acta Trop, 2000 Feb 5, 74(2-3), 115 - 9
Melioidosis as an emerging global problem; Dance DA; There is remarkably little known about the incidence of melioidosis outside a few countries (Thailand, Australia, Singapore and Malaysia) . Presumably it is widespread in tropical south east Asia . Elsewhere there are tantalising glimpses of the tip of what may be a large iceberg . Since a specific diagnosis of melioidosis requires awareness on the part of clinicians, and the existence of a laboratory capable of isolating and identifying Burkholderia pseudomallei, a luxury not available in most rural tropical areas, the size of this iceberg is likely to remain unknown for the foreseeable future . There is mounting evidence that the disease is endemic in the Indian sub-continent and the Caribbean, and there have been unsubstantiated reports of recent cases in South Africa and the Middle East . It is unclear whether melioidosis has really spread to such areas relatively recently, or has been there but unrecognised for a long time . Almost all cases diagnosed in temperate climates have been imported from the tropics, with the exception of a unique outbreak which occurred in France in the mid-1970s . With increasing world wide travel of both humans and other animals, the potential exists for melioidosis to spread to new and fertile pastures.

Biotechnol Prog, 2000 Jan-Feb, 16(1), 26 - 30
Cloning and expression of Vitreoscilla hemoglobin gene in Burkholderia sp . strain DNT for enhancement of 2,4-dinitrotoluene degradation; Patel SM et al.; The gene (vgb) encoding the hemoglobin (VHb) of Vitreoscilla sp . was cloned into a broad host range vector and stably transformed into Burkholderia (formerly Pseudomonas) sp . strain DNT, which is able to degrade and metabolize 2,4-dinitrotoluene (DNT) . Vgb was stably maintained and expressed in functional form in this recombinant strain (YV1) . When growth of YV1, in both tryptic soy broth and minimal salts broth containing DNT and yeast extract, was compared with that of the untransformed strain, YV1 grew significantly better on a cell mass basis (A(600)) and reached slightly higher maximum viable cell numbers . YV1 also had roughly twice the respiration as strain DNT on a cell mass basis, and in DNT-containing medium, YV1 degraded DNT faster than the untransformed strain . YV1 cells pregrown in medium containing DNT plus succinate showed the fastest degradation: 100% of the initial 200 ppm DNT was removed from the medium within 3 days.

J Biotechnol, 2000 Jan 21, 76(2-3), 165 - 74
Propionic acid metabolism and poly-3-hydroxybutyrate-co-3-hydroxyvalerate (P3HB-co-3HV) production by Burkholderia sp; Silva LF et al.; Mutants of Burkholderia sp . that are unable to grow on propionic acid (prp) but still accumulate P3HB-co-3HV from carbohydrate and propionic acid were studied . In shaken flask tests, yields of 3HV from propionic acid (Y(3HV/Prop)) increased from 0.10 g g(-1) in the wild type to c.a . 0.35 g g(-1) in mutants affected in alpha-oxidation pathway or to 0.80 g g(-1) in mutants not affected in that pathway . In bioreactor tests, mutant IPT 189 showed Y(3HV/Prop) = 1.20 g g(-1), a yield very close to the theoretical maximum of 1.35 g g(-1) . Accumulation of 3HV units from unrelated carbon sources was undetectable in these mutants indicating that 3HV units are produced directly from propionic acid . Thus, the industrial use of those mutants to produce the copolymer from sucrose and propionic acid could significantly reduce the production costs . The results strongly suggest the existence of at least two pathways that are involved in the oxidation of propionic acid in Burkholderia sp . Their rates would be modulated by the availability of propionic acid.

J Clin Microbiol, 2000 Feb, 38(2), 910 - 3
Diagnostically and experimentally useful panel of strains from the Burkholderia cepacia complex; Mahenthiralingam E et al.; Two new species, Burkholderia multivorans and Burkholderia vietnamiensis, and three genomovars (genomovars I, III, and IV) currently constitute the Burkholderia cepacia complex . A panel of 30 well-characterized strains representative of each genomovar and new species was assembled to assist with identification, epidemiological analysis, and virulence studies on this important group of opportunistic pathogens.

J Clin Microbiol, 2000 Feb, 38(2), 883 - 5
Presence of type III secretion genes in Burkholderia pseudomallei correlates with Ara(-) phenotypes; Winstanley C et al.; Dot blot hybridization and PCR amplification of 14 Ara(+) and 8 Ara(-) Burkholderia pseudomallei strains showed that type III secretion (TTS) genes were present in all the Ara(-) strains but absent from all but one of the Ara(+) strains . The link between TTS genes and an Ara(-) phenotype suggests a role for TTS in virulence.

J Clin Microbiol, 2000 Feb, 38(2), 818 - 25
Specific and rapid detection by fluorescent in situ hybridization of bacteria in clinical samples obtained from cystic fibrosis patients; Hogardt M et al.; We report on the rapid and specific detection of bacteria commonly isolated from clinical specimens from cystic fibrosis (CF) patients by fluorescent in situ hybridization (FISH) . On the basis of comparative sequence analysis, we designed oligonucleotide probes complementary to species-specific 16S rRNA regions of these microorganisms and demonstrated the specificities of the probes by hybridization of different remotely related as well as closely related reference strains . Furthermore, in a pilot project we investigated 75 sputum samples and 10 throat swab specimens from CF patients by FISH and detected Pseudomonas aeruginosa, Burkholderia cepacia, Stenotrophomonas maltophilia, Haemophilus influenzae, and Staphylococcus aureus within these specimens . The specificity of FISH was 100% in comparison to the results of conventional microbial culture . In contrast, the sensitivity of standard laboratory cultivation was moderately higher, since the limit for microscopic detection of bacteria within sputum samples by FISH was approximately 4 x 10(5) CFU/ml of sputum (resulting in a 90% sensitivity for FISH) . Moreover, we demonstrated that FISH will be useful for the rapid detection of bacteria that cause acute pulmonary exacerbations in CF patients, as demonstrated in patients with H . influenzae, S . aureus, and P . aeruginosa exacerbations . Therefore, FISH is a valuable additional method for the rapid and specific detection of bacteria in clinical samples from CF patients, in particular, patients with pulmonary exacerbations.

FEMS Microbiol Lett, 2000 Feb 1, 183(1), 73 - 9
Identification of new transposable genetic elements in Burkholderia pseudomallei using subtractive hybridisation; Brown NF et al.; A subtraction library of Burkholderia pseudomallei was constructed by subtractive hybridisation of B . pseudomallei genomic DNA with Burkholderia thailandensis genomic DNA . Two clones were found to have significant sequence similarity to insertion sequences which have previously not been found in B . pseudomallei (designated ISA and ISB); and two clones showed sequence similarity to different regions of Burkholderia cepacia IS407 that has recently been detected in B . pseudomallei . The former, though possibly non-functional, represents new transposable genetic elements of B . pseudomallei . All three sequences were found to be present in multi-copy in the genomes of a number of B . pseudomallei strains and in B . thailandensis, which are the first transposable elements identified in this species.

SAR QSAR Environ Res, 1999, 10(5), 473 - 95
Quantitative structure-toxicity relationships for chlorophenols to bioluminescent lux-marked bacteria using atom-based semi-empirical molecular-orbital descriptors; Warne MA et al.; Literature data on the toxicity of chlorophenols for three luminescent bacteria (Vibrio fischeri, and the lux-marked Pseudomonas fluorescens 10586s pUCD607 and Burkholderia spp . RASC c2 (Tn4431)) have been analyzed in relation to a set of computed molecular physico-chemical properties . The quantitative structure-toxicity relationships of the compounds in each species showed marked differences when based upon semi-empirical molecular-orbital molecular and atom based properties . For mono-, di- and tri-chlorophenols multiple linear regression analysis of V . fischeri toxicity showed a good correlation with the solvent accessible surface area and the charge on the oxygen atom . This correlation successfully predicted the toxicity of the heavily chlorinated phenols, suggesting in V . fischeri only one overall mechanism is present for all chlorophenols . Good correlations were also found for RASC c2 with molecular properties, such as the surface area and the nucleophilic super-delocalizability of the oxygen . In contrast the best QSTR for P . fluorescens contained the 2nd order connectivity index and ELUMO suggesting a different, more reactive mechanism . Cross-species correlations were examined, and between V . fischeri and RASC c2 the inclusion of the minimum value of the nucleophilic susceptibility on the ring carbons produced good results . Poorer correlations were found with P . fluorescens highlighting the relative similarity of V . fischeri and RASC c2, in contrast to that of P . fluorescens.

Angew Chem Int Ed Engl, 2000 Jan, 39(1), 156 - 160
Acetyl Substitution of the O-Specific Caryan from the Lipopolysaccharide of Pseudomonas (Burkholderia) caryophylli Leads to a Block Pattern; Molinaro A et al.; An exceptionally large repeating unit has been proposed for one of the two O-specific polysaccharides of the lipopolysaccharide from Pseudomonas (Burkholderia) caryophylli, which is a homopolymer of the sugar caryose . This proposal is based on the unusual acetylation pattern of the carbohydrate (shown in the picture), as determined from the presence of eight independent spin systems observed by NMR spectroscopy . A=acetylated, D=non-acetylated caryose monomer.

Arch Microbiol, 2000 Jan, 173(1), 35 - 41
Initial in vitro characterisation of phosphonopyruvate hydrolase, a novel phosphate starvation-independent, carbon-phosphorus bond cleavage enzyme in Burkholderia cepacia Pal6; Ternan NG et al.; A novel, inducible carbon-phosphorus bond cleavage enzyme, phosphonopyruvate hydrolase, was detected in cell-free extracts of Burkholderia cepacia Pal6, an environmental isolate capable of mineralising L-phosphonoalanine as carbon, nitrogen and phosphorus source . The activity was induced only in the presence of phosphonoalanine, did not require phosphate starvation for induction and was uniquely specific for phosphonopyruvate, producing equimolar quantities of pyruvate and inorganic phosphate . The native enzyme had a molecular mass of some 232 kDa and showed activation by metal ions in the order Co2+ > Ni2+ > Mg2+ > Zn2+ > Fe2+ > Cu2+ . Temperature and pH optima in crude cell extracts were 50 degrees C and 7.5, respectively, and activity was inhibited by EDTA, phosphite, sulfite, mercaptoethanol and sodium azide . Phosphonopyruvate hydrolase is the third bacterial C-P bond cleavage enzyme reported to date that proceeds via a hydrolytic mechanism.

FEMS Microbiol Ecol, 2000 Feb 1, 31(2), 127 - 141
Effect of inoculation of a TOL plasmid containing mycorrhizosphere bacterium on development of Scots pine seedlings, their mycorrhizosphere and the microbial flora in m-toluate-amended soil; Sarand I I et al.; The purpose of this study was to evaluate the influence of introduced bacteria containing a contaminant degrading plasmid on the growth and survival of pine seedlings and mycorrhizosphere microbial flora in contaminated soil . The Pseudomonas fluorescens strain OS81, originally isolated from fungal hyphae in contaminated soil, was supplied with the TOL plasmid pWW0::Km (to generate OS81(pWW0::Km)) and inoculated in humus-soil microcosms with and without pine seedlings mycorrhized with Suillus bovinus . After 3 months of regular treatment with m-toluate (mTA) solutions, the introduced catabolic plasmid was found to be disseminated in the indigenous bacterial population of both mycorrhizosphere and soil uncolonized by the fungus . Transconjugants were represented by bacteria of the genera Pseudomonas and Burkholderia and their number correlated positively with the concentration of mTA applied . Indigenous mTA degrading bacteria with low similarity to Burkholderia species were also enriched in microcosms . They were mostly associated with mycorrhizal soil or fungal structures and virtually absent in microcosms without pines . The total number of Tol(+) bacteria was higher in mycorrhizospheric soil compared with bulk soil . Inoculation with P . fluorescens OS81(pWW0::Km) had a positive effect on the development of roots and fungus in contaminated soil . Both inoculation with the P . fluorescens OS81(pWW0::Km) and mTA contamination as well as the presence of mycorrhized pine roots and fungal hyphae had an effect on the microbial community structure of soil as measured by carbon source oxidation patterns . However, the impact of mTA on the microbial community was more prominent . The study indicates that an effect on plant and fungal development can be obtained by manipulating the mycorrhizosphere . Both introduction of the bacterium carrying the degradative plasmid and the plasmid itself are likely to have a positive effect not only on the organisms involved, but also on bioremediation of contaminated soil, a factor that was not directly monitored here.

Biosci Biotechnol Biochem, 1999 Nov, 63(11), 1927 - 33
Purification and properties of extracellular carboxyl proteases of acid-tolerant bacteria, isolated from compost; Takehana T et al.; Four strains of acid-tolerant and protein-using bacteria were isolated from compost . Two of them, Gram-negative strains MB8 and MB11, were identified as a new genus close to Stenotrophomonas species MB8 and Burkholderia species MB11, respectively . Both bacteria produced extracellular carboxyl proteases (CP) in acid-casein-starch medium . The enzymes, termed CP MB8 and CP MB11, purified through ion exchange and gel filtration chromatographies had molecular weights of 61,000 (CP MB8) and 36,000 (CP MB11) as estimated by SDS-PAGE, and showed optimum activities with hemoglobin as a substrate at pH 3.5 (CP MB8) and pH 3.7 (CP MB11) at 55 degrees C . Both of the enzymes were not inhibited by pepstatin, DAN, or EPNP . These results suggest that both enzymes are members of the pepstatin-insensitive carboxyl proteinase family (EC 3.4.23.33), except for a larger molecular weight of the CP MB8 enzyme.

J Bacteriol, 2000 Jan, 182(2), 295 - 302
In vitro analysis of roles of a disulfide bridge and a calcium binding site in activation of Pseudomonas sp . strain KWI-56 lipase; Yang J et al.; The expression of lipase from Pseudomonas sp . strain KWI-56 (recently reclassified as Burkholderia cepacia) had been found to be dependent on an activator gene (act) downstream of its structural gene (lip) . In this work, the mature lipase was synthesized in an enzymatically active form with a cell-free Escherichia coli S30 coupled transcription-translation system by expressing a recombinant lipase gene (rlip) encoding the mature lipase in the presence of its purified activator or by coexpression of rlip and act . The in vitro expression systems were used for studying the folding process of the lipase . The addition of dithiothreitol in the expression systems decreased the activity dramatically without affecting the synthesis level of the lipase, whereas the in vitro-synthesized active lipase was relatively stable even in the presence of dithiothreitol . This phenomenon was further investigated by constructing mutant lipase genes only in vitro by PCR without gene cloning . Replacements of cysteine residues (Cys190 and Cys270) forming a sole putative disulfide bond to serine residues decreased the lipase activity greatly, suggesting that the disulfide bond was essential for the proper folding of the lipase . In addition, replacing Asp242 and Asp288, which were deduced to be part of a Ca(2+) binding site, also greatly decreased the activities of the in vitro-synthesized lipases . The role of the Ca(2+) binding site in the activation of the lipase is also discussed.

Microbiology, 1999 Dec, 145 ( Pt 12), 3465 - 75
Intracellular survival of Burkholderia cepacia complex isolates in the presence of macrophage cell activation; Saini LS et al.; Strains of the Burkholderia cepacia complex have emerged as a serious threat to patients with cystic fibrosis due to their ability to infect the lung and cause, in some patients, a necrotizing pneumonia that is often lethal . It has recently been shown that several strains of the B . cepacia complex can escape intracellular killing by free-living amoebae following phagocytosis . In this work, the ability of two B . cepacia complex strains to resist killing by macrophages was explored . Using fluorescence microscopy, electron microscopy and a modified version of the gentamicin-protection assay, we demonstrate that B . cepacia CEP021 (genomovar VI), and Burkholderia vietnamiensis (previously B . cepacia genomovar V) CEP040 can survive in PU5-1.8 murine macrophages for a period of at least 5 d without significant bacterial replication . Furthermore, bacterial entry into macrophages stimulated production of tumour necrosis factor and primed them to release toxic oxygen radicals following treatment with phorbol myristoyl acetate . These effects were probably caused by bacterial LPS, as they were blocked by polymyxin B . Infected macrophages primed with interferon gamma produced less nitric oxide than interferon-gamma-primed uninfected cells . We propose that the ability of B . cepacia to resist intracellular killing by phagocytic cells may play a role in the pathogenesis of cystic fibrosis lung infection . Our data are consistent with a model where repeated cycles of phagocytosis and cellular activation without bacterial killing may promote a deleterious inflammatory response causing tissue destruction and decay of lung function.

J Microbiol Immunol Infect, 1998 Jun, 31(2), 137 - 40
{Septicemic melioidosis in Southern Taiwan: a case report}; Tsai WC et al.; The patient was a 56 year-old man, a resident of Hen-Tsueng Township in Ping-Tung County . He worked as a ranger at Ken-Ting Farm in southern Taiwan, and had been to Thailand for sight-seeing 5 years ago . He came to our outpatient department about one month prior to hospitalization for intermittent fever of one week duration . At that time, complete blood count was within normal limits and a chest roentgenogram was unremarkable . He was given erythromycin without showing any clinical improvement . Two days prior to admission, he noted pain in the left hip . The next day, severe dyspnea developed suddenly and chest x-ray film revealed bilateral nodular lesions . Physical examination on hospitalization revealed an acutely ill and jaundiced male with a temperature of 37.4 degrees C, blood pressure: 110/47 mmHg, pulse rate: 137/min, and respiratory rate: 26/min . There were rales in both lungs, but no lymphadenopathy or organ enlargement . Laboratory study showed WBC: 1,470/mm(3), platelet count: 47,000/mm(3), blood sugar: 226 mg/dL, mildly elevated transaminases and bilirubin, and BUN: 69 mg/dL, Cr: 4.3 mg/dL . Arterial blood gas analysis indicated an acute metabolic acidosis with PaO2 of 32 mmHg . Despite the initial impression of melioidosis and administration of ceftazidime plus gentamicin, his condition rapidly deteriorated and expired 18 hours after admission . Two sets of blood cultures grew Burkholderia pseudomallei . Melioidosis has been called a great imitator of diseases and culture results are frequently misinterpreted . The mortality is high even with suggested therapy with ceftazidime, cotrimoxazole, amoxicillin-clavulanate, chloramphenicol, and/or tetracycline . There has been a total of 10 cases reported in southern Taiwan and 2 of them were clearly indigenous . Melioidosis should be included in the reportable diseases, and its prevalence in Taiwan also should be investigated.

Clin Diagn Lab Immunol, 2000 Jan, 7(1), 86 - 90
Detection of Francisella tularensis in biological specimens using a capture enzyme-linked immunosorbent assay, an immunochromatographic handheld assay, and a PCR; Grunow R et al.; The early detection of Francisella tularensis, the causative agent of tularemia, is important for adequate treatment by antibiotics and the outcome of the disease . Here we describe a new capture enzyme-linked immunosorbent assay (cELISA) based on monoclonal antibodies specific for lipopolysaccharide (LPS) of Francisella tularensis subsp . holarctica and Francisella tularensis subsp . tularensis . No cross-reactivity with Francisella tularensis subsp . novicida, Francisella philomiragia, and a panel of other possibly related bacteria, including Brucella spp., Yersinia spp., Escherichia coli, and Burkholderia spp., was observed . The detection limit of the assay was 10(3) to 10(4) bacteria/ml . This sensitivity was achieved by solubilization of the LPS prior to the cELISA . In addition, a novel immunochromatographic membrane-based handheld assay (HHA) and a PCR, targeting sequences of the 17-kDa protein (TUL4) gene of F . tularensis, were used in this study . Compared to the cELISA, the sensitivity of the HHA was about 100 times lower and that of the PCR was about 10 times higher . All three techniques were successfully applied to detect F . tularensis in tissue samples of European brown hares (Lepus europaeus) . Whereas all infected samples were recognized by the cELISA, those with relatively low bacterial load were partially or not detected by PCR and HHA, probably due to inhibitors or lack of sensitivity . In conclusion, the HHA can be used as a very fast and simple approach to perform field diagnosis to obtain a first hint of an infection with F . tularensis, especially in emergent situations . In any suspect case, the diagnosis should be confirmed by more sensitive techniques, such as the cELISA and PCR.

J Clin Microbiol, 2000 Jan, 38(1), 282 - 5
Species-specific PCR as a tool for the identification of Burkholderia gladioli; Whitby PW et al.; Burkholderia gladioli colonizes the respiratory tracts of patients with cystic fibrosis and chronic granulomatous disease . However, due to the high degree of phenotypic similarity between this species and closely related species in the Burkholderia cepacia complex, accurate identification is difficult . Incorrect identification of these species may have serious repercussions for the management of patients with cystic fibrosis . To develop an accurate procedure for the identification of B . gladioli, a molecular method to discriminate between this species and other species commonly isolated from the sputa of patients with cystic fibrosis was investigated . The 23S ribosomal DNA was cloned from several clinical isolates of B . gladioli, and the nucleotide sequence was determined . Computer-assisted sequence comparisons indicated four regions of the 23S rRNA specific for this species; these regions were used to design three primer pairs for species-specific PCR . Two of the primer pairs showed 100% sensitivity and specificity for B . gladioli when tested against a panel of 47 isolates comprising 19 B . gladioli isolates and 28 isolates of 16 other bacterial species . One of the primer pairs was further assessed for species specificity by using a panel of 102 isolates obtained from the Burkholderia cepacia Research Laboratory and Repository . The species-specific PCR was positive for 70 of 74 isolates of B . gladioli and was negative for all other bacterial species examined . Overall, this primer pair displayed a sensitivity and specificity of 96% (89 of 93) and 100%, respectively . These data demonstrate the potential of species-specific PCR for the identification of B . gladioli.

Appl Environ Microbiol, 2000 Jan, 66(1), 290 - 6
Detection and characterization of plasmid pJP4 transfer to indigenous soil bacteria; Newby DT et al.; Prior to gene transfer experiments performed with nonsterile soil, plasmid pJP4 was introduced into a donor microorganism, Escherichia coli ATCC 15224, by plate mating with Ralstonia eutropha JMP134 . Genes on this plasmid encode mercury resistance and partial 2, 4-dichlorophenoxyacetic acid (2,4-D) degradation . The E . coli donor lacks the chromosomal genes necessary for mineralization of 2,4-D, and this fact allows presumptive transconjugants obtained in gene transfer studies to be selected by plating on media containing 2,4-D as the carbon source . Use of this donor counterselection approach enabled detection of plasmid pJP4 transfer to indigenous populations in soils and under conditions where it had previously not been detected . In Madera Canyon soil, the sizes of the populations of presumptive indigenous transconjugants were 10(7) and 10(8) transconjugants g of dry soil(-1) for samples supplemented with 500 and 1,000 microg of 2,4-D g of dry soil(-1), respectively . Enterobacterial repetitive intergenic consensus PCR analysis of transconjugants resulted in diverse molecular fingerprints . Biolog analysis showed that all of the transconjugants were members of the genus Burkholderia or the genus Pseudomonas . No mercury-resistant, 2, 4-D-degrading microorganisms containing large plasmids or the tfdB gene were found in 2,4-D-amended uninoculated control microcosms . Thus, all of the 2,4-D-degrading isolates that contained a plasmid whose size was similar to the size of pJP4, contained the tfdB gene, and exhibited mercury resistance were considered transconjugants . In addition, slightly enhanced rates of 2,4-D degradation were observed at distinct times in soil that supported transconjugant populations compared to controls in which no gene transfer was detected.

J Bacteriol, 2000 Jan, 182(1), 135 - 42
Identification of a new class of 5'-adenylylsulfate (APS) reductases from sulfate-assimilating bacteria; Bick JA et al.; A gene was cloned from Burkholderia cepacia DBO1 that is homologous with Escherichia coli cysH encoding 3'-phosphoadenylylsulfate (PAPS) reductase . The B . cepacia gene is the most recent addition to a growing list of cysH homologs from a diverse group of sulfate-assimilating bacteria whose products show greater homology to plant 5'-adenylylsulfate (APS) reductase than they do to E . coli CysH . The evidence reported here shows that the cysH from one of the species, Pseudomonas aeruginosa, encodes APS reductase . It is able to complement an E . coli cysH mutant and a cysC mutant, indicating that the enzyme is able to bypass PAPS, synthesized by the cysC product . Insertional knockout mutation of P . aeruginosa cysH produced cysteine auxotrophy, indicating its role in sulfate assimilation . Purified P . aeruginosa CysH expressed as a His-tagged recombinant protein is able to reduce APS, but not PAPS . The enzyme has a specific activity of 5.8 micromol . min(-1) . mg of protein(-1) at pH 8.5 and 30 degrees C with thioredoxin supplied as an electron donor . APS reductase activity was detected in several bacterial species from which the novel type of cysH has been cloned, indicating that this enzyme may be widespread . Although an APS reductase from dissimilatory sulfate-reducing bacteria is known, it shows no structural or sequence homology with the assimilatory-type APS reductase reported here . The results suggest that the dissimilatory and assimilatory APS reductases evolved convergently.

Pediatr Pulmonol, 2000 Jan, 29(1), 8 - 10
Pulmonary function, serum markers of inflammation, and IgG antibodies to core lipopolysaccharide of Burkholderia cepacia in adults with cystic fibrosis, following colonization with Burkholderia cepacia; Hendry J et al.; Eight patients with cystic fibrosis {CF} colonized with Pseudomonas aeruginosa (P . aeruginosa) had serial lung function, peripheral blood inflammatory markers, and serum IgG antibodies to Burkholderia cepacia (B . cepacia) lipopolysaccharide measured in the months preceding and following colonisation with B . cepacia . One patient experienced a fall in FEV(1) from 33% to 19% of predicted values, coinciding with the first sputum isolation of B . cepacia, and he died 12 weeks later . He had a rise in inflammatory markers preterminally, and this change was refractory to antibiotic therapy . There was no significant fall in FEV(1) % of predicted values in the remaining seven patients, and no significant changes in their serum markers of inflammation following colonization with B . cepacia over a median (range) period of 10.9 (7.3-12.0) months .

Respirology, 1999 Dec, 4(4), 419 - 22
An exotic pulmonary infection in Thailand: melioidosis; Maneechotesuwan K; Melioidosis is an infectious disease from Burkholderia pseudomallei and is confined in specific geographic areas such as Southeast Asia . Its highest prevalence in Thailand is in the north-eastern part . Most infected patients had worked paddy fields or had underlying diseases such as diabetes mellitus . Melioidosis can manifest clinically, with either disseminated or localized features . In the disseminated form patients developed an acute and progressive course with septicaemia . In contrast, patients with the localized form usually presented with prolonged fever, and symptoms of one or more organ involvement, in particular the lung and the liver . Definite diagnosis of melioidosis is made by an isolation of Burkholderia pseudomallei from a variety of clinical specimens . Treatment of choice for the septicaemic patients is an initial combination of ceftrazidime and trimethoprime-sulfamethoxazole, followed by trimethoprime-sulfamethoxazole for up to 6-12 months depending on the result of clinical specimen culture . Treatment for the localized form requires simultaneous antibiotic therapy and surgical drainage . However, optimum duration of antibiotic therapy remains unknown so further research is required . Melioidosis is an important disease in terms of mortality rate and it requires rapid diagnosis and treatment . To prevent recurrence, it is necessary to continue oral doxycycline or trimethoprime-sulfamethoxazole for 6-12 months.

Appl Microbiol Biotechnol, 1999 Sep, 52(3), 440 - 5
Continuous degradation of mixtures of 4-nitrobenzoate and 4-aminobenzoate by immobilized cells of Burkholderia cepacia strain PB4; Peres CM et al.; Although isolated on 4-aminobenzoate, Burkholderia cepacia strain PB4 is also able to grow on 4-nitrobenzoate . Degradation of an equimolar mixture of the nitroaromatic compound 4-nitrobenzoate and its corresponding aminoaromatic derivative 4-aminobenzoate by this strain was investigated . Batch experiments showed that, irrespective of preculturing conditions, both compounds were degraded simultaneously . The mixture-degrading ability of B . cepacia strain PB4 was subsequently tested in continuous packed bed reactors (PBR) with the strain immobilized on Celite grade R-633 or R-635 . Higher degradation rates were achieved with the larger particles of Celite R-635 . Maximum simultaneous degradation rates per liter of packed bed of 0.925 mmol 1(-1) h(-1) 4-nitrobenzoate and 4-aminobenzoate were obtained for an applied loading rate of the same value (0.925 mmol 1(-1) h(-1) of each compound) . Even when the applied load was not removed in its entirety, neither of the two compounds was degraded preferentially but a percentage of both of them was mineralized . The present study shows the possibility for a pure strain to biodegrade not only a nitroaromatic compound (4-nitrobenzoate) but also its corresponding amino derivative (4-aminobenzoate) continuously and simultaneously.

Microbiol Immunol, 1999, 43(11), 995 - 1001
Stable marker on flagellin gene sequences related to arabinose non-assimilating pathogenic Burkholderia pseudomallei; Wajanarogana S et al.; Using PCR-based isolation and sequence analysis of the flagellin gene from two distinct biotypes of Burkholderia pseudomallei, a 15-bp deletion was found within the variable domain of the gene in isolates capable of assimilating arabinose (Ara+) . This finding led to the development of a PCR-based method in order to differentiate and identify pathogenic B . pseudomallei for epidemiological study . A pair of specific primers was designed covering the 15-bp deletion region at the variable domain . PCR-amplification products of 176 and 191 bp in size were detected from 41 Ara+ isolates and 39 Ara - isolates of B . pseudomallei, respectively . Moreover, flagellin gene fragments of other bacterial species tested in this study were not amplified using these primers . The results suggest that the flagellin gene sequences of both B . pseudomallei biotypes in this region are stable and distinct . This method can be applied and useful for the epidemiological study of B . pseudomallei.

Infect Immun, 2000 Jan, 68(1), 24 - 9
Invasion and intracellular survival of Burkholderia cepacia; Martin DW et al.; Burkholderia cepacia has emerged as an important pulmonary pathogen in immunocompromised patients and in patients with cystic fibrosis (CF) . Little is known about the virulence factors and pathogenesis of B . cepacia, although the persistent and sometimes invasive infections caused by B . cepacia suggest that the organism possesses mechanisms for both cellular invasion and evasion of the host immune response . In this study, cultured human cells were used to analyze the invasion and intracellular survival of B . cepacia J2315, a highly transmissible clinical isolate responsible for morbidity and mortality in CF patients . Quantitative invasion and intracellular growth assays demonstrated that B . cepacia J2315 was able to enter, survive, and replicate intracellularly in U937-derived macrophages and A549 pulmonary epithelial cells . Transmission electron microscopy of infected macrophages confirmed the presence of intracellular B . cepacia and showed that intracellular bacteria were contained within membrane-bound vacuoles . An environmental isolate of B . cepacia, strain J2540, was also examined for its ability to invade and survive intracellularly in cultured human cells . J2540 entered cultured macrophages with an invasion frequency similar to that of the clinical strain, but it was less invasive than the clinical strain in epithelial cells . In marked contrast to the clinical strain, the environmental isolate was unable to survive or replicate intracellularly in either cultured macrophages or epithelial cells . Invasion and intracellular survival may play important roles in the ability of virulent strains of B . cepacia to evade the host immune response and cause persistent infections in CF patients.

Immunopharmacology, 1999 Nov, 44(3), 267 - 72
Burkholderia cepacia is resistant to the antimicrobial activity of airway epithelial cells; Baird RM et al.; There has been much interest recently in the antimicrobial properties of cationic peptides called beta-defensins from epithelial cells . Human beta-defensin (hBD)-1 and -2 have been particularly implicated in cystic fibrosis (CF) patients, where their inhibition by high salt concentrations may explain in part the susceptibility of the CF lung to bacterial infection . In this work, we have employed a simple co-culture system using the 16-HBE human bronchial epithelial cell line to assess growth inhibitory activity against Pseudomonas aeruginosa and Burkholderia cepacia . In medium alone, P . aeruginosa proliferated more than 100,000-fold, whereas in the presence of 16-HBE cells or 16-HBE-conditioned medium, bacterial proliferation was less than 100-fold . Raising the salt concentration of cell-free 16-HBE conditioned medium to approximately 200 mM significantly reduced this growth inhibitory activity . In contrast, there was no evidence of epithelial-derived growth inhibitory activity against two strains of B . cepacia . RT-PCR analysis indicated expression of the hBD-2 mRNA in 16-HBE cells, but not hBD-1 . These data demonstrate for the first time that B . cepacia is resistant to epithelial-derived antimicrobial substances and argue against them being important in the defense against this organism in the lung.

Microbes Infect, 1999 Feb, 1(2), 157 - 62
Current studies on the pathogenesis of melioidosis; Woods DE et al.; Burkholderia pseudomallei is a major cause of bacterial septicemias in many parts of the world, particularly Thailand; the known geographic range of the organism appears to be enlarging as awareness of the organism and the disease it causes--melioidosis--increases . B . pseudomallei is intrinsically resistant to most antibiotics, and our knowledge of B . pseudomallei pathogenesis is lacking . Thus, the long-term objective of our research is to define at a molecular level the pathogenesis by combining genetic, immunologic, and biochemical approaches with animal model studies . Basic studies on B . pseudomallei pathogenesis are acutely needed to provide a knowledge base to rationally design new modes of therapy directed against this organism.

Phytother Res, 1999 Dec, 13(8), 675 - 9
Antibacterial activities of extracts from Nigerian chewing sticks; Taiwo O et al.; Ten aqueous extracts from wooden chewing sticks widely used in Nigeria for teeth cleaning were studied for antibacterial activities against 25 different bacteria using an agar diffusion assay . The extracts from five sticks, namely Garcinia kola, Anogeissus leiocarpus, Terminalia glaucescens, Sorindeia warneckei and Vitex doniana, exhibited strong activities against a wide spectrum of bacteria including medically and dentally relevant bacteria . Notably, these five chewing stick extracts showed potent activities against methicillin-resistant Staphylococcus aureus, vancomycin-resistant Enterococcus, and multidrug-resistant Burkholderia cepacia and Pseudomonas aeruginosa . Extracts from Vernonia amygdalina, Fagara zanthoxyloides and Massularia acuminata also showed activities against bacteria significant to periodontal disease . Methanol extracts prepared from G . kola, A . leiocarpus and V . doniana were further fractionated by solvent extraction . Results showed that the antibacterial activities were distributed into different fractions suggesting that the sticks contain different active antibacterial principles . In conclusion, the results showed that most of the Nigerian chewing sticks do contain antibacterial activities which may contribute to the reported anticaries effect of chewing sticks . These sticks may be sources for new lead antibacterial agents for therapeutic or preventive applications .

Microb Ecol, 1999 Oct, 38(3), 273 - 284
Soil Type and Maize Cultivar Affect the Genetic Diversity of Maize Root-Associated Burkholderia cepacia Populations; Dalmastri C et al.; Abstract Burkholderia cepacia populations associated with the Zea mays root system were investigated to assess the influence of soil type, maize cultivar, and root localization on the degree of their genetic diversity . A total of 180 B . cepacia isolates were identified by restriction analysis of the amplified 16S rDNA (ARDRA technique) . The genetic diversity among B . cepacia isolates was analyzed by the random amplified polymorphic DNA (RAPD) technique, using the 10-mer primer AP5 . The analysis of molecular variance (AMOVA) method was applied to estimate the variance components for the RAPD patterns . The results indicated that, among the factors studied, the soil was clearly the dominant one in affecting the genetic diversity of maize root-associated B . cepacia populations . In fact, the percentage of variation among populations was significantly higher between B . cepacia populations recovered from maize planted in different soils than between B . cepacia populations isolated from different maize cultivars and from distinct root compartments such as rhizoplane and rhizosphere . The analysis of the genetic relationships among B . cepacia isolates resulted in dendrograms showing bacterial populations with frequent recombinations and a nonclonal genetic structure . The dendrograms were also in agreement with the AMOVA results . We were able to group strains obtained from distinct soils on the basis of their origin, confirming that soil type had the major effect on the degree of genetic diversity of the maize root-associated B . cepacia populations analyzed . On the other hand, strains isolated from distinct root compartments exhibited a random distribution which confirmed that the rhizosphere and rhizoplane populations analyzed did not significantly differ in their genetic structure.http://link.springer-ny.com/link/service/journals/00248/bibs/38n3p273.html</hea

Clin Infect Dis, 1999 Oct, 29(4), 813 - 8
Antilipopolysaccharide II: an antibody protective against fatal melioidosis; Charuchaimontri C et al.; This was a study of IgG antibody responses to two S-type lipopolysaccharides (LPS I and LPS II) and flagellin of Burkholderia pseudomallei in patients with melioidosis . The specificity of these antibodies was 91.7%, 90.3%, and 93.8%, respectively, when compared to responses in a population where the organism is not endemic . Only the level of antibody to LPS II (anti-LPS II) was significantly higher in patients who survived than in those who died, as well as in patients with nonsepticemic vs . septicemic melioidosis . Results of logistic regression analysis, controlled for confounding factors such as duration of illness before treatment and bacteremic status, confirmed that a high level of anti-LPS II was a significant factor protective against fatal melioidosis . Thus, LPS II of B . pseudomallei would be a potentially useful component of a vaccine developed against fatal melioidosis . Further studies are in progress to determine the level of this antibody among those with asymptomatic infection in areas where melioidosis is endemic.

Am J Trop Med Hyg, 1999 Nov, 61(5), 735 - 7
Short report: evaluation of a monoclonal antibody-based latex agglutination test for rapid diagnosis of septicemic melioidosis; Samosornsuk N et al.; A monoclonal antibody (MAb)-based latex agglutination (MAb-LA) test was developed to rapidly identify Burkholderia pseudomallei in hemoculture of patients with septicemic melioidosis . The method was evaluated in a clinical situation on 396 hemocultures positive for bacterial growth, of which 75 cultures were positive for B . pseudomallei by conventional biochemical tests . The sensitivity and specificity of the MAb-LA test were 95% and 100%, respectively . The positive and negative predictive values were 100% and 99% . The method is highly reliable and suitable for rapid diagnosis of septicemic melioidosis, reducing the time normally required from a minimum of 3-4 days by conventional methods to less than 30 hr . Most of these 30 hr are involved in growing up enough bacteria to perform the MAb-LA test, which itself takes only 1 min.

Biometals, 1999 Sep, 12(3), 265 - 74
Sequence heterogeneity of the ferripyoverdine uptake (fpvA), but not the ferric uptake regulator (fur), genes among strains of the fluorescent pseudomonads Pseudomonas aeruginosa, Pseudomonas aureofaciens, Pseudomonas fluorescens and Pseudomonas putida; Thupvong T et al.; Pseudomonas aeruginosa, Pseudomonas aureofaciens, Pseudomonas fluorescens and Pseudomonas putida are of importance to medicine, agriculture and biocycling . These microbes acquire ferric ion via the use of the siderophores pyochelin and the family known as the pyoverdines or pseudobactins . The ferric uptake regulator (fur) gene is responsible, at least in part, for the regulation of siderophore synthesis and uptake in P . aeruginosa . To determine whether the organisms contain single or multiple homologues of the siderophore-related genes fpvA (ferripyoverdine uptake) and fur, and whether these homologues displayed sequence heterogeneity, their chromosomal DNAs were probed with fur and fpvA sequences . As a representative of a non-fluorescent pseudomonad, the bacterium Burkholderia (Pseudomonas) cepacia was also examined . The pseudomonads all contained fpvA- and fur-like homologues, and heterogeneity was observed among the different species . The presence of two or more fpvA-like genes is indicated in all of the fluorescent pseudomonads surveyed . In contrast, B . cepacia DNA either did not hybridize to these probes, or did so only very weakly, suggesting that fur- and fpvA-like homologues are either absent or significantly different in B . cepacia compared to the fluorescent pseudomonads examined.

Respiration, 1999 Nov-Dec, 66(6), 551 - 4
Successful treatment with methylprednisolone pulse therapy for a life-threatening pulmonary insufficiency in a patient with chronic granulomatous disease following pulmonary invasive aspergillosis and Burkholderia cepacia infection; Okano M et al.; A 14-year-old boy with X-linked chronic granulomatous disease developed severe invasive pulmonary aspergillosis . He was treated with itraconazole and amphotericin B . However, he deteriorated with progressive pulmonary lesions . Burkholderia cepacia was isolated from his bronchoalveolar lavage . Finally, he was given granulocyte transfusions . Following this procedure, his condition rapidly worsened leading to respiratory failure . His lung biopsy demonstrated organizing pneumonia at his right middle lobe . Then, a methylprednisolone pulse therapy was initiated together with the administration of appropriate antibiotics and adequate amounts of amphotericin B . Dramatically, his condition improved . Therefore, a methylprednisolone pulse therapy with appropriate antimicrobial drugs seems to be beneficial for severe pulmonary insufficiency in this type of patients . Copyright

J Infect Dis, 1999 Dec, 180(6), 1878 - 85
Elevated plasma concentrations of interferon (IFN)-gamma and the IFN-gamma-inducing cytokines interleukin (IL)-18, IL-12, and IL-15 in severe melioidosis; Lauw FN et al.; Interferon (IFN)-gamma plays an important role in the pathogenesis of sepsis . Production of IFN-gamma is stimulated by synergistic effects of interleukin (IL)-18, IL-12, and IL-15 . To investigate the regulation of IFN-gamma production during severe gram-negative infection, the plasma concentrations of IFN-gamma, IL-18, IL-12, and IL-15 were measured in 83 patients with suspected melioidosis . The diagnosis was confirmed in 62 patients, 31 of whom had blood cultures positive for Burkholderia pseudomallei, of whom 12 died . Compared with healthy controls, patients had elevated levels of IFN-gamma, IL-18, IL-12p40, and IL-15 on admission, with significantly higher levels in blood culture-positive patients, and these levels remained elevated during the 72-h study period . In whole blood stimulated with heat-killed B . pseudomallei, anti-IL-12 had a stronger inhibitory effect than anti-IL-18 and anti-IL-15 on IFN-gamma production . This effect of anti-IL-12 was further enhanced by anti-IL-18 . These data suggest that during gram-negative sepsis, IFN-gamma production is controlled at least in part by endogenous IL-18, IL-12, and IL-15.

J Clin Microbiol, 1999 Dec, 37(12), 3809 - 14
Molecular epidemiological investigation using a randomly amplified polymorphic DNA assay of Burkholderia cepacia isolates from nosocomial outbreaks; Okazaki M et al.; We experienced two Burkholderia cepacia outbreaks over a 1-year period . During this period, 28 B . cepacia isolates were obtained from clinical specimens, and 2 were obtained from environmental specimens (i.e., from a nebulizer solution and a nebulizer tube) . These 30 isolates were subjected to the PCR-based randomly amplified polymorphic DNA (RAPD) assay as well as to pulsed-field gel electrophoresis (PFGE) . In the first outbreak, in which eight patients hospitalized in the Trauma and Critical Care Center were involved, the RAPD assay revealed that all 20 isolates obtained from clinical specimens and the 2 isolates from environmental specimens had identical DNA profiles . These RAPD data enabled us to pinpoint a possible source and to take countermeasures to prevent further spread of the epidemic-causing strain . In the second outbreak, two consecutive B . cepacia infection/colonization cases were seen in the surgery ward . The RAPD profiles of four isolates obtained were again identical, but they were distinct from those seen in the first outbreak, clearly indicating that the second outbreak was not related to the first . Thus, our experience demonstrated that the RAPD assay is a useful and reliable tool for epidemiological studies of B . cepacia isolates from nosocomial outbreaks . Since the RAPD assay could provide discriminatory potential and reproducibility comparable to those of the widely used PFGE assay with less complexity and in a shorter time, the introduction of the RAPD assay into hospital microbiology laboratories as a routine technique may help prevent nosocomial outbreaks.

Am J Respir Crit Care Med, 1999 Nov, 160(5 Pt 1), 1572 - 7
Burkholderia cepacia in cystic fibrosis . Variable disease course; Frangolias DD et al.; Variable clinical course has been reported with the acquisition of Burkholderia cepacia in patients who have cystic fibrosis (CF) . We hypothesized that the perceived worsening with B . cepacia may reflect the underlying severity of pulmonary disease at the time of acquisition . To test this hypothesis, we matched CF patients colonized with B . cepacia with CF patients not colonized with the organism . Two-year pre- and postacquisition data and long-term data were compared . Patients were matched for gender, age (+/- 1 yr), height (+/- 5 cm), weight (+/- 8 kg), percent predicted forced expiratory volume in one second (% pred FEV(1)) (+/- 10%), and pancreatic sufficiency status . Differences in rates of change pre- and postacquisition for FEV(1), FVC, weight, and frequency of intravenous courses were compared within pairs with the Wilcoxon signed rank test . Two-year and long-term survival was compared within pairs with the McNemar test . No significant differences were observed in mean annual rates of change in weight (0.33 and -0.28 kg/yr), % pred FEV(1) (-0.36 and -1.74%/yr), and percent predicted forced vital capacity (% pred FVC) (-3.80 and -2.32%/yr) between B . cepacia and control pairs in 2-yr and long-term postacquisition interval, respectively . Similar rates of change were noted for pre- to postacquisition intervals within pairs for weight (0.17 kg/yr), % pred FEV(1) (-0.16%/yr), % pred FVC (5.02 %/yr) . There was a significantly higher rate of intravenous antibiotic courses in B . cepacia cases in the 2-yr and long-term postacquisition interval . Higher mortality was observed in the B . cepacia cases in the long term (p < 0.05) . We conclude that colonization with B . cepacia does not necessarily adversely affect pulmonary status, but is associated with reduced long term survival . Whereas previous associations may be attributed to a propensity to colonize those who had more advanced disease, specific strain types of B . cepacia may have enhanced pathogenicity.

Int J Syst Bacteriol, 1999 Oct, 49 Pt 4, 1657 - 66
Identification of Burkholderia species and genomovars from cystic fibrosis patients by AFLP fingerprinting; Coenye T et al.; AFLP is a genomic fingerprinting technique based on the selective amplification of restriction fragments from a total double-digest of genomic DNA . The applicability of this method to differentiate between species and genomovars of the genus Burkholderia was tested, with particular emphasis on taxa occurring in cystic fibrosis patients . In this study, 78 well-characterized strains and field isolates were investigated by two methods of AFLP fingerprinting . In the manual procedure, a radioactively labelled primer was used, amplified fragments were separated by conventional PAGE and the patterns were revealed by autoradiography . In the automated procedure, a fluorescently labelled primer was used in combination with electrophoresis and on-line data collection by means of an automated DNA sequencer . Overall, there was good agreement between the two AFLP procedures and the data were mostly consistent with results obtained from SDS-PAGE of whole-cell proteins and DNA-DNA hybridization experiments . The automated AFLP procedure has considerable technical advantages compared with the manual AFLP procedure, but a thorough visual analysis of the DNA profiles was required to avoid misidentification of some Burkholderia cepacia genomovar III strains.

Am J Trop Med Hyg, 1999 Oct, 61(4), 658 - 62
Rapid identification of Burkholderia pseudomallei in blood cultures by latex agglutination using lipopolysaccharide-specific monoclonal antibody; Dharakul T et al.; Melioidosis, an infection caused by Burkholderia pseudomallei, is endemic in Southeast Asia . The septicemic form of melioidosis is the leading cause of death due to community-acquired bacteremia in the northeastern part of Thailand . The delay in isolation and identification of the causative organism is a major contributing factor to the high mortality . The present study describes the evaluation of a latex agglutination test for rapid identification of the bacteria directly from blood cultures . The Bps-L1 monoclonal antibody recognized the lipopolysaccharide antigen of 96.8% of B . pseudomallei clinical isolates and was highly specific for B . pseudomallei . The diagnostic value of the latex agglutination test based on Bps-L1 monoclonal antibody was prospectively evaluated in an area endemic for melioidosis . The agglutination test kit was evaluated in 88 blood cultures with gram-negative bacteria identified with Gram staining . The sensitivity and specificity of the test kit were both 100% . These results indicated that the detection of B . pseudomallei lipopolysaccharide by specific monoclonal antibody in a latex agglutination format is clinically useful for the rapid identification of the bacteria in blood cultures in areas endemic for melioidosis.

FEMS Microbiol Lett, 1999 Nov 1, 180(1), 39 - 44
Conservation of the pyrrolnitrin biosynthetic gene cluster among six pyrrolnitrin-producing strains; Hammer PE et al.; The prnABCD gene cluster from Pseudomonas fluorescens encodes the biosynthetic pathway for pyrrolnitrin, a secondary metabolite derived from tryptophan which has strong anti-fungal activity . We used the prn genes from P . fluorescens strain BL915 as a probe to clone and sequence homologous genes from three other Pseudomonas strains, Burkholderia cepacia and Myxococcus fulvus . With the exception of the prnA gene from M . fulvus59% similar among the strains, indicating that the biochemical pathway for pyrrolnitrin biosynthesis is highly conserved . The prnA gene from M . fulvus is about 45% similar to prnA from the other strains and contains regions which are highly conserved among all six strains.

Antimicrob Agents Chemother, 1999 Nov, 43(11), 2773 - 5
In vitro susceptibilities of Burkholderia mallei in comparison to those of other pathogenic Burkholderia spp; Kenny DJ et al.; The in vitro antimicrobial susceptibilities of isolates of Burkholderia mallei to 16 antibiotics were assessed and compared with the susceptibilities of Burkholderia pseudomallei and Burkholderia cepacia . The antibiotic susceptibility profile of B . mallei resembled that of B . pseudomallei more closely than that of B . cepacia, which corresponds to their similarities in terms of biochemistry, antigenicity, and pathogenicity . Ceftazidime, imipenem, doxycycline, and ciprofloxacin were active against both B . mallei and B . pseudomallei . Gentamicin was active against B . mallei but not against B . pseudomallei . Antibiotics clinically proven to be effective in the treatment of melioidosis may therefore be effective for treating glanders.

Antimicrob Agents Chemother, 1999 Nov, 43(11), 2648 - 56
Isolation of polymyxin B-susceptible mutants of Burkholderia pseudomallei and molecular characterization of genetic loci involved in polymyxin B resistance; Burtnick MN et al.; Burkholderia pseudomallei is a gram-negative bacterium that causes the disease known as melioidosis . This pathogen is endemic to Southeast Asia and northern Australia and is particularly problematic in northeastern Thailand . It has been previously reported that B . pseudomallei is resistant to the killing action of cationic antimicrobial peptides, including human neutrophil peptide, protamine sulfate, poly-L-lysine, magainins, and polymyxins . Recently, we have also found that the virulent clinical isolate B . pseudomallei 1026b is capable of replicating in media containing polymyxin B at concentrations of >100 mg/ml . In order to identify genetic loci that are associated with this particular resistance phenotype, we employed a Tn5-OT182 mutagenesis system in coordination with a replica plating screen to isolate polymyxin B-susceptible mutants . Of the 17,000 Tn5-OT182 mutants screened via this approach, five polymyxin B-susceptible mutants were obtained . Three of these mutants harbored Tn5-OT182 insertions within a genetic locus demonstrating strong homology to the lytB gene present in other gram-negative bacteria . Of the remaining two mutants, one contained a transposon insertion in a locus involved in lipopolysaccharide core biosynthesis (waaF), while the other contained an insertion in an open reading frame homologous to UDP-glucose dehydrogenase genes . Isogenic mutants were also constructed via allelic exchange and used in complementation analysis studies to further characterize the relative importance of each of the various genetic loci with respect to the polymyxin B resistance phenotype exhibited by B . pseudomallei 1026b.

Microbiology, 1999 Oct, 145 ( Pt 10), 2821 - 34
Occurrence and expression of glutathione-S-transferase-encoding bphK genes in Burkholderia sp . strain LB400 and other biphenyl-utilizing bacteria; Bartels F et al.; The gene bphK of Burkholderia sp . strain LB400 has previously been shown to be located within the bph locus, which specifies the degradation of biphenyl (BP) and chlorobiphenyls, and to encode a glutathione S-transferase (GST) which accepts 1-chloro-2,4-dinitrobenzene (CDNB) as substrate . The specific physiological role of this gene is not known . It is now shown that the gene is expressed in the parental organism and that GST activity is induced more than 20-fold by growth of the strain on BP relative to succinate when these compounds serve as sole carbon source . Approximately the same induction factor was observed for 2,3-dihydroxybiphenyl 1,2-dioxygenase activity, which is encoded by the 5'-adjacent bphC gene . This suggests that the expression of bphK is coregulated with the expression of genes responsible for the catabolism of BP . A bphK probe detected only a single copy of the gene in strain LB400 . A spontaneous BP- mutant of the organism neither gave a signal with the bphK probe nor showed CDNB-accepting GST activity, suggesting that this activity is solely encoded by bphK . Complementation of the mutant with a bph gene cluster devoid of bphK restored the ability to grow on BP, indicating that bphK is not essential for utilization of this carbon source . BphK activity proved to be almost unaffected by up to 100-fold differences in proton concentration or ionic strength . The enzyme showed a narrow range with respect to a variety of widely used electrophilic GST substrates, accepting only CDNB . A number of established laboratory strains as well as novel isolates able to grow on BP as sole carbon and energy source were examined for BphK activity and the presence of a bphK analogue . CDNB assays, probe hybridizations and PCR showed that several, but not all, BP degraders possess this type of GST activity and/or a closely related gene . In all bacteria showing BphK activity, this was induced by growth on BP as sole carbon source, although activity levels differed by up to 10-fold after growth on BP and by up to 60-fold after growth on succinate . This resulted in a variation of induction factors between 2 and 30 . In the majority of bphK+ bacteria examined, the gene appeared to be part of LB400-like bph gene clusters . DNA sequencing revealed almost complete identity of bphK genes from five different bph gene clusters . These results suggest that bphK genes, although not essential, fulfill a strain-specific function related to the utilization of BPs by their host organisms . The usefulness of BphK as a reporter enzyme for monitoring the expression of catabolic pathways is discussed.

Clin Infect Dis, 1999 Sep, 29(3), 490 - 4
Changing epidemiology of infections in patients with neutropenia and cancer: emphasis on gram-positive and resistant bacteria; Zinner SH; Over the past 3 decades, considerable changes have occurred in the types of bacteria causing infection in febrile patients with neutropenia and cancer . Twenty years ago, gram-negative bacteria caused approximately 70% of bloodstream infections . As a probable consequence of long-dwelling intravascular devices, fluoroquinolone prophylaxis, and high-dose chemotherapy-induced mucositis, there has been a shift toward gram-positive coccal bacteremia . In most centers today, approximately 70% of bacteremic isolates are gram-positive cocci . Of potential concern is that antimicrobial-resistant gram-positive organisms are becoming increasingly frequent in patients with neutropenia . Fluoroquinolone-resistant Escherichia coli are being isolated from several cancer centers . Several "new" organisms, such as Stomatococcus mucilaginosus, Bacillus cereus, Leuconostoc species, Corynebacterium jeikeium, Rhodococcus species, Stenotrophomonas maltophilia, Moraxella catarrhalis, Burkholderia cepacia, and Bartonella species, now cause infections in these patients . Careful application of infection-control principles, judicious prophylaxis, appropriate evaluation of new antibiotics, and prompt effective therapy will maximize benefits for these patients.

Microbiol Immunol, 1999, 43(7), 625 - 30
Differences in genomic macrorestriction patterns of arabinose-positive (Burkholderia thailandensis) and arabinose-negative Burkholderia pseudomallei; Chaiyaroj SC et al.; We reported previously two biochemically and antigenically distinct biotypes of Burkholderia pseudomallei . These two distinct biotypes could be distinguished by their ability to assimilate L-arabinose . Some B . pseudomallei isolated from soil samples could utilize this substrate (Ara+), whereas the other soil isolates and all clinical isolates could not (Ara-) . Only the Ara isolates were virulent in animals and reacted with monoclonal antibody directed at the surface envelope, most likely the exopolysaccharide component . In the present study, pulsed-field gel electrophoresis was employed for karyotyping of these previously identified B . pseudomallei strains . We demonstrate here that the DNA macrorestriction pattern allows the differentiation between B . pseudomallei, which can assimilate L-arabinose, and the proposed B . thailandensis, which cannot do so . Bacterial strains from 80 melioidosis patients and 33 soil samples were examined by genomic DNA digestion with NcoI . Two major reproducible restriction patterns were observed . All clinical (Ara-) isolates and 9 Ara- soil isolates exhibited macrorestriction pattern I (MPI), while 24 soil isolates (Ara+) from central and northeastern Thailand displayed macrorestriction pattern II (MPII) . The study here demonstrated pulsed-field gel electrophoresis to be a useful tool in epidemiological investigation possibly distinguishing virulent B . pseudomallei from avirulent B . thailandensis or even identifying closely related species of Burkholderia.

Curr Microbiol, 1999 Dec, 39(6), 336 - 0341
Regulation of expression of the nonhemolytic phospholipase C of Burkholderia cepacia; Weingart CL et al.; Burkholderia cepacia is an opportunistic pathogen that causes serious pulmonary infections in cystic fibrosis patients . We have purified and partially characterized one potential virulence factor for the organism-a nonhemolytic phospholipase C-and we studied the effect of iron restriction and choline and phosphate concentrations on the expression of phospholipase C . Iron limitation did not affect expression, the effect of choline was variable, and high phosphate concentrations repressed expression . Experiments with heat-treated spent culture supernatants suggested that autoinducers affected the expression of the phospholipase and two other potential virulence factors, a protease and a lipase . We screened 26 B . cepacia isolates for autoinducer activity: 11 induced violacein production in the autoinducer-deficient mutant Chromobacterium violaceum CV026 . Spent supernatants from two strains, one that was positive in the C . violaceum assay and one that was negative, were tested for inducing early expression of phospholipase C, protease, and lipase in homologous and heterologous cultures . Expression of all three enzymes was increased or induced at an earlier stage in the growth curve in every case, suggesting not only that autoinducers were involved in the regulation of the expression of these enzymes, but also that the autoinducers were of two different classes.

Clin Infect Dis, 1999 Nov, 29(5), 1323 - 6
Melioidosis in Southern Vietnam: clinical surveillance and environmental sampling; Parry CM et al.; From 1992-1998, Burkholderia pseudomallei was isolated from only 9 (0.25%) of 3653 cultures of blood from febrile patients admitted to the Centre for Tropical Diseases in Ho Chi Minh City, an infectious disease referral center for southern Vietnam . Soil was sampled from 407 sites in 147 rice fields along the 5 major roads radiating from Ho Chi Minh City . B . pseudomallei was isolated from 73 sites (18%) in 39 rice fields (27%), but only 15 (21%) of the 71 isolates from 9 (6%) of 147 fields were the virulent l-arabinose (ara)-negative biotype . All except 1 of the fields with the ara-negative biotype were close to the homes of the patients with melioidosis . The low incidence of melioidosis in the provinces around Ho Chi Minh City may be explained by the restricted distribution of ara-negative B . pseudomallei in the soil in this area.

J Clin Microbiol, 1999 Nov, 37(11), 3742 - 5
Cloning and characterization of a nonhemolytic phospholipase C gene from Burkholderia pseudomallei; Korbsrisate S et al.; We cloned and characterized a phosphatidylcholine-hydrolyzing phospholipase C (PC-PLC) gene from Burkholderia pseudomallei . DNA sequence analysis of the gene indicated an open reading frame coding for 700 amino acids with a 34-amino-acid signal peptide . When cleaved, this yields a secreted 73-kDa mature protein . The deduced amino acid sequence exhibited 48% similarity to that of a nonhemolytic PLC from Pseudomonas aeruginosa . The expressed PC-PLC was heat stable, nonhemolytic for sheep erythrocytes, and active between pH 2 and 8 . Western blot analysis with sera from melioidosis patients indicated that they produced immunoglobulin M antibodies against this PC-PLC protein.

J Clin Microbiol, 1999 Nov, 37(11), 3662 - 7
Rapid identification of Burkholderia pseudomallei in blood cultures by a monoclonal antibody assay; Pongsunk S et al.; Burkholderia pseudomallei is the causative agent of melioidosis . In northeast Thailand, this gram-negative bacterium is a major cause of mortality from septicemia . The definitive diagnosis of this disease is made by bacterial culture . In this study, we produced a monoclonal antibody (MAb) specific to the 30-kDa protein of B . pseudomallei by in vivo and in vitro immunization of BALB/c mice with a crude culture filtrate antigen . The MAb could directly agglutinate with all 243 clinical isolates of B . pseudomallei but not with other gram-negative bacteria, except for one strain of Burkholderia mallei . However, the MAb cross-reacted with the gram-positive Bacillus sp . and Streptococcus pyogenes . B . pseudomallei in brain heart infusion broth (BHIB) subcultured from a BacT/Alert automated blood culture system could be identified by simple agglutination with this MAb assay . The sensitivity and specificity of direct agglutination compared to the "gold standard," the culture method, were 94.12 and 98.25%, respectively . However, the MAb adsorbed to polystyrene beads or latex particles directly identified the bacterium in blood culture specimens and in BHIB subcultured from a BacT/Alert automated blood culture system . The sensitivity of the latex agglutination test was 100% for both blood culture and BHIB specimens . The specificity was 85.96 and 96.49% for the blood culture and BHIB specimens, respectively . The specificity could be increased if the nonspecific materials in the blood culture broths were eradicated by centrifugation at low speeds . Thus, a combination of blood culture and the agglutination method could be used for the rapid diagnosis of melioidosis in the routine bacteriological laboratory . This method could speed up detection of the bacterium in blood culture by at least 2 days, compared to the conventional bacterial culture method . In addition, the MAb is stable at room temperature for 2 weeks and at 4, -20, and -70 degrees C for at least 1 year . The latex reagent was stable for at least 6 months at 4 degrees C.

J Microbiol Methods, 1999 Oct, 38(1-2), 63 - 7
Convenient and versatile DNA extraction using agarose plugs for ribotyping of problematic bacterial species; Nair S et al.; We describe a convenient, versatile and safe method for preparing bacterial DNA for ribotyping analysis . In this method, extraction of bacterial DNA from Salmnonella typhi and Burkholderia pseudomallei . and subsequent restriction endonuclease digestion, was performed in agarose blocks/plugs thus minimizing shearing and loss of DNA, problems commonly associated with liquid phase phenol extraction . Digested DNA in the plugs was then electrophoresed directly, transferred to nylon membranes and hybridized with labeled rDNA probes in the usual manner to provide reproducible restriction patterns . This method is particularly useful for bacterial species where standard DNA extraction in the liquid phase using phenol has been problematic (e.g . B . pseudomallei) but can be used for any bacterial species . The DNA extracted within the agarose plugs can be stored for long periods and can be used in other, widely-used typing methods such as pulsed-field gel electrophoresis (PFGE) and PCR-based techniques . Embedding live cells directly in agarose plugs also minimizes the risk of exposure to these virulent human pathogens among laboratory workers.

FEMS Microbiol Lett, 1999 Oct 15, 179(2), 203 - 8
Transformation of polychlorinated biphenyls by a novel BphA variant through the meta-cleavage pathway; Bruhlmann F et al.; The transformation of 20 polychlorinated biphenyls (PCBs) through the meta-cleavage pathway by recombinant Escherichia coli cells expressing the bphEFGBC locus from Burkholderia cepacia LB400 and the bphA genes from different sources was compared . The analysis of PCB congeners for which hydroxylation was observed but no formation of the corresponding yellow meta-cleavage product demonstrated that only lightly chlorinated congeners including one tetrachlorobiphenyl (2,2',4,4'-CB) were transformed into their corresponding yellow meta-cleavage products . Although many other tetrachlorobiphenyls (2, 2',5,5'-CB, 2,2',3,5'-CB, 2,4,4',5-CB, 2,3',4',5-CB, 2,3',4,4'-CB) and one pentachlorobiphenyl (2,2',4,5,5'-CB) tested were depleted from resting cell suspensions, no yellow meta-cleavage products were observed . For most of these congeners, dihydrodiol compounds accumulated as the endproducts, indicating that the bphB-encoded biphenyl-2,3-dihydrodiol-2,3-dehydrogenase is a key limiting step for further degradation of highly chlorinated congeners . These results suggest that engineering the biphenyl dioxygenase alone is insufficient for an improved removal of PCB . Rather, improved degradation of PCBs is more likely to be achieved with recombinant strains containing metabolic pathways not only specifically engineered for expanding the initial dioxygenation but also for the mineralization of PCBs.

Eur Respir J, 1999 Aug, 14(2), 435 - 8
Cystic fibrosis: inflammatory response to infection with Burkholderia cepacia and Pseudomonas aeruginosa; Hendry J et al.; Pulmonary colonization by Burkholderia cepacia in cystic fibrosis (CF) may be associated with enhanced deterioration of pulmonary function . This may be due to a more florid host inflammatory response than in colonization by Pseudomonas aeruginosa, leading to greater lung injury . Circulating markers of inflammation were determined during infective exacerbations and periods of clinical stability in an 18 month prospective study in adults with CF colonized by P . aeruginosa (n=41) . B . cepacia (n=13) and in adults who intermittently grew B . cepacia (n=6) . There were no differences between the levels of the inflammation markers measured in the three groups (P . aeruginosa, B . cepacia, B . cepacia intermittent) at any of the assessment points . When clinically stable, levels of inflammatory markers in all groups were elevated compared to a matched non-CF population, indicating, continuous inflammation and the potential for lung damage between infective exacerbations . This study does not support the hypothesis that pulmonary colonization with Burkholderia cepacia is associated with a heightened inflammatory response compared with Pseudomonas aeruginosa colonization.

Pharmacotherapy, 1999 Oct, 19(10), 1159 - 66
Cost of care for individuals with cystic fibrosis: a regression approach to determining the impact of recombinant human DNase; Johnson JA et al.; We estimated direct medical costs of care and important determinants of the costs in patients with cystic fibrosis (CF), including therapy with recombinant human DNase (rhDNase) . Costs were estimated with resource use data from the Epidemiologic Study of Cystic Fibrosis . Ordinary least squares regression was used to determine the effect of clinical and demographic variables on individual cost of care . The estimated cost of caring for 303 patients in Alberta was $2,279,801 in 1996 . The mean cost of care was $7524 (range $386-92,376)/patient . Regression results indicated that age and forced expiratory volume predicted had a negative association with costs . Being female, receiving rhDNase, and having Pseudomonas aeruginosa or Burkholderia cepacia were all associated with high costs . Our estimates indicated large interindividual variation in cost of care for patients with CF.

J Antimicrob Chemother, 1999 Sep, 44(3), 385 - 7
The effect of human lactoferrin on the MICs of doxycycline and rifampicin for Burkholderia cepacia and Pseudomonas aeruginosa strains; Alkawash M et al.; The presence of lactoferrin at the concentration found in cystic fibrosis (CF) sputum (0.9 g/L) reduced MICs and MBCs of doxycycline for Burkholderia cepacia and Pseudomonas aeruginosa strains . MICs for B . cepacia fell by 32- to 64-fold, from highly resistant to clinically achievable values . Rifampicin MICs for B . cepacia strains were reduced by lactoferrin and for some strains MBCs were reduced . These findings suggest new therapeutic approaches to infections and question the relevance of standard sensitivity tests for CF pathogens . Addition of lactoferrin to media for the routine sensitivity testing of CF isolates might give more relevant results.

Pathology, 1999 Aug, 31(3), 264 - 7
Combination antimicrobial therapy of acute Burkholderia pseudomallei infection in a mouse model; Ulett GC et al.; Burkholderia pseudomallei is the causative agent of melioidosis, a disease endemic in tropical and subtropical regions of South-East Asia and Northern Australia . Antimicrobial therapy regimens for treatment of acute septicemic melioidosis are of variable efficacy . Ceftazidime is the current antibiotic of choice and is commonly administered with other agents such as cotrimoxazole or doxycycline . The emergence of resistant strains of B . pseudomallei and the persistence of high mortality rates prompted the present study . Using an established mouse model of acute disseminated B . pseudomallei infection, we compared the efficacy of ceftazidime versus cefpirome in combination with cotrimoxazole or chloramphenicol therapy in vivo . Control mice that were infected but did not receive antibiotic therapy died within 96 hours of infection . No deaths occurred in treatment groups receiving either cephalosporin or cotrimoxazole, despite the demonstrated resistance of B . pseudomallei to cotrimoxazole in vitro . The mortality rate in treatment groups receiving either cephalosporin and chloramphenicol was 66% . These results demonstrate a comparable level of efficacy between ceftazidime and cefpirome for treatment of acute B . pseudomallei infection in mice.

J Bacteriol, 1999 Oct, 181(19), 6197 - 9
Characterization of the phthalate permease OphD from Burkholderia cepacia ATCC 17616; Chang HK et al.; The ophD gene, encoding a permease for phthalate transport, was cloned from Burkholderia cepacia ATCC 17616 . Expression of the gene in Escherichia coli results in the ability to transport phthalate rapidly into the cell . Uptake inhibition experiments show that 4-hydroxyphthalate, 4-chlorophthalate, 4-methylphthalate, and cinchomeronate compete for the phthalate permease . An ophD knockout mutant of 17616 grows slightly more slowly on phthalate but is still able to take up phthalate at rates equivalent to that of the wild-type strain . This means that 17616 must have a second phthalate-inducible phthalate uptake system.

J Bacteriol, 1999 Oct, 181(19), 6003 - 9
Cloning and characterization of a cryptic haloacid dehalogenase from Burkholderia cepacia MBA4; Tsang JS et al.; Burkholderia cepacia MBA4 has been shown to produce a single dehalogenase batch culture . Moreover, other cryptic dehalogenases were also detected when the cells were grown in continuous culture . In this paper, we report the cloning and characterization of one of the cryptic dehalogenases in MBA4 . This cryptic haloacid dehalogenase, designated Chd1, was expressed constitutively in Escherichia coli . This recombinant Chd1 had a relative molecular weight of 58,000 and existed predominantly as a dimer . The subunits had a relative molecular weight of 27,000 . Chd1 exhibited isomer specificity, being active towards the L-isomer of 2-monochloropropionic acid only . The structural gene, chd1, was isolated on a 1.7-kb PstI fragment . This fragment contains a functional promoter, because expression of chd1 in E . coli is orientation independent . The nucleotide sequence of this fragment was determined and characterized . An open reading frame of 840 bp encoding a putative peptide of 280 amino acids was identified . This corresponds closely with the size of the subunit . The nucleotide sequence of chd1 did not show any homology with those of other dehalogenase genes . Comparison of the predicted amino acid sequence, however, shows significant homology, ranging from 42 to 50%, with the amino acid sequences of many other dehalogenases . Chd1 is unusual in having a long leader sequence, a property of periplasmic enzymes.

Am J Trop Med Hyg, 1999 Sep, 61(3), 473 - 5
Mediation of attachment of Burkholderia pseudomallei to human pharyngeal epithelial cells by the asialoganglioside GM1-GM2 receptor complex; Gori AH et al.; Melioidosis is the term given to any infection caused by Burkholderia pseudomallei . This bacteria is one of the important causative agents of life-threatening pulmonary infections in the tropical and subtropical areas . The initiation of respiratory infections is attachment of this bacteria to pharyngeal cells . The precise mechanism of attachment of B . pseudomallei is not known . In this study, we found that asialoganglioside GM1 at concentrations of 25, 12.5, and 5 microg/ml significantly decreased the attachment of B . pseudomallei strain Sp-186 in a dose-dependent manner . On the other hand, asialoganglioside GM2 decreased the attachment of B . pseudomallei, but only at a concentration of 25 microg/ml . At a concentration of 1 mg/ml, glucose, N-acetyl-galactosamine, and galactose caused a significant decrease in attachment . However, at concentrations of 250 microg/ml, no decrease in attachment was observed in B . pseudomallei treated with these carbohydrates . Mannose and fucose at concentrations of 1 mg/ml had no effects on the inhibition of attachment of B . pseudomallei . Four other isolates of B . pseudomallei showed a significant decrease in attachment after treatment with asialoganglioside GM1 . We conclude that asialogangliosides GM1 and GM2 are part of the receptor complex for B . pseudomallei on human pharyngeal epithelial cells.

Mil Med, 1999 Sep, 164(9), 658 - 62
Melioidosis, an environmental and occupational hazard in Thailand; Thummakul T et al.; Melioidosis is a tropical environmental hazard that causes acute and chronic pulmonary disease, abscesses of the skin and internal organs, meningitis, brain abscess and cerebritis, and acute fulminant rapidly fatal sepsis . It is more common among adults, individuals with diabetes, and individuals with chronic renal disease, but it can occur in normal hosts and children . Burkholderia pseudomellei is the most prevalent cause of community-acquired pneumonia, liver and splenic abscess, and sepsis in northeastern Thailand . Melioidosis can reactivate years after primary infection and result in chronic or acute life-threatening disease . With increasing worldwide travel and migration, patients may present in nonendemic countries with reactivation melioidosis decades after leaving an endemic region . We discuss seven selected patients presenting with this disease to a tertiary care facility in Bangkok between 1995 and 1997 . Awareness should allow early diagnosis and treatment, which can lead to decreased morbidity and mortality.

J Clin Microbiol, 1999 Oct, 37(10), 3167 - 70
Development of rRNA-based PCR assays for identification of Burkholderia cepacia complex isolates recovered from cystic fibrosis patients; LiPuma JJ et al.; PCR assays targeting rRNA genes were developed to identify species (genomovars) within the Burkholderia cepacia complex . Each assay was tested with 177 bacterial isolates that also underwent taxonomic analysis by whole-cell protein profile . These isolates were from clinical and environmental sources and included 107 B . cepacia complex strains, 23 Burkholderia gladioli strains, 20 Ralstonia pickettii strains, 10 Pseudomonas aeruginosa strains, 8 Stenotrophomonas maltophilia strains, and 9 isolates belonging to nine other species . The sensitivity and specificity of the 16S rRNA-based assay for Burkholderia multivorans (genomovar II) were 100 and 99%, respectively; for Burkholderia vietnamiensis (genomovar V), sensitivity and specificity were 87 and 92%, respectively . An assay based on 16S and 23S rRNA gene analysis of B . cepacia ATCC 25416 (genomovar I) was useful in identifying genomovars I, III, and IV as a group (sensitivity, 100%, and specificity, 99%) . Another assay, designed to be specific at the genus level, identified all but one of the Burkholderia and Ralstonia isolates tested (sensitivity, 99%, and specificity, 96%) . The combined use of these assays offers a significant improvement over previously published PCR assays for B . cepacia.

J Med Microbiol, 1999 Sep, 48(9), 825 - 32
Lipopolysaccharide chemotypes of Burkholderia cepacia; Evans E et al.; Burkholderia cepacia is an important pathogen in patients with cystic fibrosis (CF) and much is now known of its epidemiology . In contrast, its virulence mechanisms are poorly understood . The lipopolysaccharide (LPS) of B . cepacia, a well-recognised virulence factor of other gram-negative bacteria, is known to be strongly endotoxic in vitro . The aim of this study was to observe if there were any links between the structure of B . cepacia LPS and virulence . This has been investigated by polyacrylamide gel electrophoresis and immunoblotting to define the chemotype and antigenic cross reactivity of B . cepacia LPS . Strains (16) belonging to different genomovars of the B . cepacia complex were selected to represent epidemic and non-epidemic clinical isolates and environmental strains . All strains belonging to genomovars I and II (the latter now renamed B . multivorans) had smooth LPS . However, isolates belonging to genomovar III, the group to which most of the epidemic CF isolates belong - including the highly transmissible strain (ET 12) which has been found in both the UK and North America - were of either rough or smooth LPS chemotype . In this study, B . cepacia J2315 represents the ET 12 lineage, and has a rough chemotype . Rabbit antiserum raised to strain J2315 revealed that the LPS core of this strain was antigenically related to some but not all other genomovar III strains, but it also cross-reacted strongly with all B . multivorans (genomovar II) and most genomovar I strains . Intra-strain phenotypic variation was demonstrated between bacteria grown in broth or on solid agar with a concomitant variation in antigenic cross reactivity . There was no clear evidence to associate any particular LPS phenotype with epidemic or non-epidemic strains, but changes in phenotype in vitro may provide clues to the survival and adaptability of B . cepacia in hostile environments and possibly to its ability to produce an inflammatory response in vivo.

Scand J Infect Dis, 1999, 31(3), 293 - 8
Endemic burkholderia cepacia bacteraemia: clinical features and antimicrobial susceptibilities of isolates; Yu WL et al.; Burkholderia cepacia has emerged as a nosocomial pathogen, causing numerous outbreaks, particularly among cystic fibrosis (CF) patients . Reports of clinical features of endemic B . cepacia bacteraemia in non-CF patients are rare . Twenty-five patients with B . cepacia bacteraemia were matched with 25 controls with nosocomial Escherichia coli bacteraemia at China Medical College Hospital, Taichung, Taiwan, over a period of 3 y . Case-patients included 16 men and 9 women, from 13 to 75 y . All had severe underlying diseases, most commonly malignancy (44%) . Twenty-four patients (96%) had nosocomial infections . Five patients (20%) had polymicrobial bacteraemia . Our controls included 11 men and 14 women, age range 18-80 y . The most common underlying disease was malignancy (44%) . Multivariate analysis revealed that indwelling central venous catheter was the significant risk factor predisposing to B . cepacia bacteraemia (p= 0.025) . Eleven case-patients met the definition of catheter-related bloodstream infection . Fifteen patients (60%) received appropriate antimicrobial therapy after notification of positive blood cultures and susceptibility patterns . The overall case-fatality rate was 12% (3/25), only 1 of whom died of B . cepacia bacteraemia . There was no statistically significant difference in overall mortality rate between case-patients and controls . All isolates were susceptible to ceftazidime, piperacillin and minocycline and 84% of the isolates were susceptible to imipenem . B . cepacia should be considered a potential pathogen in hospitalized patients with severe underlying diseases, particularly those with indwelling central venous catheters.

Am J Trop Med Hyg, 1999 Mar, 60(3), 458 - 61
Epidemiology of Burkholderia pseudomallei in Thailand; Vuddhakul V et al.; The distribution of Burkholderia pseudomallei in soil collected from four regions of Thailand and the frequency of B . pseudomallei infections in patients attending government hospitals throughout Thailand in 1997 were surveyed . A total of 3,585 soil samples collected from 896 sites in four regions of Thailand were cultured for B . pseudomallei using selective enrichment broth and modified Ashdown's agar . The organism was recovered in 4.4%, 6.1%, 20.4%, and 5.9% of the soil samples collected from the northern, central, northeastern, and southern regions, respectively, of Thailand (P < 0.0001) . Burkholderia pseudomallei was cultured from 50.1% of the sites in the northeastern region compared with 13.8%, 24.5%, and 18.4% in the northern, central, and southern regions, respectively (P < 0.0001) . The infection rate in patients attending government hospitals in the northeastern region (137.9 per 100,000 inpatients) was significantly higher than those in the northern (18 per 100,000 inpatients), central (13.4 per 100,000 inpatients), and southern (14.4 per 100,000 inpatients) regions, respectively (P < 0.0001) . It is suggested that melioidosis, which is endemic in Thailand, is associated with the presence of B . pseudomallei in soil.

Int J Antimicrob Agents, 1999 Aug, 12(3), 263 - 5
Imipenem for the treatment of melioidosis; Minassian MA et al.; Melioidosis is a protean disease caused by Burkholderia pseudomallei . It is rare in the UK and is generally only seen in patients with a travel history to endemic areas such as Thailand, Singapore and Malaysia . Cases may present with disseminated bacteraemic, non-disseminated bacteraemic, multi-focal bacteraemic or localized disease . Subclinical infections also occur . Following acquisition of the organism a patient may remain asymptomatic for several years before infection becomes clinically apparent . Factors such as diabetes, renal failure or other causes for a decrease in host immunity may precipitate the appearance of overt disease . The current treatment choice for severe melioidosis is parenteral ceftazidime followed by oral amoxycillin-clavulanic acid or a combination of co-trimoxazole, doxycycline and chloramphenicol . We report a case of melioidosis in a 59-year-old male diabetic from Bangladesh who initially responded to piperacillin-tazobactam but was changed to ceftazidime when a definitive diagnosis was made . His condition deteriorated on the latter antibiotic . He subsequently responded to imipenem . The patient's long-term outcome is still not known.

Infect Immun, 1999 Sep, 67(9), 4443 - 55
Role of ornibactin biosynthesis in the virulence of Burkholderia cepacia: characterization of pvdA, the gene encoding L-ornithine N(5)-oxygenase; Sokol PA et al.; Burkholderia cepacia is a frequent cause of respiratory infections in cystic fibrosis patients . B . cepacia has been shown to produce at least four siderophores which may play a role in the virulence of this organism . To characterize genes involved in the synthesis of siderophores, Tn5-OT182 mutants were isolated in strain K56-2, which produces two siderophores, salicylic acid (SA) and ornibactins . Two mutants were characterized that did not produce zones on Chrome Azurol S agar in a commonly used assay to detect siderophore activity . These mutants were determined to produce sevenfold more SA than K56-2 yet did not produce detectable amounts of ornibactins . These mutants, designated I117 and T10, had a transposon insertion in genes with significant homology to pyoverdine biosynthesis genes of Pseudomonas aeruginosa . I117 contained an insertion in a pvdA homolog, the gene for the enzyme L-ornithine N(5)-oxygenase, which catalyzes the hydroxylation of L-ornithine . Ornibactin synthesis in this mutant was partially restored when the precursor L-N(5)-OH-Orn was added to the culture medium . T10 contained an insertion in a pvdD homolog, which is a peptide synthetase involved in pyoverdine synthesis . beta-Galactosidase activity was iron regulated in both I117 and T10, suggesting that the transposon was inserted downstream of an iron-regulated promoter . Tn5-OT182 contains a lacZ gene that is expressed when inserted downstream of an active promoter . Both I117 and T10 were deficient in uptake of iron complexed to either ornibactins or SA, suggesting that transposon insertions in ornibactin biosynthesis genes also affected other components of the iron transport mechanism . The B . cepacia pvdA homolog was approximately 47% identical and 59% similar to L-ornithine N(5)-oxygenase from P . aeruginosa . Three clones were identified from a K56-2 cosmid library that partially restored ornibactin production, SA production, and SA uptake to parental levels but did not affect the rate of (59)Fe-ornibactin uptake in I117 . A chromosomal pvdA deletion mutant was constructed that had a phenotype similar to that of I117 except that it did not hyperproduce SA . The pvdA mutants were less virulent than the parent strain in chronic and acute models of respiratory infection . A functional pvdA gene appears to be required for effective colonization and persistence in B . cepacia lung infections.

Hum Genet, 1999 Jun, 104(6), 511 - 5
Two novel mutations in a cystic fibrosis patient of Chinese origin; Wagner JA et al.; Cystic fibrosis is rare in non-Caucasian populations, and in such populations little is known about the spectrum of mutations and polymorphisms in the CFTR gene . We studied a 23-year-old patient of Chinese ethnicity with sweat chloride values of 104 mM/l, pancreatic sufficiency, an FEV1 60% of normal, sputum cultures positive for Staphylococcus aureus and Burkholderia cepacia, and a history of allergic bronchopulmonary aspergillosis . Genetic screening for 31 common CFTR mutations was negative, leading us to search for unknown mutations using single-strand conformation polymorphism and heteroduplex analysis (SSCP/HA) . Two novel mutations were detected . In exon 4, a deletion of 8 bp (451458, deltaGCTTCCTA) causes a frameshift and immediately creates a stop codon . In exon 16, mutation 3041G-->A causes the missense change G970D . Functional analysis using an isotopic flux assay indicated that the G970D mutation retains partial function; western blotting indicated that the protein is glycosylated . The patient is heterozygous for the common polymorphisms (2694T/G) in exon 14a and (GATT)6/7 in intron 6a, indicating that these variants arose in ancestors common to Caucasians and Chinese.

J Clin Pathol, 1999 Mar, 52(3), 173 - 6
Evaluation of three oligonucleotide primer sets in PCR for the identification of Burkholderia cepacia and their differentiation from Burkholderia gladioli; Clode FE et al.; AIMS: To evaluate three oligonucleotide primer pairs--two specific for 16S and 23S rRNA sequences of Burkholderia cepacia, and the third specific for internal transcribed spacer region of 16S-23S sequences of B gladioli--for the identification and differentiation of reference and clinical strains of these and other species . METHODS: The three primers sets were applied in polymerase chain reaction (PCR) to a collection of 177 clinical isolates submitted for identification from diagnostic laboratories as presumed B cepacia . RESULTS: At an annealing temperature of 63 degrees C, all eight B cepacia and four B gladioli reference strains reacted with their specific primers . B vandii was the only other species that was positive with both B cepacia primers but five Burkholderia or Ralstonia species reacted with one of these primers . Seventy eight isolates were typical of B cepacia in biochemical tests and 75 of these reacted with specific primers; three, however, were positive with the B gladioli primers . Fifteen asaccharolytic isolates were confirmed as B cepacia by PCR but other non-fermenting Gram negative species were negative with each of the primers . CONCLUSIONS: PCR using 16S rRNA sequences is recommended for identification of B cepacia that give atypical results in biochemical tests.

J Clin Invest, 1999 Aug, 104(4), 431 - 7
Association of mannose-binding lectin gene heterogeneity with severity of lung disease and survival in cystic fibrosis; Garred P et al.; Mannose-binding lectin (MBL) is a key factor in innate immunity, and lung infections are the leading cause of morbidity and mortality in cystic fibrosis (CF) . Accordingly, we investigated whether MBL variant alleles, which are associated with recurrent infections, might be risk factors for CF patients . In 149 CF patients, different MBL genotypes were compared with respect to lung function, microbiology, and survival to end-stage CF (death or lung transplantation) . The lung function was significantly reduced in carriers of MBL variant alleles when compared with normal homozygotes . The negative impact of variant alleles on lung function was especially confined to patients with chronic Pseudomonas aeruginosa infection . Burkholderia cepacia infection was significantly more frequent in carriers of variant alleles than in homozygotes . The risk of end-stage CF among carriers of variant alleles increased 3-fold, and the survival time decreased over a 10-year follow-up period . Moreover, by using a modified life table analysis, we estimated that the predicted age of survival was reduced by 8 years in variant allele carriers when compared with normal homozygotes . Presence of MBL variant alleles is therefore associated with poor prognosis and early death in patients with CF.

Biochem Biophys Res Commun, 1999 Aug 19, 262(1), 308 - 14
Conserved and hybrid meta-cleavage operons from PAH-degrading Burkholderia RP007; Laurie AD et al.; We have compared the sequence and gene order of meta-cleavage pathway operons from alpha- and gamma-subgroups of the Proteobacteria with operons from Burkholderia sp . strain RP007 which belongs to the beta-subgroup of the Proteobacteria . Burkholderia RP007 was isolated for its ability to degrade phenanthrene and contains two meta-cleavage operons . One exhibits a comparable gene order to previously characterised gamma-subgroup Proteobacterial (Pseudomonas) meta operons, whilst the other has distinctive features present in both alpha- and gamma-subgroup Proteobacterial (Sphingomonas and Pseudomonas) meta operons . Gene sequence conservation, highlighted by examining the phylogeny of Proteobacterial catechol 2,3-dioxygenase sequences, reveals that sequences generally cluster in a manner which correlates with the taxonomic grouping of the Proteobacterial subgroup from which they originated .

Eur J Clin Microbiol Infect Dis, 1999 Jun, 18(6), 428 - 31
Comparative in vitro activity of gatifloxacin against Stenotrophomonas maltophilia and Burkholderia species isolates including evaluation of disk diffusion and E test methods; Biedenbach DJ et al.; The in vitro activity of gatifloxacin, a new fluoroquinolone, was compared to that of five other fluoroquinolones against 105 Stenotrophomonas maltophilia isolates and 52 Burkholderia spp . isolates . The gatifloxacin MICs were determined using the broth microdilution method and the E test (AB Biodisk, Sweden); these methods were compared for test accuracy, and 5 microg disk zone diameters were compared for interpretive accuracy using the standardized disk diffusion method . In terms of potency, gatifloxacin was most similar to sparfloxacin and trovafloxacin against Stenotrophomonas maltophilia (MIC50, 0.5-1 mg/l) and Burkholderia spp . (MIC50, 1-2 mg/l) . This activity was greater than that of ciprofloxacin, levofloxacin or ofloxacin (MIC50, > or = 2 mg/l) against Stenotrophomonas maltophilia isolates but comparable to that of levofloxacin against the Burkholderia spp . (60% susceptible at < or = 2 mg/l) . The E test results compared well with the reference dilution test results (81-97% at +/- 1 log2 dilution) . The disk diffusion test using previously suggested breakpoints for other bacteria (> or = 18 mm or < or = 2 mg/l for susceptible and < or = 14 mm or > or = 8 mg/l for resistant) also performed well, at > 90% categorical agreement . The activity of gatifloxacin is comparable to that of other newer quinolones against isolates of Stenotrophomonas maltophilia and Burkholderia spp., and susceptibility testing using simple qualitative and quantitative methods appears to function well with these drug/organism combinations.

Microbiology, 1999 Jul, 145 ( Pt 7), 1509 - 17
Intracellular survival and saprophytic growth of isolates from the Burkholderia cepacia complex in free-living amoebae; Marolda CL et al.; Members of the taxonomically diverse Burkholderia cepacia complex have become a major health risk for patients with cystic fibrosis (CF) . Although patient-to-patient transmission of B . cepacia strains has been well-documented, very little is known about possible vehicles of transmission and reservoirs for these micro-organisms . In this work, it is shown that strains of the B . cepacia complex can survive within different isolates of the genus Acanthamoeba . Trophozoites containing bacteria developed profuse cytoplasmic vacuolization . Vacuolization was not detected in trophozoites infected with live Escherichia coli or heat-killed B . cepacia, or by incubation of trophozoites with filter-sterilized culture supernatants, indicating that metabolically active intracellular bacteria are required for the formation of vacuoles . Experiments with two different B . cepacia strains and two different Acanthamoeba isolates revealed that bacteria display a low level of intracellular replication approximately 72-96 h following infection . In contrast, extracellular bacteria multiplied efficiently on by-products released by amoebae . The findings suggest that amoebae may be a reservoir for B . cepacia and possibly a vehicle for transmission of this opportunistic pathogen among CF patients.

Food Addit Contam, 1999 Feb, 16(2), 63 - 9
The effect of lipids on bongkrekic (Bongkrek) acid toxin production by Burkholderia cocovenenans in coconut media; Garcia RA et al.; Tempe bongkrek is an Indonesian food made by fermentation of coconut presscake or coconut milk residue Rhizopus oligosporus . Consumption of tempe bongkrek is associated with a food-borne human intoxication and significant numbers of deaths annually . The bacterium Burkholderia cocovenenans, which is the causative organism, produces two toxins, toxoflavin and bongkrekic acid (also commonly referred to as bongkrek acid) . The reasons why these poisonings occur only in a very limited number of foods and only in isolated regions of the world are unclear . Our preliminary experiments in defined media and coconut investigated several compositional and environmental factors and suggested that lipid type and/or concentration were important . The effect of lipid concentration and fatty acid type on the production of bongkrekic acid by B . cocovenenans was examined by adding different amounts of coconut fat or individual free fatty acids to defatted and sterilized Rich Coconut Media (dRCM) . The dRCM with added lipid was inoculated with B . cocovenenans, incubated at 30 degrees C for 5 days and the amount of bongkrekic acid formed quantified by HPLC . Coconut fat concentrations of 10% (dry basis) or less did not result in detectable amounts of bongkrekic acid even though the B . cocovenenans grew to high levels . Forty and 50% coconut fat resulted in as much as 1.4 mg/g bonkrekic acid (dry weight) at the same level of growth . Of eight saturated fatty acids tested, only lauric (12:0), myristic (14:0), and palmitic (16:0) acids stimulated the production of detectable amounts of toxin . When four 18-carbon free fatty acids with different degrees of saturation were compared, significant amounts of bongkrekic acid (2.62 mg/g dry weight) were produced only with oleic acid (18:1) . These data indicate that the concentration and type of lipid in the substrate is critical for bongkrekic acid formation . This may explain why bongkrekic acid intoxication is limited to certain foods . Outbreaks associated with foods containing less than 20% fat may be a result of toxoflavin formation and not bongkrekic acid formation.

Am J Trop Med Hyg, 1999 Jul, 61(1), 34 - 6
Melioidosis with adrenal gland abscess; Lee SC et al.; We report a case of melioidosis with left adrenal gland abscess in a 51-year-old man from Taiwan who traveled to Rangoon, Burma for a four-day tour on July 15, 1997 . The patient developed fever and left upper abdominal pain upon returning to Taiwan on July 19, 1997 . Ten days after returning to Taiwan, he was admitted to Chang Gung Memorial Hospital in Keelung, Taiwan and blood culture on admission was positive for Burkholderia pseudomallei . Computerized tomography of the abdomen revealed left adrenal gland swelling and suppuration . Treatment with parenteral ceftazidime and cotrimoxazole for three weeks followed by two months of oral cotrimoxazole cured the infection . The patient remained asymptomatic at 12 months follow-up.

Mol Med Today, 1999 Aug, 5(8), 351 - 8
Cystic fibrosis: an inherited susceptibility to bacterial respiratory infections; Tummler B et al.; Cystic fibrosis is a severe monogenic disorder of ion transport in exocrine glands . The basic defect predisposes to chronic bacterial airway infections with Staphylococcus aureus, Haemophilus influenzae, Pseudomonas aeruginosa and Burkholderia cepacia . The Pseudomonas infections in cystic fibrosis are a paradigm of how versatile environmental bacteria can conquer, adapt and persist in an atypical habitat and successfully evade defence mechanisms and chemotherapy in a susceptible host . Regular chemotherapy with aerosol and systemic antipseudomonal drugs has improved the course and prognosis of the disease, and research for effective vaccines is on the wayPublication Types:

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