J Appl Microbiol, 1997 May, 82(5), 589 - 96 Isolation and identification of Burkholderia pseudomallei from soil using selective culture techniques and the polymerase chain reaction; Brook MD et al.; An environmental soil survey to detect Burkholderia pseudomallei was performed during the dry and wet seasons in Darwin, Northern Territory, Australia . Soil was sampled at regular intervals during a 15-month period at different depths from areas which were representative of the local, soil environment . Selective culture techniques using Ashdown's and Galimand and Dodin's methods and the polymerase chain reaction (PCR) using specific 16S rRNA primers were used to detect and identify the organism and determine its distribution within the soil stratum over the change in seasons . Results showed that Ashdown's method gave higher isolation rates in the dry season, and Galimand and Dodin's method gave higher isolation rates during the wet season . PCR of the soil enrichment proved to be a more sensitive method than culture and was also a useful confirmatory test in determining the identification of isolates where biochemical tests gave inconsistent results . The PCR primers were specific and able to detect 10(1) cfu g-1 soil and 10(4) cfu g-1 of soil using Ashdown's enrichment broth and Galimand and Dodin's broth, respectively . Overall the isolation of B . pseudomallei was greatest during the dry season and at the higher and lower soil depths, which is contradictory to epidemiological evidence that melioidosis occurs primarily during the wet season among patients exposed to contaminated surface soil and water. Am J Respir Crit Care Med, 1997 May, 155(5), 1699 - 704 The effects of panresistant bacteria in cystic fibrosis patients on lung transplant outcome; Aris RM et al.; The number of cystic fibrosis (CF) patients undergoing lung transplant continues to rise as long term survival improves . One major contraindication to this potentially life-saving intervention is infection with multi-drug-resistant bacteria . We undertook this retrospective study in 66 transplanted patients over 6 yr to determine the influence of panresistant bacteria on transplant outcome . The in vitro antibiotic susceptibility pattern of respiratory tract bacteria obtained pre- and/or intraoperatively was used to categorize patients into panresistant (n = 27) (Burkholderia cepacia, n = 6, and Pseudomonas aeruginosa, n = 21) or sensitive (n = 39) groups . Postoperative ventilator days, hospital length of stay, and antibiotic days were similar for both groups (p > 0.2) . The incidence of bacterial bronchitis (28% and 33%, respectively) and pneumonia (28% and 38%, respectively) did not differ between these groups (p > 0.2) at 6 mo . Likewise, one-year (81% and 83%, respectively) survival was similar for both groups (p > 0.2) . As expected, panresistant B . cepacia patients had a lower 1-yr survival (50% versus 90%, p < 0.05) and had a higher mortality attributable to B . cepacia (50% versus 0%, p < 0.01) compared with panresistant P . aeruginosa patients . Our results indicate that CF patients infected with panresistant P . aeruginosa have similar transplant outcomes as patients with sensitive bacteria and should not be excluded from lung transplant based solely on this criterion. Eur J Epidemiol, 1997 Apr, 13(3), 323 - 7 Microbiologic data overview of Italian cystic fibrosis patients; Taccetti G et al.; The aim of our study was to determine the frequency of microbiological culturing and prevalence of colonization by principal pathogens of the respiratory tract of Italian cystic fibrosis patients . Data on all Italian cystic fibrosis patients were collected using a questionnaire sent to all Italian CF Centers . Results were obtained of microbiological cultures from 2,521 patients . Information was gained regarding the method of gathering biological samples, the percentage of patients undergoing microbiological culturing regularly, the procedures used to isolate bacteria and types of culture media used which were selective for Burkholderia cepacia . Ninety-four percent of Italian CF patients are regularly tested microbiologically . Sputum and pharyngeal cultures are most often carried out . 49% of Italian patients are colonized by Staphylococcus aureus, 5.4% by Haemophilus influenzae, 48.9% by Pseudomonas aeruginosa and 3.8% by Burkholderia cepacia . In Italy there is a high prevalence of CF patients colonized by Staphylococcus aureus and a low prevalence of patients colonized by Haemophilus influenzae . The prevalence of Burkholderia cepacia and Pseudomonas aeruginosa does not differe significantly from other countries examined. Am J Trop Med Hyg, 1997 Apr, 56(4), 418 - 23 Diagnostic value of an antibody enzyme-linked immunosorbent assay using affinity-purified antigen in an area endemic for melioidosis; 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 from nonhospital-acquired septicemia in the northeastern part of Thailand . A major factor that contributes to the high mortality is the delay in isolation and identification of the causative organism . The present study was undertaken to evaluate the use of enzyme-linked immunosorbent assays based on an immunoaffinity-purified antigen for detecting specific IgG and IgM antibodies to this organism as a rapid serodiagnostic method for melioidosis . The diagnostic value of these tests was evaluated in an actual clinical situation in an area endemic for melioidosis . The specificity of specific IgG test (82.5%) and the specific IgM test (81.8%) were significantly better than that of the indirect hemagglutination (IHA) test (74.7%) . The sensitivity of the specific IgG assay (85.7%) was higher than that of the IHA test (71.0%) and the specific IgM test (63.5%) . Specific IgG antibody was detected in a majority of septicemic melioidosis (87.8%), as well as in localized forms (82.6%) . The specific IgG test was also better than the specific IgM test and the IHA test in identifying acute melioidosis cases in the first five days after admission . In addition, the IgG antibody level to this antigen remained high over a period of more than five years in those who had recovered from melioidosis and remained clinically free of the disease . These results indicate that the detection of specific IgG antibody is clinically useful for the diagnosis of acute melioidosis in an endemic area. J Clin Microbiol, 1997 Apr, 35(4), 808 - 16 Identification and characterization of a novel DNA marker associated with epidemic Burkholderia cepacia strains recovered from patients with cystic fibrosis; Mahenthiralingam E et al.; Burkholderia cepacia is a problematic pathogen that may spread among patients with cystic fibrosis (CF) . One highly infectious CF strain that causes epidemics in both the United Kingdom and eastern Canada has been shown to possess both the cable pilin subunit gene (cblA) and a unique combination of insertion sequences . However, no genetic markers linking this strain type with other types epidemic at various centers have been identified . Using a randomly amplified polymorphic DNA (RAPD) typing scheme, we identified an apparently conserved 1.4-kb fragment in the DNA fingerprint of epidemic B . cepacia strains . Conservation of the DNA marker among epidemic strains was demonstrated by Southern hybridization, and its prevalence was assessed in a collection of chromosomal DNAs extracted from 627 isolates representative of 132 RAPD-defined B . cepacia strain types . The marker was specifically associated with seven epidemic CF strains, was absent among nonepidemic strains infecting individual patients with CF, and rare among strains recovered from the natural environment . Only one of the seven epidemic CF strain types possessed DNA homologous to cblA . The RAPD marker was designated the "B . cepacia epidemic strain marker" (BCESM) . Sequence analysis of chromosomal DNA corresponding to the 1.4-kb RAPD marker revealed the presence of a putative open reading frame (ORF) with significant homology to several negative transcriptional regulators; the ORF was designated the "epidemic strain marker regulator," or esmR . The BCESM DNA is the first genetic marker that has been identified to be specifically associated with and conserved among several epidemic B . cepacia strains which infect multiple patients with CF. Zentralbl Bakteriol, 1997 Apr, 285(4), 541 - 57 Isolation of an Acanthamoeba strain with intracellular Burkholderia pickettii infection; Michel R et al.; Burkholderia pickettii is a facultative pathogen that has been isolated from patient sources and environmental sources including respiratory therapy solutions, deionized water and aqueous disinfectants . The organism has been associated with septicemia and respiratory tract infections . In our investigation, Burkholderia pickettii (biovar 2) was for the first time isolated from Acanthamoeba sp . (group II), a free living amoeba species recovered from the wet area of a physiotherapy unit . Pathogenic strains of acanthamoebae may cause amoebic-encephalitis (AE) and keratitis . Light and electron microscopic examinations showed that in a first step, the bacterial were phagocytized by the amoebae . In contrast to Enterobacter cloacae and Escherichia coli that were used as food organisms and digested within food vacuoles, Burkholderia pickettii caused the amoebae to develop large vacuoles filled with completely intact and motile bacteria . 3-5 days after infection, Pseudomonas pickettii had multiplied within the enlarging parasitophorous vacuoles . Ultrastructural changes in the host cells occurred and the amoebae finally underwent rupture or lysis . In cocultivation assays we could not only reinfect the original host amoeba but Acanthamoeba strains from other habitats could successfully be infected with Burkholderia pickettii as well. Epidemiol Infect, 1997 Apr, 118(2), 137 - 48 Characterization of Burkholderia pseudomallei and Burkholderia pseudomallei-like strains; Brett PJ et al.; Previous reports in the literature suggest that Burkholderia pseudomallei strains can be differentiated on the basis of animal virulence . Twenty environmentally and clinically derived isolates of Burkholderia pseudomallei were examined for the production of exoenzymes, morphological and biochemical phenotypes and virulence for Syrian golden hamsters . The partial sequence of the 16S ribosomal RNA {rRNA} genes from a number of these strains was also determined . Based upon these observations, it is suggested that highly virulent Burkholderia pseudomallei strains are true Burkholderia pseudomallei strains . The DNA sequences of the 16S rRNA genes of the true Burkholderia pseudomallei strains were identical to the published sequences for Burkholderia pseudomallei while differences were revealed between the published sequences and those of the lowly virulent strains . Thus, these latter strains have been designated as Burkholderia pseudomallei-like organisms since they demonstrate significant differences in exoenzyme production, hamster virulence and 16S rRNA gene sequences. Blood, 1997 Apr 1, 89(7), 2268 - 75 Enhanced host defense after gene transfer in the murine p47phox-deficient model of chronic granulomatous disease; Mardiney M 3rd et al.; The p47phox-/- mouse exhibits a phenotype similar to that of human chronic granulomatous disease (CGD) and, thus, is an excellent model for the study of gene transfer technology . Using the Moloney murine leukemia virus-based retroviral vector MFG-S encoding the human form of p47phox, we performed ex vivo gene transfer into Sca-1+ p47phox-/- marrow progenitor cells without conditioning of donors with 5-fluorouracil . Transduced progenitors were transplanted into moderately irradiated (500 cGy), G-CSF preconditioned sibling p47phox-/- mice . Using the fluorescent probe dihydrorhodamine 123 (DHR), in vivo biochemical correction of the superoxide-generating NADPH oxidase system was detected by flow cytometry in 12.3% +/- 0.9% of phorbol myristate acetate-stimulated peripheral blood neutrophils at 4 weeks and 2.6% +/- 1.0% at 14 weeks after transplantation . Following gene therapy, mice were challenged with the CGD pathogen Burkholderia (formerly Pseudomonas) cepacia and bacteremia levels were assessed at 24 hours and 7 days after inoculation . At both time points, bacteremia levels in gene corrected p47phox-/- mice were significantly lower than untreated p47phox-/- mice (0.89 +/- 0.30 colonies v 237.7 +/- 83.6 colonies at 24 hours, P < .02; 4.0 +/- 2.0 colonies v 110.2 +/- 26.5 colonies at 7 days, P < .0014) . More importantly, Kaplan-Meier survival analysis showed a significant survival advantage of gene corrected versus untreated p47phox-/- mice (P < .001) . Thus, stem-cell-directed ex vivo gene therapy is capable of restoring phagocyte oxidant-dependent host-defense function in this mouse model of a human immune-system disorder. Am J Respir Crit Care Med, 1997 Apr, 155(4), 1436 - 40 Nosocomial acquisition of Burkholderia gladioli in patients with cystic fibrosis; Wilsher ML et al.; Burkholderia gladioli has been reported as colonizing the airways of patients with cystic fibrosis (CF) but has not previously been associated with adverse outcome . We describe six patients with CF in whom the same strain of B . gladioli, on the basis of ribotyping and biochemical characteristics, was grown in their sputum . Acquisition of this organism was followed by a fatal outcome in all six patients; one had a rapid decline in respiratory status and another developed fulminant B . gladioli bacteremia . Evidence suggests that patient-to-patient transmission of the organism occurred, and supports nosocomial infection in the ward and/or outpatient clinic despite general and stringent infection-control measures . This is the first report of adverse clinical outcome following sputum colonization with B . gladioli, and the first to demonstrate person-to-person transmission. Mol Plant Microbe Interact, 1997 Apr, 10(3), 369 - 79 Molecular cloning, characterization, and mutagenesis of a pel gene from Pseudomonas syringae pv . lachyrmans encoding a member of the Erwinia chrysanthemi pelADE family of pectate lyases; Bauer DW et al.; The pelS gene from Pseudomonas syringae pv . lachrymans 859 was cloned by heterologous expression in nonpectolytic P . syringae pv . syringae BUVS1, using genomic DNA libraries constructed with two novel broad-host-range cosmid vectors, pCPP34 and pCPP47 . Screening of P . syringae pv . syringae transconjugants for the ability to pit pectate media at pH 6.0 and 8.5 yielded several overlapping clones of the same DNA region . Ultrathin-layer isoelectric focusing gels, activity-stained with diagnostically buffered substrate overlays, revealed that this region encoded a single pectate lyase (PelS) with a pI of 9.4 . pelS was subcloned from cosmid pCPP5020 and sequenced, revealing it to encode a member of the Erwinia chrysanthemi PelADE family, with highest similarity to Pseudomonas viridiflava PelV . A pelS probe hybridized at high stringency in DNA gel blots with total DNA from P . syringae pv . lachrymans strains 859 and Pla5, P . syringae pv . tabaci, P . syringae pv . phaseolicola, P . syringae pv . glycinea, P . fluorescens (marginalis), P . viridiflava, and Xanthomonas campestris pv . campestris, but not with P . syringae pv . pisi, P . syringae pv . syringae, P . syringae pv . tomato, P . syringae pv . papulans, E . chrysanthemi, or Ralstonia (Pseudomonas or Burkholderia) solanacearum . The PelS sequence revealed an N-terminal signal peptide, whose processing in Escherichia coli was confirmed by protein sequence analysis . PelS was similar to E . chrysanthemi PelE in its substrate preference and ability to reduce the viscosity of pectate and to macerate potato tuber tissue . A pelS:: omega Kmr mutation was marker-exchanged into P . syringae pv . lachrymans Pla5, pelS was also subcloned into the broad-host-range expression vector pML122 under control of the vector nptII promoter, and then transformed into P . syringae pv . lachrymans Pla5 to produce a strain overproducing PelS . Necrotic lesions developed in cotyledons following inoculation with all of the P . syringae pv . lachrymans Pla5 derivatives, regardless of their Pel phenotype . However, only cotyledons infected with pelS+ strains showed evidence of maceration and yielded Pel activity upon extraction . In contrast, pelS+ P . syringae pv . syringae BUVS1(pCPP5020) produced no symptoms in cucumber cotyledons . Thus, PelS in P . syringae pv . lachrymans appears to alter the final symptoms in infected cucumber cotyledons without contributing to pathogenicity or altering host range. J Bacteriol, 1997 Apr, 179(8), 2717 - 23 A fusion promoter created by a new insertion sequence, IS1490, activates transcription of 2,4,5-trichlorophenoxyacetic acid catabolic genes in Burkholderia cepacia AC1100; Hubner A et al.; Transposition and transcriptional activation by insertion sequences in Burkholderia cepacia AC1100 were investigated . Two closely related new elements, IS1413 and IS1490, were identified and characterized . These elements are not highly related to other insertion sequences identified in AC1100 or other B . cepacia isolates . Based on their structures and the sequences of the inverted terminal repeats and the putative transposase protein, the insertion elements (IS elements) are similar to IST2 of Thiobacillus ferrooxidans and several related elements . All the IS elements that have been identified in this strain are found in multiple copies (10 to 40), and they have high-level promoter activity capable of stimulating transcription from a distance up to 500 bp from a target gene . Strain AC1100 was originally isolated after prolonged selection for the ability to utilize the herbicide 2,4,5-trichlorophenoxyacetic acid (2,4,5-T) as a sole carbon source . Three IS elements are located near the first gene of the 2,4,5-T catabolic pathway, tftA . IS1490 inserted 110 bp upstream of tftA and created a fusion promoter responsible for constitutive transcription of the gene . Our results confirm the hypothesis that IS elements play a central role in transcription of 2,4,5-T genes and likely have stimulated rapid evolution of the metabolic pathway. Antimicrob Agents Chemother, 1997 Apr, 41(4), 853 - 6 Conservation of the multidrug resistance efflux gene oprM in Pseudomonas aeruginosa; Bianco N et al.; An intragenic probe derived from the multidrug resistance gene oprM hybridized with genomic DNA from all 20 serotypes of Pseudomonas aeruginosa and from all 34 environmental and clinical isolates tested, indicating that the MexA-MexB-OprM multidrug efflux system is highly conserved in this organism . The oprM probe also hybridized with genomic DNA from Pseudomonas aureofaciens, Pseudomonas chlororaphis, Pseudomonas syringae, Burkholderia pseudomallei, and Pseudomonas putida, suggesting that efflux-mediated multidrug resistance mechanisms may be somewhat broadly distributed. J Bacteriol, 1997 Apr, 179(7), 2116 - 25 Mutagenesis of Burkholderia pseudomallei with Tn5-OT182: isolation of motility mutants and molecular characterization of the flagellin structural gene; DeShazer D et al.; Burkholderia pseudomallei is a human and animal pathogen in tropical regions, especially Southeast Asia and northern Australia . Currently little is known about the genetics and molecular biology of this organism . In this report, we describe the mutagenesis of B . pseudomallei with the transposon Tn5-OT182 . B . pseudomallei 1026b transposon mutants were obtained at a frequency of 4.6 x 10(-4) per initial donor cell, and the transposon inserted randomly into the chromosome . We used Tn5-OT182 to identify the flagellin structural gene, fliC . We screened 3,500 transposon mutants and identified 28 motility mutants . Tn5-OT182 integrated into 19 unique genetic loci encoding proteins with homology to Escherichia coli and Salmonella typhimurium flagellar and chemotaxis proteins . Two mutants, MM35 and MM36, contained Tn5-OT182 integrations in fliC . We cloned and sequenced fliC and used it to complement MM35 and MM36 in trans . The fliC transcriptional start site and a sigmaF-like promoter were identified by primer extension analysis . We observed a significant difference in the expression of two distinct fliC-lacZ transcriptional fusions during bacterial growth, suggesting the presence of a latent intragenic transcriptional terminator in fliC . There was no significant difference in the virulence of 1026b compared to that of MM36 in diabetic rats or Syrian hamsters, suggesting that flagella and/or motility are probably not virulence determinants in these animal models of B . pseudomallei infection . A phylogenetic analysis based on the flagellins from a variety of bacterial species supported the recent transfer of B . pseudomallei from the genus Pseudomonas to Burkholderia. Curr Microbiol, 1997 Apr, 34(4), 250 - 7 Probing of Pseudomonas aeruginosa, Pseudomonas aureofaciens, Burkholderia (Pseudomonas) cepacia, Pseudomonas fluorescens, and Pseudomonas putida with the ferripyochelin receptor A gene and the synthesis of pyochelin in Pseudomonas aureofaciens, Pseudomonas fluorescens, and Pseudomonas putida; Castignetti D; The ferripyochelin receptor A (fptA) gene codes for the transport of the ferrisiderophore ferripyochelin in Pseudomonas aeruginosa . A P . aeruginosa fptA internal fragment was used to probe chromosomal DNA from P . aureofaciens, B . cepacia, P . fluorescens, P . putida, and five strains of P.aeruginosa . These bacteria all contained DNA that hybridized to the fptA fragment . Four of the five P . aeruginosa strains displayed marked and identical patterns, indicating a high degree of sequence similarities among these strains . DNA from the non-P . aeruginosa bacteria, in contrast, hybridized less to the fptA fragment . Pseudomonas aeruginosa and B . cepacia synthesize pyochelin . Experiments were performed to confirm P . fluorescens pyochelin synthesis and to determine if pyochelin, cepabactin or salicylic acid were made by P . aureofaciens, P . putida, and P . fluorescens . Only pyochelin was isolated and identified from P . fluorescens . P . aureofaciens and P . putida produced none of these compounds . While all of these bacteria contain chromosomal DNA that hybridized to the fptA fragment probe, pyochelin synthesis did not occur in all, indicating that fptA fragment hybridization cannot always be correlated with pyochelin biosynthesis. Zh Mikrobiol Epidemiol Immunobiol, 1997 Mar-Apr, (2), 60 - 4 {The pathomorphology and pathogenesis of glanders in laboratory animals}; Diadishchev NR et al.; The pathomorphology and cell-mediated response of the body to Burkholderia mallei in laboratory animals, highly sensitive and resistant to these bacteria . In the comparative study of the pathomorphology and pathogenesis of glanders in golden hamsters and white rats quantitative and qualitative differences in the histogenesis of response reaction and the morphology of immunocompetent organs were established . Cell-mediated reactions play a greater role in the limitation of the early spread of B . mallei in the host body than antigen-mediated mechanisms. Am J Respir Crit Care Med, 1997 Mar, 155(3), 984 - 9 PCR ribotyping and endonuclease subtyping in the epidemiology of Burkholderia cepacia infection; Shreve MR et al.; Because of conflicting data about hospital-based transmission of Burkholderia (Pseudomonas) cepacia, an important respiratory pathogen in cystic fibrosis (CF), we compared strains found in sputum, lung, or blood of 29 CF patients in our center from 1988 to 1994, studying the relationship between strain and hospital exposure of incident and that of prevalent cases . Exposure was defined as a concurrent hospital stay between a prevalent and an incident case . B . cepacia strains were determined by polymerase chain reaction (PCR) ribotyping and endonuclease subtyping . The 16S to 23S spacer regions of the bacterial ribosomal RNA (rRNA) genes were amplified by PCR, and the product-size patterns used to type each B . cepacia isolate . Endonuclease digestion of the PCR products provided length polymorphisms for subtyping . There were 17 incident events during the period from 1988 to 1994, 16 of which involved a single ribotype . These 16 ribotypes could be divided into five subtypes by endonuclease mapping . Four patients grew B . cepacia from the blood, with the organism being the same strain as found in the lung in each case . Case controls were obtained to evaluate risk factors for B . cepacia acquisition . Concurrent hospitalization with a prevalent case significantly increased the risk of acquisition . There was no association between length of hospitalization, length of exposure, or FEV1 and the risk of B . cepacia acquisition. Thorax, 1997 Mar, 52(3), 291 - 3 Exercise testing and prognosis in adult cystic fibrosis; Moorcroft AJ et al.; BACKGROUND: The assessment of prognosis is an important issue in cystic fibrosis . The prognostic value of exercise testing in comparison with other predictors of mortality was examined . METHODS: Ninety two adult patients with cystic fibrosis performed progressive maximal exercise tests and outcome was assessed at five years . The results of exercise testing were examined along with spirometric values, age, sex, body mass index (BMI), and sputum culture . RESULTS: Twenty two subjects died during the five year follow up period and 67 survived . Five subjects received a lung transplant and were excluded from the analysis . There were significant differences between those who survived and those who died: mean (SE) forced expiratory volume in one second (FEV1) 68.9 (2.7) versus 39.7 (3.5)% predicted, BMI 19.0 (0.3) versus 17.1 (0.4) kg/m2, peak oxygen uptake (VO2 peak) 66.6 (2.2) versus (53.7) (3.7)% predicted, peak work rate (Wpeak) 89.4 (3.8) versus 71.2 (5.5)% predicted, peak minute ventilation (VEpeak) 51.3 (2.0) versus 43.3 (3.1) 1/min, and ventilatory equivalent for oxygen (VE/VO2) 32.4 (0.6) versus 38.7 (1.7) . Age, sex, oxygen saturation and Burkholderia cepacia colonisation were not found to be significant predictors of mortality . When significant independent factors were entered into a multivariate logistic regression model only FEV1 was found to be a significant correlate of mortality . A cutoff for FEV1 of 55% predicted gave the best combination of specificity and sensitivity with 54% of those below this value dying within five years and 96% of those above it surviving . CONCLUSIONS: The results of maximal exercise testing are correlated with survival but they are not better than the FEV1 as prognostic indicators. Chemotherapy, 1997 Mar-Apr, 43(2), 100 - 7 Substituted amines synergistic with tobramycin against Burkholderia cepacia in vitro; Cohn RC et al.; Antibiotic-resistant Burkholderia cepacia is an important pathogen in cystic fibrosis . We have previously shown enhanced tobramycin susceptibility of most strains in the presence of the drug amiloride . Others have shown similar findings in the presence of cationic agents with amine groups . To examine the relationship of substituted amine drug structure and synergy potential with tobramycin, the in vitro activity of 45 non-antibiotic substituted amine and related compounds in the presence and absence of tobramycin was studied . Very few agents had intrinsic antimicrobial action but many were very synergistic with tobramycin . Dichloroisoproterenol and propranolol were the most synergistic . Sympathomimetic, imidazoline agents, phenothiazenes, and thioxanthenes varied greatly . Exploration of these compounds may provide an avenue for treating antibiotic-resistant B . cepacia. Gene, 1997 Feb 20, 186(1), 113 - 8 Cloning and expression of the major porin protein gene opcP of Burkholderia (formerly Pseudomonas) cepacia in Escherichia coli; Tsujimoto H et al.; The outer membrane protein OpcP1 of Burkholderia (formerly Pseudomonas) cepacia is one of the subunits forming the porin oligomer OpcPO by non-covalent association . OpcP1 was cleaved with lysyl endopeptidase and the N-terminal amino acid (aa) sequences of the polypeptide fragments were determined . Based on the sequence information, we cloned the opcP gene on a 10-kb EcoRI DNA fragment of the B . cepacia ATCC25416 chromosome . Nucleotide (nt) sequencing revealed a 1086-bp open reading frame (ORF), encoding a 361-aa polypeptide with a signal sequence of 20 residues . The predicted opcP gene encoded a mature protein of Mr 35,696, which agrees well with the value observed previously on SDS-PAGE . The opcP was sub-cloned into pTrc99A and introduced into Escherichia coli . Immunoblot analysis using murine antiserum specific to OpcP1 visualized the protein expressed in the E . coli cells after induction by isopropyl beta-D-thiogalactopyranoside (IPTG). Med J Aust, 1997 Feb 17, 166(4), 197 - 9 Fatal human melioidosis in south-eastern Queensland; Scott IA et al.; Two simultaneous human cases of fatal melioidosis in temperate south-eastern Queensland involved patients who had had pre-existing multisystem illnesses, had sustained cutaneous lesions before illness onset, and died from overwhelming sepsis . Onset of disease was preceded by unseasonably heavy rainfall . These and other features of these cases suggest that the source of infection was local, in which case the endemicity of Burkholderia pseudomallei in temperate regional Australia may be broader than is currently recognised, and melioidosis may need to be considered in at-risk patients in these areas, as well as in tropical and subtropical areas, who present with severe pneumonia and septicaemia. Mol Cell Probes, 1997 Feb, 11(1), 25 - 31 Detection of Burkholderia pseudomallei in blood samples using polymerase chain reaction; Rattanathongkom A et al.; A highly sensitive, specific, rapid and simple method to detect Burkholderia pseudomallei in blood samples was developed . Two 22-base oligonucleotide primers, based on sequences from a specific DNA probe, were used for amplification of bacterial DNA by the polymerase chain reaction (PCR) . Amplification with these primers yielded a 178-base pair product in 100 clinical isolates of B . pseudomallei . As little as 0.5 fg of B . pseudomallei DNA was detectable by this method . Experiments involving inoculation of the organism into uninfected blood samples showed that the method could be used to detect as few as 1 bacterial cell ml-1 of whole blood . Non-specific amplification of other bacterial DNAs from 18 samples of bacteria was not observed . Blood samples from seven patients proven to have melioidosis by haemoculture were positive using these primers . The total time required for sample processing, amplification and visualization was approximately 3.5 h . The high sensitivity, rapidity and simplicity of this method should make it valuable for diagnosis, monitoring of drug treatment and for epidemiological studies of the melioidosis. J Antimicrob Chemother, 1997 Feb, 39(2), 169 - 75 Isolation from clinical sources of Burkholderia cepacia possessing characteristics of Burkholderia gladioli; Baxter IA et al.; Further characterization of 32 clinical isolates originally identified as Burkholderia cepacia by biochemical and fatty acid profiling revealed the presence of 12 strains bearing partial resemblance to the closely related species Burkholderia gladioli . These strains were highly resistant to a wide range of antibiotics including ticarcillin (with or without clavulanic acid), cefsulodin, imipenem, the aminoglycosides, colistin and fosfomycin . They typically produced a red-brown pigment and possessed distinct, although quite diverse biochemical profiles . This reinforces the previous opinion that hybrids between B . cepacia and B . gladioli exist and may possess a significant pathogenic role . It also suggests that further taxonomic clarification is required in the genus Burkholderia. FEMS Immunol Med Microbiol, 1997 Feb, 17(2), 87 - 94 Serum IgG response to an outer membrane porin protein of Burkholderia cepacia in patients with cystic fibrosis; Lacy DE et al.; Early and accurate diagnosis of Burkholderia cepacia infection is important, particularly if segregation is to prevent patient-to-patient transmission . We have examined the serum response to a B . cepacia-specific 80-kDa outer membrane protein . 21 patients colonised with B . cepacia and Pseudomonas aeruginosa for 2-51 months (mean 11 months) were age- and sex-matched with 21 patients colonised with P . aeruginosa but not B . cepacia . The 80-kDa protein was recovered by electroelution from outer membrane proteins, separated by SDS-PAGE, coated onto ELISA plates, reacted with patient sera diluted 1:200, protein A-peroxidase and chromogenic substrate . We found that 19/24 patients colonised with B . cepacia and P . aeruginosa had high values, 2/24 patients had intermediate values, and 2/24 patients had a low value . 20/21 patients colonised with P . aeruginosa alone had low values and 1/21 had an intermediate value . We found that in the longitudinal serum samples studied from four patients only one patient developed high values after the first isolation of B . cepacia suggesting that seroconversion does not occur immediately after the first sputum culture of B . cepacia . We conclude that an ELISA test using B . cepacia-specific 80-kDa outer membrane protein can distinguish B . cepacia colonised and non-colonised patients and may be useful in the early diagnosis of B . cepacia infection. Pediatr Res, 1997 Feb, 41(2), 161 - 5 Identifying treatments that halt progression of pulmonary disease in cystic fibrosis; Davis PB et al.; Rapid progress in cystic fibrosis research affords the possibility of halting the progress of the lung disease . We used data from 215 patients who had sputum cultures negative for Burkholderia cepacia, at least one outpatient pulmonary function test during 1990, and at least one test a year later to estimate the number of subjects and study duration required to demonstrate that a hypothetical treatment reduces the rate of decline of forced expiratory volume in 1 s (FEV1) to zero . Mean rate of decline of FEV1 (percent predicted) was about 2% predicted per year . Variability decreases with increasing time of observation . For a 1-y study, with alpha = 0.05 and beta = 0.20, over 550 patients must complete the study in each group to show that a treatment halts pulmonary decline . For a 2-y study, 86 subjects in each group are required, and for 4 y, 65 . Increasing the number of data points used to determine the rate of decline of FEV1 had only small effect on sample size . Use of pulmonary function data collected at regular intervals for research purposes did not alter these conclusions . Higher initial FEV1 was associated with a greater rate of decline, and among patients with initial FEV1 > 60% predicted, younger subjects had a faster decline than did older subjects . Thus, fewer subjects will be required to detect a complete halt in progression of lung disease if the patients are young and have mild pulmonary disease. Infect Immun, 1997 Feb, 65(2), 617 - 22 Induction of biologically active interleukin-8 from lung epithelial cells by Burkholderia (Pseudomonas) cepacia products; Palfreyman RW et al.; The frequency of isolation of Burkholderia cepacia from the sputum of cystic fibrosis (CF) patients is increasing . Using the human A549 lung epithelial cell line, we have investigated the ability of B . cepacia exoproducts to stimulate interleukin-8 (IL-8) release . Cell-free supernatants from a panel of CF clinical, non-CF clinical, and nonclinical B . cepacia isolates were found to stimulate IL-8 release, with levels ranging from 11.8 +/- 2.8 to 80.0 +/- 3.5 ng/ml . A similar pattern was seen at the level of the IL-8 mRNA . The bioactivity of the IL-8 was confirmed by examining its effect on the intracellular free calcium in neutrophils and inhibition by a neutralizing anti-IL-8 antibody . B . cepacia lipopolysaccharide, which was able to stimulate IL-8 release from monocytes, did not release IL-8 from the A549 cells . Furthermore, the stimulating ability of the bacterial cell-free supernatant was not diminished by polymyxin B, was markedly reduced by boiling, and appeared unrelated to N-acylhomoserine lactones . The ability of B . cepacia to elicit IL-8 release from epithelial cells may be important in the pathology of CF. Infect Immun, 1997 Feb, 65(2), 472 - 7 Identification of neutralizing epitopes on Pseudomonas aeruginosa elastase and effects of cross-reactions on other thermolysin-like proteases; Kooi C et al.; Monoclonal antibodies (MAbs) to a Burkholderia (Pseudomonas) cepacia 36-kDa protease (PSCP) which neutralize PSCP and Pseudomonas aeruginosa elastase but not P . aeruginosa alkaline protease have been isolated (C . Kooi et al., Infect . Immun . 62:2811-2817, 1994) . These MAbs, designated 36-6-6 and 36-6-8, react with N-chlorosuccinimide cleavage products of P . aeruginosa elastase, consistent with the recognition of a 13.9-kDa fragment which contains the active site . Overlapping 9-mer peptides that span this region were synthesized . Neutralizing MAbs to PSCP reacted strongly with two peptides (341HGFTEQNSG349 and 395RYM DQPSRD403) . Peptide 341HGFTEQNSG349 overlaps the motif 337HEXXH341, which has been found in many zinc-dependent endopeptidases . Peptide 395RYMDQPSRD403 lies between E361, which binds a zinc atom, and H420, which acts as a proton donor at the active site . Polyclonal rabbit sera raised against these peptides reacted with elastase on Western immunoblots and by enzyme-linked immunosorbent assay . With hide powder azure as the substrate, antisera to either HGFTEQNG and RYMDQPSRD completely neutralized the activities of elastase, thermolysin, Vibrio cholerae hemagglutinin/protease, and PSCP but had no effect on P . aeruginosa alkaline protease or the Serratia marcescens major protease . These results suggest that the MAbs recognize two different epitopes on P . aeruginosa elastase and that antibodies raised against synthetic peptides corresponding to either of these epitopes neutralize proteolytic activity. Scand J Infect Dis, 1997, 29(2), 199 - 201 Two patients with recurrent melioidosis after prolonged antibiotic therapy; Silbermann MH et al.; Melioidosis is a tropical infectious disease caused by Burkholderia (Pseudomonas) pseudomallei . Clinically manifest melioidosis occurs mostly in people with underlying disorders . Melioidosis is a disease with protein manifestations and a high rate of relapse . Two patients with infection due to Burkholderia pseudomallei after a visit to Thailand are described . Both patients presented with sepsis and appropriate therapy was initiated and continued for several months . Despite such long-term antimicrobial treatment, both patients had a relapse after cessation of therapy . It is unclear if recurrent melioidosis can be prevented by prolonging the treatment even further. Ann Acad Med Singapore, 1997 Jan, 26(1), 13 - 7 Burkholderia pseudomallei infection in the Singapore Armed Forces from 1987 to 1994--an epidemiological review; Lim MK et al.; Between 1987 and 1994, twenty-three cases of Burkholderia pseudomallei infection (melioidosis) were diagnosed in persons serving in the Singapore Armed Forces . There were four deaths resulting from complications of the infection . Unlike the situation in the general population, where the affected are mainly the elderly with underlying illness, the majority of cases in the Singapore Armed Forces were otherwise fit and healthy young servicemen . Serological surveys have shown the prevalence of the infection in Singapore to be 0.2% in the military as well as civilian population . As physical contact with soil is an unavoidable part of military training, military personnel continue to be at risk of exposure to this soil-related disease. Int J Syst Bacteriol, 1997 Jan, 47(1), 19 - 27 Systematic study of the genus Vogesella gen . nov . and its type species, Vogesella indigofera comb . nov; Grimes DJ et al.; A blue-pigmented colony that had a metallic copper-colored sheen was isolated in 1973 from a standard spread plate count preparation of oxidation pond sediment . Over the next 11 years, an additional 12 strains of blue-pigmented bacteria were isolated from freshwater samples and compared to several reference strains of bacteria . Morphological and biochemical tests revealed that these 13 isolates were very similar to {Pseudomonas} indigofera ATCC 19706T (T = type strain) and ATCC 14036 . A numerical analysis (in which simple matching similarity coefficients were clustered by the unweighted pair group mathematical averaging method) of morphological and biochemical characteristics revealed 90.0% relatedness between the 13 isolates and {P.} indigofera ATCC 19706T and ATCC 14036 and 73.6% relatedness between the 13 isolates and a cluster containing Burkholderia cepacia ATCC 25416T, Janthinobacterium lividum ATCC 12473T, and the Pseudomonas species tested . A phylogenetic analysis, in which both 5S rRNA and 16S rRNA were used, also revealed that the 13 isolates were closely related to each other and to strains ATCC 19706T and ATCC 14036 . In addition, both 5S rRNA and 16S rRNA analyses demonstrated that the isolates and strains ATCC 19706T and ATCC 14036 were members of the beta subdivision of the Proteobacteria and were closely related to Chromobacterium violaceum ATCC 12742T but sufficiently distinct to warrant placement in a new genus . Accordingly, we propose that the 13 isolates and strains ATCC 19706T and ATCC 14306 be placed in the genus Vogesella gen . nov., which is named in honor of Otto Voges, who first isolated and described this blue-pigmented eubacterium in 1893 . We also propose that {P.} indigofera be renamed Vogesella indigofera comb . nov . and designated the type species of the genus; strain ATCC 19706 is the type strain of this species. J Bacteriol, 1997 Jan, 179(1), 115 - 21 2-Naphthoate catabolic pathway in Burkholderia strain JT 1500; Morawski B et al.; Burkholderia strain (JT 1500), able to use 2-naphthoate as the sole source of carbon, was isolated from soil . On the basis of growth characteristics, oxygen uptake experiments, enzyme assays, and detection of intermediates, a degradation pathway of 2-naphthoate is proposed . The features of this pathway are convergent with those for phenanthrene . We propose a pathway for the conversion of 2-naphthoate to 1 mol (each) of pyruvate, succinate, and acetyl coenzyme A and 2 mol of CO2 . During growth in the presence of 2-naphthoate, six metabolites were detected by thin-layer chromatography, high-performance liquid chromatography, and spectroscopy . 1-Hydroxy-2-naphthoate accumulated in the culture broth during growth on 2-naphthoate . Also, the formation of 2'-carboxybenzalpyruvate, phthalaldehydate, phthalate, protocatechuate, and beta-carboxy-cis,cis-muconic acid was demonstrated . (1R,2S)-cis-1,2-Dihydro-1,2-dihydroxy-2-naphthoate was thus considered an intermediate between 2-naphthoate and 1-hydroxy-2-naphthoate, but it was not transformed by whole cells or their extracts . We conclude that this diol is not responsible for the formation of 1-hydroxy-2-naphthoate from 2-naphthoate but that one of the other three diastereomers is not eliminated as a potential intermediate for a dehydration reaction. J Bacteriol, 1997 Jan, 179(1), 90 - 6 Trichloroethylene oxidation by purified toluene 2-monooxygenase: products, kinetics, and turnover-dependent inactivation; Newman LM et al.; Trichloroethylene is oxidized by several types of nonspecific bacterial oxygenases . Toluene 2-monooxygenase from Burkholderia cepacia G4 is implicated in trichloroethylene oxidation and is uniquely suggested to be resistant to turnover-dependent inactivation in vivo . In this work, the oxidation of trichloroethylene was studied with purified toluene 2-monooxygenase . All three purified toluene 2-monooxygenase protein components and NADH were required to reconstitute full trichloroethylene oxidation activity in vitro . The apparent Km and Vmax were 12 microM and 37 nmol per min per mg of hydroxylase component, respectively . Ten percent of the full activity was obtained when the small-molecular-weight enzyme component was omitted . The stable oxidation products, accounting for 84% of the trichloroethylene oxidized, were carbon monoxide, formic acid, glyoxylic acid, and covalently modified oxygenase proteins that constituted 12% of the reacted {14C}trichloroethylene . The stable oxidation products may all derive from the unstable intermediate trichloroethylene epoxide that was trapped by reaction with 4-(p-nitrobenzyl)pyridine . Chloral hydrate and dichloroacetic acid were not detected . This finding differs from that with soluble methane monooxygenase and cytochrome P-450 monooxygenase, which produce chloral hydrate . Trichloroethylene-dependent inactivation of toluene 2-monooxygenase activity was observed . All of the protein components were covalently modified during the oxidation of trichloroethylene . The addition of cysteine to reaction mixtures partially protected the enzyme system against inactivation, most notably protecting the NADH-oxidoreductase component . This suggested the participation of diffusible intermediates in the inactivation of the oxidoreductase. Carbohydr Res, 1996 Dec 13, 295, 179 - 84 Structure of the O-specific polysaccharide from Burkholderia vietnamiensis strain LMG 6998; Gaur D et al.; The putative O-specific polymer containing D-mannose and L-rhamnose was isolated from the lipopolysaccharide obtained from cells walls of Burkholderia vietnamiensis strain LMG 6998 . NMR and degradative studies showed that the polymer has a linear trisaccharide repeating-unit of the structure shown . The same polymer carrying an O-acetyl group at position 3 of the 4-substituted mannose residue has previously been found as the O antigen in the related species Burkholderia cepacia serogroup J . {formula: see text} Br J Biomed Sci, 1996 Dec, 53(4), 249 - 53 The laboratory diagnosis of melioidosis; Walsh AL et al.; The recognition of unusual, but important, pathogens such as Burkholderia pseudomallei is essential for the rapid implementation of appropriate antimicrobial therapy--delays can be fatal . Melioidosis should be considered as a potential diagnosis for any patient with exposure to areas of endemicity, and thus laboratories should be aware of the differential features of the disease and the causative organism . Isolation of B . pseudomallei is readily achieved using standard culture media such as blood, MacConkey or cystine-lactose-electrolyte-deficient (CLED) agars, and routine blood culture broths . Selective media, Ashdown's agar and selective broth, are required for respiratory tract specimens to ensure reliable isolation from amongst the normal or contaminating flora . These media are easily prepared from common media constituents . Colonial morphology and simple biochemical tests will suggest the identity of the organism, which can then be confirmed by additional tests for 'non-fermenters', such as the API 20NE. Mol Cell Probes, 1996 Dec, 10(6), 397 - 403 Polymerase chain reaction for the detection of Pseudomonas aeruginosa, Stenotrophomonas maltophilia and Burkholderia cepacia in sputum of patients with cystic fibrosis; Karpati F et al.; Occurrence of Pseudomonas aeruginosa, Stenotrophomonas (Xanthomonas) maltophilia and Burkholderia (Pseudomonas) cepacia in sputum of cystic fibrosis (CF) patients was demonstrated with a simple and rapid polymerase chain reaction (PCR) technique . The PCR was performed with a set of three primer pairs based on 16S rRNA sequences after sputum preparation with dithiothreitol and NaOH lysis . All three pathogens could be individually detected by the use of this technique . To prevent carry-over contamination, dUTP and uracil-N-glycosylase were included in the reaction . The amplicons were visualized by agarose gel electrophoresis . Sputum culture was performed on all samples . Ninety specimens from CF patients were analysed . The sensitivity for the detection of P . aeruginosa was 37/40 (93%) compared to culture . Bacterial growth of P . aeruginosa was found in three cases, where PCR amplicons were not detected, while PCR was positive in five cases, where culture did not reveal the presence of this bacterium . For this reason, the specificity was 45/50 (90%) . For S . maltophilia, the PCR was less sensitive than culture (positive in three of six cases) . In our series, B . cepacia was detected by culture in one case and this was also detected by PCR . There were no false-positive PCR results regarding S . maltophilia or B . cepacia . Thus, combined PCR-based detection of these three clinically relevant bacteria in sputum samples from CF patients can be performed by a reliable technique in a relatively simple manner . The present data indicate a high sensitivity and specificity for P . aeruginosa . The lower sensitivity observed for the detection of S . maltophilia in sputum and B . cepacia, as estimated from laboratory strains, may depend on PCR conditions and genetic heterogeneity, respectively . The greatest gains with this method can be made when it is used for the early detection of P . aeruginosa in sputum-producing CF patients. Eur Respir J, 1996 Dec, 9(12), 2612 - 7 Bacterial colonization as a potential source of nosocomial respiratory infections in two types of spirometer; Burgos F et al.; The potential risk of spirometers in the transmission of respiratory infections has not been yet established . We performed a prospective cross-sectional study to determine the rate of colonization of a water-sealed spirometer and a pneumotachograph, and the potential risk of cross-transmission of microorganisms to patients using each of these devices . Fifty four patients (aged 51 +/- 18 (mean +/- SD) yrs) were included in the study . All of them had undergone forced spirometry with bronchodilator response by means of the water-sealed spirometer (n = 36) or the pneumotachograph (n = 18) . None had a clinically apparent respiratory infection at the time of the study . Routine hygiene measures for respiratory equipment were performed before the study protocol . Samples for microbiological cultures of different parts both of the water-sealed spirometer (proximal and distal tubing, bell and water-bell) and pneumotachograph (proximal and distal tubing) were taken daily before and after the usual series of lung function tests during a 5 day period . Pharyngeal swab cultures were obtained before spirometry and 7 days later in each subject . Thirty six out of a total of 40 (90%) culture samples from the water-sealed spirometer showed microbial growth compared to 4 out of 30 (13%) samples obtained from the pneumotachograph (p < 0.0001) . Significant colonization of the water-sealed spirometer was apparent after the third day of the study . The microorganisms most frequently isolated were penicillium sp . (62%), Pseudomonas fluorescens (32%), and Burkholderia cepacea (48%) . Distal tubing, water and water-bell were the parts of the water-sealed spirometer that showed higher colonization counts (> or = 10(4) colony-forming units (cfu).mL-1) . No transmission sequence of potentially pathogenic microorganisms from equipment to patients or vice versa could be demonstrated . In summary, the water-sealed spirometer frequently became colonized by microorganisms . The potential hazard of such equipment as reservoirs of microorganisms suggests a need for the implementation of new hygiene measures for their maintenance. J Med Microbiol, 1996 Dec, 45(6), 408 - 12 Biochemical characteristics of clinical and environmental isolates of Burkholderia pseudomallei; Wuthiekanun V et al.; The biochemical characteristics of 213 isolates of Burkholderia pseudomallei from patients with melioidosis and 140 isolates from the soil in central and northeastern Thailand were compared . Whereas the biochemical profiles of all the clinical isolates were similar, all soil isolates from the central area and 25% of isolates from northeastern Thailand comprised a different phenotype . This was characterised by the ability to assimilate L-arabinose (100%), adonitol (100%), 5-keto-gluconate (90%) and D-xylose (84%), but failure to assimilate dulcitol (0%), erythritol (0%) and trehalose (10%) . Compared with clinical isolates, these organisms had similar antibiotic susceptibility profiles and were also recognised by a specific polyclonal antibody against B . pseudomallei . As melioidosis is rare in central Thailand, but common in the northeast, this raises the possibility that this biochemical phenotype may be less virulent, or may even represent a different species. J Med Microbiol, 1996 Dec, 45(6), 395 - 407 Burkholderia cepacia: medical, taxonomic and ecological issues; Govan JR et al.; The increasing challenge posed by multiresistant saprophytes in medical microbiology is strikingly demonstrated by the emergence of Burkholderia (formerly Pseudomonas) cepacia as an opportunist pathogen in immunocompromised patients, particularly individuals with chronic granulomatous disease and cystic fibrosis (CF) . Best known previously as a phytopathogen and the cause of soft rot of onions, B . cepacia presents three major problems for the CF community: innate multiresistance to antimicrobial agents; person-to-person transmission of epidemic strains through nosocomial or social contacts; and 'cepacia syndrome', a fulminating fatal pneumonia, sometimes associated with septicaemia, that occurs in approximately 20% of colonised patients, including those with previously mild disease . Accumulated evidence to dispel earlier suggestions that the organism is avirulent and merely a marker of existing lung disease includes: case-controlled studies in CF patients; reports of serious infections in non-CF patients; in-vitro and in-vivo evidence that B . cepacia induces production of pro-inflammatory markers, including the major cytokine TNFalpha; and histopathological evidence that exposure of transgenic CF mice to B . cepacia results in pneumonia . By the early 1990s, the use of selective culture media and DNA-based bacterial fingerprinting confirmed suspicions of epidemic person-to-person spread of B . cepacia . This evidence provided scientific justification for draconian and controversial measures for infection control, in particular, segregation of B . cepacia-colonised patients during treatment at CF centres and their exclusion from social gatherings and national conferences . Recently, molecular analyses of type strains and clinical isolates have revealed that isolates identified previously as B . cepacia belong to at least three distinct species and have increased concern regarding the reliability of current laboratory detection and identification systems . Clarification of the taxonomy of B . cepacia-like organisms and the pathogenic potential of environmental isolates remains a high priority, particularly when the organism's antifungal and degradative properties have created interest in its potential use as a biological control agent to improve crop yields and its use for the bioremediation of contaminated soils. Infect Immun, 1996 Dec, 64(12), 4952 - 9 Burkholderia pseudomallei activates complement and is ingested but not killed by polymorphonuclear leukocytes; Egan AM et al.; The mechanism by which Burkholderia pseudomallei is resistant to lysis by human serum is unknown but may include interference with complement activation, effective opsonization, or complement-mediated lysis . We investigated the interaction of B . pseudomallei with complement in the presence and absence of specific antibody to determine potential mechanisms of serum resistance . We demonstrated rapid activation and consumption of complement by B . pseudomallei which, in the absence of specific antibody, occurred predominantly via the alternative pathway . Complement activation was associated with deposition of the opsonically active C3b and iC3b fragments on the bacterial surface . C5b-9, detected on the bacterial surface after opsonic periods of 1 to 60 min, was susceptible to elution by 1 M NaCl, indicating that resistance to complement-mediated lysis may result from deposition of the membrane attack complex in a nonmicrobicidal location . To define the role of opsonins, we investigated the ability of polymorphonuclear leukocytes (PMNL) to phagocytose B . pseudomallei . Phagocytosis of bacteria by PMNL, and the observed oxidative response, was significantly increased by opsonization of organisms with complement and/or specific antibody . Despite opsonophagocytosis by PMNL and the production of an oxidative response, no significant bacterial killing was observed. J Clin Microbiol, 1996 Dec, 34(12), 2914 - 20 Epidemiology of Burkholderia cepacia infection in patients with cystic fibrosis: analysis by randomly amplified polymorphic DNA fingerprinting; Mahenthiralingam E et al.; We fingerprinted a collection of 627 Burkholderia cepacia isolates from 255 patients with cystic fibrosis (CF) and 43 patients without CF and from the environment, by a PCR-based randomly amplified polymorphic DNA (RAPD) method with primers selected for their ability to produce discriminatory polymorphisms . The RAPD typing method was found to be reproducible and discriminatory, more sensitive than PCR ribotyping, and able to group epidemiologically related B . cepacia strains previously typed by both pulsed-field gel electrophoresis and conventional ribotyping . Seven strain types infecting multiple CF patients were found at several different CF treatment centers in Canada, the United States, the United Kingdom, France, and Australia, indicating the presence of epidemic strain types . Most CF patients were each colonized with a single strain type, and several patients harbored the same strain type for 5 or more years . B . cepacia isolates recovered from other clinical sources (44 isolates examined) and from the environment (58 isolates examined) possessed RAPD fingerprints that were generally distinct from CF-associated strain types (525 isolates examined) . RAPD is a versatile fingerprinting method for studying the epidemiology of B . cepacia. Gene, 1996 Nov 7, 179(1), 1 - 8 Biodegradation of 2,4,5-trichlorophenoxyacetic acid by Burkholderia cepacia strain AC1100: evolutionary insight; Daubaras DL et al.; Many microorganisms in nature have evolved new genes which encode catabolic enzymes specific for chlorinated aromatic substrates, allowing them to utilize these compounds as sole sources of carbon and energy . An understanding of the evolutionary mechanisms involved in the acquisition of such genes may facilitate the development of microorganisms with enhanced capabilities of degrading highly chlorinated recalcitrant compounds . A number of studies have been based on microorganisms isolated from the environment which utilize simple chlorinated substrates . In our laboratory, a selective technique was used to isolate microorganisms capable of degrading highly chlorinated compounds, such as 2,4,5-trichlorophenoxyacetic acid (2,4,5-T), as sole sources of carbon and energy . This article summarizes the genetic and biochemical information obtained regarding the pathway of degradation, the mechanism of recruitment of new genes, and the organization of the degradative genes . In addition, we discuss the potential practical application of such microorganisms in the environment. Infect Control Hosp Epidemiol, 1996 Nov, 17(11), 741 - 3 An outbreak of Burkholderia cepacia lower respiratory tract infection associated with contaminated albuterol nebulization solution; Reboli AC et al.; An outbreak of Burkholderia cepacia lower respiratory tract colonization and infection occurred in the adult intensive-care units in various geographic locations throughout our hospital . Forty-four patients became colonized or infected over an 11-month period, whereas B cepacia had been isolated from only 13 patients in the preceding 48 months . Environmental cultures revealed the source to be extrinsically contaminated albuterol nebulization solution . Polymerase chain reaction-ribotyping confirmed the genetic relatedness of the B cepacia patient isolates and the contaminated albuterol . After extensive infection control training for the respiratory therapy staff, including attention to nebulization technique, washing and drying the nebulizer cup, and good handwashing, there have not been any new cases. Infect Control Hosp Epidemiol, 1996 Nov, 17(11), 737 - 40 Three-year outbreak of pseudobacteremia with Burkholderia cepacia traced to a contaminated blood gas analyzer; Gravel-Tropper D et al.; Between November 1990 and June 1993, Burkholderia cepacia was isolated from the blood cultures of 13 neonates born at the Ottawa General Hospital . Eight of the 13 neonates appeared symptomatic, and only 4 were treated with appropriate antimicrobial therapy, but all improved clinically . In August 1993, the blood gas analyzer in the neonatal intensive-care unit was found to be contaminated heavily with B cepacia . Eight available patient isolates were identical to the isolates recovered from the blood gas analyzer by ribotyping analysis . Infection control measures were implemented to prevent future contamination of the analyzer, and no further cases have been identified. Anal Biochem, 1996 Nov 1, 242(1), 73 - 6 Transformation of Burkholderia pseudomallei by electroporation; Mack K et al.; Conditions were defined for the successful transformation of the human pathogen Burkholderia pseudomallei 4845 by electroporation, using the incQ plasmid pKT230 . We have obtained frequencies of up to 8 x 10(3) transformants per microgram plasmid DNA with freshly prepared electrocompetent B . pseudomallei . The method also allows for easy and reproducible production of frozen cell suspensions which can produce efficiencies up to 2.5 x 10(2) transformants per microgram of plasmid DNA . Kanamycin had to be added to the culture growth medium at a minimum concentration of 20 micrograms ml-1 and a maximum concentration of 50 micrograms ml-1 for the bacteria to become electrocompetent . Bacteria grown in medium containing a final concentration of 30 micrograms ml-1 kanamycin produced the highest numbers of transformants, although the transformation frequency at this concentration was not as efficient as that at 50 micrograms ml-1 . Electron microscopy indicated that the kanamycin affects the integrity of the bacterial outer membrane, which becomes loose and wrinkled in appearance . The electrocompetence does not result in a permanent morphological change. Chest, 1996 Nov, 110(5), 1150 - 4 Bronchiolitis obliterans organizing pneumonia (BOOP) in lung transplant recipients; Chaparro C et al.; We reviewed all tissue specimens from 163 transplant patients (108 double lung transplant {DLT}, 55 single lung transplant {SLT}) between November 1983 and January 1994 for abnormalities indicating bronchiolitis obliterans organizing pneumonia (BOOP) and found 17 cases (14 DLT and 3 SLT) . Of the three SLTs, BOOP was diagnosed by open lung biopsy (OLB) in two and one was found at autopsy . Of the 14 DLTs, BOOP was diagnosed by transbronchial biopsy (TBB) specimens (9), OLB specimens (2), autopsy (1), TBB and OLB specimens (1), and OLB specimens and autopsy (1) . BOOP was found between 1 and 43 months posttransplantation; time of survival from diagnosis was between 2 and 36 months with 9 patients presently alive . Concurrent pathologic diagnosis at the time of BOOP findings were as follows: acute rejection (7) (grade 1 {4} and grade 2 {3}), BO and grade 1 rejection (2), BO and grade 2 rejection (2), BO and Aspergillus infection (1), acute alveolar injury (1), acute alveolar injury and pulmonary embolus (1), acute rejection (grade 1) and Burkholderia cepacia pneumonia (1) . No other pathologic diagnosis was found in 1 patient . In total, 11 of 17 patients (65%) had associated acute rejection . Of the 17 patients, 7 subsequently developed BO and 3 had BO before the finding of BOOP . Death occurred in 8 patients (5 DLT and the 3 SLT) between 2 and 6 months after the diagnosis . We conclude that BOOP is an important complication after lung transplantation; it was present in 13% of DLTs and 5% of SLTs . BOOP was most often associated with acute rejection. FEMS Microbiol Lett, 1996 Nov 1, 144(2-3), 203 - 6 Identification and sequencing of a novel insertion sequence, IS1471, in Burkholderia cepacia strain 2a; Xia XS et al.; A novel insertion sequence (IS), IS1471, has been identified which is inserted into IS element IS1071 possessed by plasmid pIJB1 in Burkholderia cepacia strain 2a . Nucleotide sequencing has revealed that IS1471 is 1112 bp in length and is flanked by 22/21-bp imperfect inverted repeats with a 3-bp duplication of the target sequence IS1471 contains a single open reading frame encoding a putative polypeptide of 345 amino acids with molecular mass of 39406 Da . Searches of DNA and protein sequence databases did not result in the detection of any homologous IS elements, suggesting that IS1471 is novel and may not belong to any known IS groups. FEMS Microbiol Lett, 1996 Nov 1, 144(2-3), 117 - 28 Genomic complexity and plasticity of Burkholderia cepacia; Lessie TG et al.; Burkholderia cepacia has attracted attention because of its extraordinary degradative abilities and its potential as a pathogen for plants and for humans . This bacterium was formerly considered to belong to the genus Pseudomonas in the gamma-subclass of the Proteobacteria, but recently has been assigned to the beta-subclass is based on rrn gene sequence analyses and other key phenotypic characteristics . The B . cepacia genome is comprised of multiple chromosomes and is rich in insertion sequences . These two features may have played a key role in the evolution of novel degradative functions and the unusual adaptability of this bacterium. Appl Environ Microbiol, 1996 Nov, 62(11), 4276 - 9 Purification of hydroxyquinol 1,2-dioxygenase and maleylacetate reductase: the lower pathway of 2,4,5-trichlorophenoxyacetic acid metabolism by Burkholderia cepacia AC1100; Daubaras DL et al.; The enzyme hydroxyquinol 1,2-dioxygenase, which catalyzes ortho cleavage of hydroxyquinol (1,2,4-trihydroxybenzene) to produce maleylacetate, was purified from Escherichia coli cells containing the tftH gene from Burkholderia cepacia AC1100 . Reduction of the double bond in maleylacetate is catalyzed by the enzyme maleylacetate reductase, which was also purified from E . coli cells, these cells containing the tftE gene from B . cepacia AC1100 . The two enzymes together catalyzed the conversion of hydroxyquinol to 3-oxoadipate . The purified hydroxyquinol 1,2-dioxygenase was specific for hydroxyquinol and was not able to use catechol, tetrahydroxybenzene, 6-chlorohydroxyquinol, or 5-chlorohydroxyquinol as its substrate . The native molecular mass of hydroxyquinol 1,2-dioxygenase was 68 kDa, and the subunit size of the protein was 36 kDa, suggesting a dimeric protein of identical subunits. J Bacteriol, 1996 Nov, 178(21), 6327 - 37 Cascade regulation of the toluene-3-monooxygenase operon (tbuA1UBVA2C) of Burkholderia pickettii PKO1: role of the tbuA1 promoter (PtbuA1) in the expression of its cognate activator, TbuT; Byrne AM et al.; Burkholderia pickettii PKO1 metabolizes toluene and benzene via a chromosomally encoded toluene-3-monooxygenase pathway . Expression of the toluene-3-monooxygenase operon (tbuA1UBVA2C) is activated by the regulator, TbuT, in the presence of toluene . We have identified the TbuT coding region downstream of the toluene-3-monooxygenase structural genes by nucleotide sequence analysis and have shown that although TbuT is similar to XylR and DmpR, two members of the NtrC family of transcriptional activators which control toluene-xylene and (methyl)phenol catabolism, respectively, it is significantly different in the domain associated with effector specificity . Using a tbuA1-lacZ fusion reporter system, we determined that TbuT is activated not only by aromatic effectors but also the chlorinated aliphatic hydrocarbon trichloroethylene . Expression of tbuT and that of the tbuA1UBVA2C operon were found to be linked by readthrough transcription of tbuT from the toluene-3-monooxygenase promoter . As a result, transcription of tbuT is low when the toluene-3-monooxygenase operon is uninduced and high when expression of tbuA1UBVA2C is induced by toluene . Thus, the toluene-3-monooxygenase promoter drives the cascade expression of both the toluene-3-monooxygenase operon and tbuT, resulting in a positive feedback circuit . Examination of the nucleotide sequence upstream of the toluene-3-monooxygenase operon for promoter-like sequences revealed a -24 TGGC, -12 TTGC sequence, characteristic of sigma54 (rpoN)-dependent promoters . Primer extension and tbuA1-lacZ fusion analyses demonstrated that this -24, -12 promoter sequence, referred to as PtbuA1, was the toluene-3-monooxygenase promoter . Upstream of PtbuA1, a DNA region with dyad symmetry exhibited homology with the XylR-binding site present upstream of the Pu promoter . Deletions within this DNA sequence resulted in complete loss of expression from PtbuA1, suggesting that this region may serve as the TbuT-binding site. J Biol Chem, 1996 Oct 18, 271(42), 25754 - 61 Identification, characterization, and cloning of a phosphonate monoester hydrolase from Burkholderia caryophilli PG2982; Dotson SB et al.; The glyphosate-degrading bacterium, Burkholderia caryophilli PG2982, was observed to utilize glyceryl glyphosate as a sole phosphorus source . The hydrolysis of glyceryl glyphosate to glyphosate by a phosphonate ester hydrolase (PEH) was identified as the first metabolic step in the mineralization pathway . This observation provides the first biological role for a phosphonate ester hydrolase activity . Purified PEH enzyme hydrolyzed several phosphonate esters including p-nitrophenyl phenylphosphonate, beta-naphthyl phenylphosphonate, and 5-bromo-4-chloro-3-indolyl phenylphosphonate . The purified PEH also hydrolyzed some phosphodiesters including p-nitrophenyl 5'-thymidine monophosphate and p-nitrophenyl phosphorylcholine . The most catalytically efficient substrate identified was bis-(p-nitrophenyl) phosphate with a Km of 0.9 mM and a kcat of 6.2 x 10(2) min-1, suggesting that the enzyme may also function in vivo as a phosphodiesterase . The native enzyme was a homotetramer of 58-kDa subunits and exhibited a pI of 4.2 . The enzyme activity had a pH activity optimum of 9.0 and was stimulated 14-fold by Mn2+ ions, but a metal cofactor was not essential for activity . N-terminal and tryptic fragment amino acid sequences were obtained from the purified PEH protein and used to clone the B . caryophilli PG2982 gene, designated pehA . The unique substrate specificity of the enzyme and potential use as a novel conditional lethal gene in plants are discussed. Gene, 1996 Oct 10, 175(1-2), 43 - 8 Identification and nucleotide sequence of Rhizobium meliloti insertion sequence ISRm6, a small transposable element that belongs to the IS3 family; Zekri S et al.; The insertion sequence ISRm6 is a small transposable element identified in Rhizobium meliloti strain GR4 by sequence analysis . Two copies of this IS element were found in strain GR4, one of them is linked to the nfe genes located on plasmid pRmeGR4b . ISRm6 seems to be widespread in R . meliloti . Data suggest that ISRm6 is active in transposition at an estimated frequency of 2 x 10(-5) per generation per cell in strain GR4 . This 1269-bp element carries 27/26-bp terminal imperfect inverted repeats with six mismatches and a direct target site duplication of 4 bp . The IR terminate with the dinucleotide 5'-TG as all the members of the IS3 family . In addition, as other IS belonging to the IS3 family, ISRm6 carries two open reading frames (ORFA and ORFB) with a characteristic translational frame-shifting window in the overlapping region . Furthermore, ISRm6 putative transposase contains the triad of amino acids called DDE motif . Comparison of the ISRm6 DNA sequence and the putative proteins encoded with sequences derived from the EMBL, GenBank, PIR and Swissprot databases showed significant similarity to IS that belongs to the IS3 family with a highest homology to a subclass containing IS476 from Xanthomonas campestris, IS407 from Burkholderia cepacia, and ISR1 from Rhizobium lupini. Gene, 1996 Oct 3, 174(2), 191 - 4 A genetic analysis system of Burkholderia cepacia: construction of mobilizable transposons and a cloning vector; Abe M et al.; A genetic analysis system of Burkholderia cepacia (Bc) was developed which included transposon mutagenesis and complementation of mutation with the cloned genes of interest . To deliver the transposon in this multidrug-resistant microorganism, two plasmids, pKN30 and pKN31, were constructed which contained Tn5 derivatives, Tn5-30Tp and Tn5-31Tp, respectively, carrying KmR and TpR genes . The plasmids have the origin of ColE1 replication and the mobilization gene of RP4 . Tn5-31Tp was mobilized to Bc KF1, a strain isolated from a pneumonia patient, by the transfer system of RP4 integrated in the chromosome of Escherichia coli (Ec) . Selection with trimethoprim resulted in generation of a number of transposants of Bc KF1 . Fourteen protease-deficient mutants were isolated, all of which contained a single transposon marker in the chromosome . Thirteen protease-deficient mutants were also lipase deficient . An Ec-Bc shuttle plasmid, pTS1209, was constructed that consists of oriColE1, oripSa, ApR and CmR genes, and several unique restriction sites for cloning . Plasmid pTS1209 was successfully employed for cloning genes of Bc involved in protease production. J Biomol NMR, 1996 Oct, 8(3), 311 - 8 Measurement of three-bond coupling constants in the osmoregulated periplasmic glucan of Burkholderia solanacearum; Lippens G et al.; The cyclic osmoregulated periplasmic glucan produced by Burkholderia solanacearum contains 13 glucose units, all beta-(1-2) linked except for one alpha-(1-6) linkage . We report here the measurement of the 3J(C1-H2') and 3J(H1-C2') coupling constants, characterizing the glycosidic linkages, through the use of a 13C/12C double half-filtered NOESY experiments . The values obtained give information about the (phi, psi) angles of the different linkages . The results presented from an important step towards a detailed experimental model of the cyclic glucan, which might allow us to clarify its biological role and establish whether the cavity of these molecules is compatible with the capability of complexing host molecular signals. Infect Immun, 1996 Oct, 64(10), 4378 - 80 The dsbB gene product is required for protease production by Burkholderia cepacia; Abe M et al.; Burkholderia cepacia KF1, isolated from a pneumonia patient, produces a 37-kDa extracellular metalloprotease . A protease-deficient and lipase-proficient mutant, KFT1007, was complemented by a clone having an open reading frame coding for a 170-amino-acid polypeptide which showed significant homology to Escherichia coli DsbB . KFT1007, a presumed dsbB mutant, also failed to show motility, and both protease secretion and motility were restored by the introduction of the cloned dsbB gene of B . cepacia . The mutant KFT1007 excreted a 43-kDa polypeptide that is immunologically related to the 37-kDa mature protease . These results suggested that the dsbB mutant secretes a premature and catalytically inactive form of protease and that disulfide formation is required for the production of extracellular protease by B . cepacia. Infect Immun, 1996 Oct, 64(10), 4054 - 9 Invasion of respiratory epithelial cells by Burkholderia (Pseudomonas) cepacia; Burns JL et al.; Pulmonary infections caused by Burkholderia (Pseudomonas) cepacia are an important cause of morbidity and mortality in cystic fibrosis (CF) patients . Several features suggestive of cellular invasion and intracellular sequestration of B . cepacia in CF are persistence of infection in the face of antibiotic therapy to which the organism demonstrates in vitro susceptibility and a propensity to cause bacteremic infections in patients with CF . Epithelial cell invasion was demonstrated in vitro in A549 cells by a modified gentamicin protection assay . The kinetics of invasion appear to be saturable . Electron microscopy of invaded monolayers showed intracytoplasmic bacteria enclosed by membrane-bound vacuoles . No lysosomal fusion with these vacuoles was observed . Intraepithelial cell replication was suggested by electron microscopy and confirmed by both a quantitative assay and a visual assay . Cytochalasin D, but not colchicine, inhibited invasion, suggesting a role for microfilaments but not microtubules . The invasion phenotype in B . cepacia may be an important virulence factor for CF infections. Am J Infect Control, 1996 Oct, 24(5), 389 - 95 Microbial contamination of antiseptics and disinfectants; Oie S et al.; BACKGROUND: There have been a number of reports on microbial contamination of antiseptics and disinfectants . At present, however, the necessity of measures to prevent contamination do not seem to be fully appreciated . We investigated microbial contamination of antiseptics and disinfectants that are used in our hospital . METHODS: Fifty-one samples of benzalkonium chloride and chlorhexidine gluconate that were being used in the hospital were examined . Viability of the contaminants detected in these samples was also tested in the agents . Then we examined measures to prevent contamination of these agents . RESULTS: Microbial contamination was detected at 10(2) to 10(7) CFU/ml in the following samples: 6 of 23 samples of cotton balls soaked in 0.02% benzalkonium chloride kept in a canister for antisepsis and disinfection (26.1%); 7 of 13 samples of 0.02%, benzalkonium chloride or 0.02% chlorhexidine gluconate in an irrigation apparatus kept at 37 degrees C for vaginal douching (53.8%); and 9 of 15 samples of 0.02% benzalkonium chloride or 0.05% chlorhexidine gluconate for storage of suction catheters in a plastic bottle (60%) . The major contaminants were Burkholderia cepacia, Pseudomonas aeruginosa, Xanthomonas maltophilia, and Pseudomonas fluorescens . The first two organisms examined grew in the agents . After improvements in the handling of the antiseptics and disinfectants, no microbial contamination was observed . CONCLUSIONS: It is necessary to check microbial contamination of diluted benzalkonium chloride and diluted chlorhexidine gluconate that are in use . Such products are not recommended as antiseptics. J Bacteriol, 1996 Oct, 178(20), 6019 - 24 Purification and sequence analysis of 4-methyl-5-nitrocatechol oxygenase from Burkholderia sp . strain DNT; Haigler BE et al.; 4-Methyl-5-nitrocatechol (MNC) is an intermediate in the degradation of 2,4-dinitrotoluene by Burkholderia sp . strain DNT . In the presence of NADPH and oxygen, MNC monooxygenase catalyzes the removal of the nitro group from MNC to form 2-hydroxy-5-methylquinone . The gene (dntB) encoding MNC monooxygenase has been previously cloned and characterized . In order to examine the properties of MNC monooxygenase and to compare it with other enzymes, we sequenced the gene encoding the MNC monooxygenase and purified the enzyme from strain DNT . dntB was localized within a 2.2-kb ApaI DNA fragment . Sequence analysis of this fragment revealed an open reading frame of 1,644 bp with an N-terminal amino acid sequence identical to that of purified MNC monooxygenase from strain DNT . Comparison of the derived amino acid sequences with those of other genes showed that DntB contains the highly conserved ADP and flavin adenine dinucleotide (FAD) binding motifs characteristic of flavoprotein hydroxylases . MNC monooxygenase was purified to homogeneity from strain DNT by anion exchange and gel filtration chromatography . Sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed a single protein with a molecular weight of 60,200, which is consistent with the size determined from the gene sequence . The native molecular weight determined by gel filtration was 65,000, which indicates that the native enzyme is a monomer . It used either NADH or NADPH as electron donors, and NADPH was the preferred cofactor . The purified enzyme contained 1 mol of FAD per mol of protein, which is also consistent with the detection of an FAD binding motif in the amino acid sequence of DntB . MNC monooxygenase has a narrow substrate specificity . MNC and 4-nitrocatechol are good substrates whereas 3-methyl-4-nitrophenol, 3-methyl-4-nitrocatechol, 4-nitrophenol, 3-nitrophenol, and 4-chlorocatechol were not . These studies suggest that MNC monooxygenase is a flavoprotein that shares some properties with previously studied nitrophenol oxygenases. Am Fam Physician, 1996 Sep 15, 54(4), 1291 - 7 Treatment of cystic fibrosis in adults; Hamer L et al.; More patients with cystic fibrosis are surviving into adulthood . Primary care physicians need a basic understanding of adult cystic fibrosis and the evaluation of patients with acute decompensation . Respiratory decompensation is usually the result of infective agents, including Pseudomonas aeruginosa, Stentrophomonas maltophilia and Burkholderia cepacia, and requires treatment with intravenous antibiotics . If symptoms worsen, the possibility of another complication, such as pneumothorax, hemoptysis, mycobacterial infection and allergic bronchopulmonary aspergillosis, must be considered . Family physicians can play an important role in the birth to adult care of patients with cystic fibrosis. Southeast Asian J Trop Med Public Health, 1996 Sep, 27(3), 592 - 9 Evolution of cell-surface acid phosphatase of Burkholderia pseudomallei; Kondo E et al.; Acid phosphatase active fractions were obtained from cell-free extract, outermembrane fraction and culture filtrate of Burkholderia pseudomallei by column chromatography with sepharose 6B and DEAE cellulose . The comparison of the elution patterns of protein, sugar and enzymatic activity among these three components suggested that the enzyme is a glycoprotein evolving from premature proteins through glycosylation and that the enzyme is translocated during glycosylation from the cytoplasm to the outer membrane and finally excreted into the environment . When tunicamycin, a glycosylation inhibitor, was added to the culture, the peaks of sugar and enzymatic activity were lowered concomitantly leaving the protein peak unchanged in the elution pattern of the culture filtrate . The affinity of the bacterial surface to antienzyme sera was demonstrated by immuno-fluorescence microscopy. Southeast Asian J Trop Med Public Health, 1996 Sep, 27(3), 584 - 91 Affinity and response of Burkholderia pseudomallei and Burkholderia cepacia to insulin; Kanai K et al.; The cells of Burkholderia pseudomallei, B . cepacia and Pseudomonas aeruginosa grown on agar plates were stained with fluorescently-labeled insulin . The former two species were stained positively indicating insulin binding but P . aeruginosa was not . Insulin exposure reduced phospholipase C and acid phosphatase activities of B . pseudomallei but did not affect those enzymatic activities of B . cepacia in the employed experimental conditions . It is suggested that B . pseudomallei have insulin receptors which may be associated with a signal transfer system involving phospholipase and protein tyrosine phosphatase. Eur J Clin Microbiol Infect Dis, 1996 Sep, 15(9), 755 - 8 Lack of evidence of nosocomial cross-infection by Burkholderia cepacia among Danish cystic fibrosis patients; Ryley HC et al.; Burkholderia cepacia isolates from nine of the ten Danish cystic fibrosis (CF) patients known between 1975 and the present day to carry this organism were investigated . Eight distinct genotypes were found with polymerase chain reaction ribotyping and pulsed-field gel electrophoresis . The results indicate that there is little patient-to-patient cross-infection with Burkholderia cepacia within the Danish CF population, even though the majority of patients attend the same CF clinic on a regular basis. J Hosp Infect, 1996 Sep, 34(1), 59 - 69 An outbreak of pseudobacteraemia caused by Burkholderia pickettii: the critical role of an epidemiological link; Luk WK; A protracted outbreak of pseudobacteraemia caused by Burkholderia pickettii is reported . Initial investigation did not reveal the source of the outbreak . Subsequently, careful examination of patient records provided an insight to the probable source, which was further confirmed by a case control study . It was found that 48% of cases had either hepatobiliary diseases or haematological malignancies, whereas only 15% of the controls had similar problems . Furthermore, a coagulation study was performed on all of the cases on the day the blood culture was taken, but was performed on only 17% of the controls . With the establishment of this epidemiological link, a breakdown in aseptic blood culture technique was suspected . B . pickettii was isolated from citrated bottles used in the wards and from the citrate solution stocked in the haematology laboratory . The clonality of these strains was established by the pulsed-field gel electrophoresis (PFGE) and the arbitrary primed-polymerase chain reaction (AP-PCR) . The phlebotomists were notified of the findings of the outbreak investigation, and the importance of strict adherence to the aseptic blood culture technique was re-emphasized. J Hosp Infect, 1996 Sep, 34(1), 51 - 8 Bacterial contamination of commercially available ethacridine lactate (acrinol) products; Oie S et al.; Bacterial contamination of commercially available ethacridine lactate (acrinol) solutions and cotton gauze soaked in ethacridine lactate solution was investigated . Of 56 samples from ethacridine lactate solutions (eight products, seven manufacturers), seven samples (12.5%) of two products (two manufacturers) were contaminated with 10(1)-10(4) colony forming units (cfu)/mL of Burkholderia pickettii . Of 67 samples obtained from gauze soaked in ethacridine lactate solution (seven products, seven manufacturers), 41 (61.2%) of six products (six manufacturers) were contaminated with 10(2)-10(6) cfu/mL of bacteria . The major bacteria detected were Burkholderia cepacia and Burkholderia pickettii . This relatively high incidence of bacterial contamination in commercially available cotton gauze soaked in ethacridine lactate solution may be due to the presence of gauze in ethacridine lactate solution. J Hosp Infect, 1996 Sep, 34(1), 43 - 9 Antimicrobial activity of superoxidized water; Tanaka H et al.; We tested the antimicrobial activity of superoxidized water against methicillin-sensitive Staphylococcus aureus, methicillin-resistant Staphylococcus aureus, Staphylococcus epidermidis, Serratia marcescens, Escherichia coli, Pseudomonas aeruginosa and Burkholderia cepacia . The number of bacteria was reduced below detection limit following incubation in superoxidized water for 10 s . The bactericidal activity of superoxidized water was similar to that of 80% ethanol, but superior to that of 0.1% chlorhexidine and 0.02% povidone iodine . We conclude that superoxidized water is a low cost but powerful disinfectant. Microbiol Rev, 1996 Sep, 60(3), 539 - 74 Microbial pathogenesis in cystic fibrosis: mucoid Pseudomonas aeruginosa and Burkholderia cepacia; Govan JR et al.; Respiratory infections with Pseudomonas aeruginosa and Burkholderia cepacia play a major role in the pathogenesis of cystic fibrosis (CF) . This review summarizes the latest advances in understanding host-pathogen interactions in CF with an emphasis on the role and control of conversion to mucoidy in P . aeruginosa, a phenomenon epitomizing the adaptation of this opportunistic pathogen to the chronic chourse of infection in CF, and on the innate resistance to antibiotics of B . cepacia, person-to-person spread, and sometimes rapidly fatal disease caused by this organism . While understanding the mechanism of conversion to mucoidy in P . aeruginosa has progressed to the point where this phenomenon has evolved into a model system for studying bacterial stress response in microbial pathogenesis, the more recent challenge with B . cepacia, which has emerged as a potent bona fide CF pathogen, is discussed in the context of clinical issues, taxonomy, transmission, and potential modes of pathogenicity. Microbiology, 1996 Sep, 142 ( Pt 9), 2407 - 18 4-Methylphthalate catabolism in Burkholderia (Pseudomonas) cepacia Pc701: a gene encoding a phthalate-specific permease forms part of a novel gene cluster; Saint CP et al.; We have determined the entire nucleotide sequence of a 4.4 kbp fragment of pMOP, a plasmid involved in 4-methylphthalate catabolism in Burkholderia cepacia (formerly Pseudomonas cepacia) Pc701 . Two complete ORFs were identified and termed mopA and mopB . mopB encodes a 4-methylphthalate permease which is a member of a superfamily of symport proteins found in both prokaryotes and eukaryotes . Functionality was assigned to MopB by detailed analysis of the predicted amino acid sequence, resulting in the identification of 12 hydrophobic membrane-spanning domains and motifs associated with this class of protein . An assay was developed to demonstrate MopB function in substrate uptake . Of 4-methylphthalate, 4-hydroxyisophthalate, benzoate, p-toluate and phthalate, only uptake of 4-methylphthalate and phthalate was demonstrated, suggesting that two carboxyl groups in the ortho position are essential for substrate recognition . The predicted protein MopA showed significant levels of homology to reductase proteins implicated in aromatic and aliphatic catabolism, and contained motifs recognized as binding the ADP and flavin moieties of FAD/NAD . Northern hybridization experiments determined that mopA and mopB are contranscribed, but expression was only seen in cells grown on 4-methylphthalate and not in cells grown on closely related structural analogues, including phthalate . mopA and mopB may be situated at the 3' terminus of a cistron about 10 kbp in size . The isolation and characterization of a 4-methylphthalate permease gene may lead to the identification of other permeases involved in bacterial biodegradation processes and possibly the construction of strains with enhanced degradative abilities. Biochem J, 1996 Aug 15, 318 ( Pt 1), 157 - 62 Molecular cloning and overexpression of a glutathione transferase gene from Proteus mirabilis; Perito B et al.; The structural gene of the Proteus mirabilis glutathione transferase GSTB1-1 (gstB) has been isolated from genomic DNA . A nucleotide sequence determination of gstB predicted a translational product of 203 amino acid residues, perfectly matching the sequence of the previously purified protein {Mignogna, Allocati, Aceto, Piccolomini, Di Ilio, Barra and Martini (1993) Eur . J . Biochem . 211, 421-425} . The P . mirabilis GST sequence revealed 56% identity with the Escherichia coli GST at DNA level and 54% amino acid identity . Similarity has been revealed also with the translation products of the recently cloned gene bphH from Haemophilus influenzae (28% identity) and ORF3 of Burkholderia cepacia (27% identity) . Putative promoter sequences with high similarity to the E . coli sigma 70 consensus promoter and to promoters of P . mirabilis cat and glnA genes preceded the ATG of the gstB open reading frame (ORF) . gstB was brought under control of the tac promoter and overexpressed in E . coli by induction with isopropyl-beta-D-thiogalactopyranoside and growth at 37 degrees C . The physicochemical and catalytic properties of overexpressed protein were indistinguishable from those of the enzyme purified from P . mirabilis extract . Unlike the GST belonging to Mu and Theta classes, GSTB1-1 was unable to metabolize dichloromethane . The study of the interaction of cloned GSTB1-1 with a number of antibiotics indicates that this enzyme actively participates in the binding of tetracyclines and rifamycin. Plant J, 1996 Aug, 10(2), 383 - 92 A phosphonate monoester hydrolase from Burkholderia caryophilli PG2982 is useful as a conditional lethal gene in plants; Dotson SB et al.; A bacterial phosphonate monoester hydrolase was evaluated in plants as a conditional lethal gene useful for cell ablation and negative selection . Glyphosate is a potent herbicide whereas its phosphonate monoester derivative, glyceryl glyphosate, is approximately 50-fold less active . A phosphonate monoesterase gene (pehA) encoding an enzyme that hydrolyzes phosphonate esters including glyceryl glyphosate to glyphosate and glycerol was cloned from the glyphosate metabolizing bacterium, Burkholderia caryophilli PG2982 . Constitutive expression of the pehA gene in Escherichia coli and Arabidopsis thaliana RLD had no observable phenotypic effects on growth and development . However, cells and plants expressing the pehA gene were killed when treated with glyceryl glyphosate . The phytotoxicity resulted from the hydrolysis of glyceryl glyphosate to glyphosate and subsequent inhibition of aromatic amino acid biosynthesis . As an example of tissue-specific cell ablation, floral sterility without vegetative toxicity was demonstrated by expressing the pehA gene using a tapetal-specific promoter and treating the mature plants with glyceryl glyphosate . A chromogenic phosphonate ester substrate, 5-bromo-4-chloro-indolyl phenylphosphonate, was used to monitor in situ expression of the pehA gene . The general utility of the pehA gene as a heterologous conditional lethal gene in plants is discussed. J Bacteriol, 1996 Aug, 178(16), 4926 - 34 2,4-Dinitrotoluene dioxygenase from Burkholderia sp . strain DNT: similarity to naphthalene dioxygenase; Suen WC et al.; 2,4-Dinitrotoluene (DNT) dioxygenase from Burkholderia sp . strain DNT catalyzes the initial oxidation of DNT to form 4-methyl-5-nitrocatechol (MNC) and nitrite . The displacement of the aromatic nitro group by dioxygenases has only recently been described, and nothing is known about the evolutionary origin of the enzyme systems that catalyze these reactions . We have shown previously that the gene encoding DNT dioxygenase is localized on a degradative plasmid within a 6.8-kb NsiI DNA fragment (W.-C . Suen and J . C . Spain, J . Bacteriol . 175:1831-1837, 1993) . We describe here the sequence analysis and the substrate range of the enzyme system encoded by this fragment . Five open reading frames were identified, four of which have a high degree of similarity (59 to 78% identity) to the components of naphthalene dioxygenase (NDO) from Pseudomonas strains . The conserved amino acid residues within NDO that are involved in cofactor binding were also identified in the gene encoding DNT dioxygenase . An Escherichia coli clone that expressed DNT dioxygenase converted DNT to MNC and also converted naphthalene to (+)-cis-(1R,2S)-dihydroxy-1,2-dihydronaphthalene . In contrast, the E . coli clone that expressed NDO did not oxidize DNT . Furthermore, the enzyme systems exhibit similar broad substrate specificities and can oxidize such compounds as indole, indan, indene, phenetole, and acenaphthene . These results suggest that DNT dioxygenase and the NDO enzyme system share a common ancestor. Appl Environ Microbiol, 1996 Aug, 62(8), 3005 - 10 An obligately endosymbiotic mycorrhizal fungus itself harbors obligately intracellular bacteria; Bianciotto V et al.; Arbuscular-mycorrhizal fungi are obligate endosymbionts that colonize the roots of almost 80% of land plants . This paper describes the employment of a combined morphological and molecular approach to demonstrate that the cytoplasm of the arbuscular-mycorrhizal fungus Gigaspora margarita harbors a further bacterial endosymbiont . Intracytoplasmic bacterium-like organisms (BLOs) were detected ultrastructurally in its spores and germinating and symbiotic mycelia . Morphological observations with a fluorescent stain revealed about 250,000 live bacteria inside each spore . The sequence for the small-subunit rRNA gene obtained for the BLOs from the spores was compared with those for representatives of the eubacterial lineages . Molecular phylogenetic analysis unambiguously showed that the endosymbiont of G . margarita was an rRNA group II pseudomanad (genus Burkholderia) . PCR assays with specifically designed oligonucleotides were used to check that the sequence came from the BLOs . Successful amplification was obtained when templates from both the spores and the symbiotic mycelia were used . A band of the expected length was also obtained from spores of a Scutellospora sp . No bands were given by the negative controls . These findings indicate that mycorrhizal systems can include plant, fungal, and bacterial cells. Appl Environ Microbiol, 1996 Aug, 62(8), 2716 - 22 Chloroform mineralization by toluene-oxidizing bacteria; McClay K et al.; Seven toluene-oxidizing bacterial strains (Pseudomonas mendocina KR1, Burkholderia cepacia G4, Pseudomonas putida F1, Pseudomonas pickettii PKO1, and Pseudomonas sp . strains ENVPC5, ENVBF1, and ENV113) were tested for their ability to degrade chloroform (CF) . The greatest rate of CF oxidation was achieved with strain ENVBF1 (1.9 nmol/min/mg of cell protein) . CF also was oxidized by P . mendocina KR1 (0.48 nmol/min/mg of cell protein), strain ENVPC5 (0.49 nmol/min/mg of cell protein), and Escherichia coli DH510B(pRS202), which contained cloned toluene 4-monooxygenase genes from P . mendocina KR1 (0.16 nmol/min/mg of cell protein) . Degradation of {14C}CF and ion analysis of culture extracts revealed that CF was mineralized to CO2 (approximately 30 to 57% of the total products), soluble metabolites (approximately 15%), a total carbon fraction irreversibly bound to particulate cellular constituents (approximately 30%), and chloride ions (approximately 75% of the expected yield) . CF oxidation by each strain was inhibited in the presence of trichloroethylene, and acetylene significantly inhibited trichloroethylene oxidation by P . mendocina KR1 . Differences in the abilities of the CF-oxidizing strains to degrade other halogenated compounds were also identified . CF was not degraded by B . cepacia G4, P . putida F1, P . pickettii PKO1, Pseudomonas sp . strain ENV113, or P . mendocina KRMT, which contains a tmo mutation. J Formos Med Assoc, 1996 Jul, 95(7), 562 - 6 Melioidosis: two indigenous cases in Taiwan; Lee SS et al.; We report the first two indigenously acquired cases of melioidosis in Taiwan, diagnosed by positive culture and biochemically identified using the ID 32 GN system (BioMerieux Vitek Inc, Hazelwood, MO, USA) . The first patient was a 75-year-old Chinese woman who had not travelled abroad since her arrival from mainland China (San-Tung province) 47 years ago . She presented with spontaneous bacterial peritonitis and hepatitis C-related liver cirrhosis with septic shock . Burkholderia pseudomallei (formerly Pseudomonas pseudomallei) was isolated from cultures of both blood and ascites fluid . The second patient, a 70-year-old Chinese man, presented with right lower lobar pneumonia complicated with empyema and septic shock . Blood cultures grew B . pseudomallei . Both patients had underlying diabetes mellitus; one also had liver cirrhosis and chronic renal failure, while the other had a renal stone . The first patient died of refractory septic shock prior to diagnosis . The second patient survived with the use of intravenous ceftazidime for 30 days, followed by oral amoxicillin-clavulanic acid for a further 3 months . These cases serve as a reminder to clinical physicians that melioidosis is now no longer exclusive to patients with a history of travel to endemic areas . A high index of clinical suspicion is required for early diagnosis and treatment in order to reduce the mortality and improve clinical outcome. J Clin Microbiol, 1996 Jul, 34(7), 1610 - 6 Identification of IS1356, a new insertion sequence, and its association with IS402 in epidemic strains of Burkholderia cepacia infecting cystic fibrosis patients; Tyler SD et al.; Burkholderia cepacia is now recognized as an important opportunistic pathogen in cystic fibrosis (CF) and other compromised patients . Epidemicity among CF patients has been attributed to at least one particularly infectious strain (strain ET12), and both genetic evidence and anecdotal evidence suggest that this strain, currently endemic in Ontario, and those causing an epidemic in the United Kingdom, are indeed the same . Our study was conducted to determine whether there was any association between the presence of various insertion sequence (IS) elements, the cable pilin subunit gene (cblA), electrophoretic type (ET), and ribotype (RT) in a collection of 97 clinical and 2 environmental isolates of B . cepacia . No apparent linkage was found for IS elements IS401, IS402, IS406, IS407, and IS408 with ET or RT . The cblA target, said to be a marker for high infectivity, was detected in 100% (38 of 38) of strains of B . cepacia ET12 and in a single strain of ET13 that differed in a single enzyme allele . A new IS, IS1356, identified during the investigation, was present in 71.7% of all isolates, and 50.7% of these isolates harbored IS1356 as a hybrid IS element inserted into IS402 . IS1356 is 1,353 bp in length, and when it is inserted into IS402 it results in a 10-bp duplication at the site of insertion . IS1356 contains one major open reading frame of 1,260 bp coding for a putative transposase which has significant homology to ISRm3 in Rhizobium meliloti (59%) and to an undesignated IS element in Corynebacterium diphtheriae (49%) . The IS402-IS1356 element was found exclusively in the epidemic strains from Ontario and the United Kingdom, being detected in 94.7% (36 of 38 isolates) of B . cepacia ET12 isolates . Of the two ET12 isolates found to be devoid of the IS402-IS1356 element, both contained IS1356 unassociated with IS402, one was temporally unrelated to the epidemic, and the other was from a CF patient in a geographic area remote from Ontario and the United Kingdom . It is evident that the IS402-IS1356 hybrid element, the cblA pilin subunit gene, and the allelic suite represented by multilocus enzyme electrophoretic type ET12 may provide useful markers for the epidemic, highly transmissible transatlantic strain isolated in Ontario and the United Kingdom. Infect Immun, 1996 Jul, 64(7), 2824 - 8 Structural and immunological characterization of Burkholderia pseudomallei O-polysaccharide-flagellin protein conjugates; Brett PJ et al.; The O-polysaccharide moiety of Burkholderia pseudomallei 319a lipopolysaccharide was covalently linked to flagellin protein isolated from the same strain . A glycoconjugate incorporating adipic acid dihydrazide as a spacer molecule elicited high-titer immunoglobulin G responses to both the protein and carbohydrate components of the construct . This immunoglobulin G was capable of protecting diabetic rats from challenge with a heterologous B . pseudomallei strain. Southeast Asian J Trop Med Public Health, 1996 Jun, 27(2), 263 - 6 Indirect hemagglutination antibodies against Burkholderia pseudomallei in normal blood donors and suspected cases of melioidosis in Malaysia; Norazah A et al.; Interpretation of the indirect hemagglutination test (IHA) for melioidosis in endemic areas is difficult because of the presence of antibodies in apparently healthy individuals . Fifty-three out of 200 healthy blood donors in Malaysia showed positive antibody titers (> or = 1 : 40) against Burkholderia pseudomallei . Seven percent had an IHA titer of 1 : 40, 11% had an IHA titer of 1 : 80 while 8.5% had a titer > or = 1 : 160 . Out of 258 sera sent for melioidosis serology, 7% of the patients had an IHA titer of 1 : 40, 9% had an IHA titer of 1 : 80 while 20% had an IHA titer of > or = 1 : 160 . If a titer of > or = 1 : 80 is taken as cut off point for positivity, 29% of the patients had positive melioidosis serology . Increasing the positivity threshold may jeopardize the sensitivity of the test . A more specific and sensitive test is needed. Asian Pac J Allergy Immunol, 1996 Jun, 14(1), 43 - 7 Production of specific monoclonal antibodies to Burkholderia pseudomallei and their diagnostic application; Pongsunk S et al.; Hybridomas secreting monoclonal antibodies (MAbs) specific to Burkholderia pseudomallei were produced by immunizing BALB/cJ mice with crude culture filtrate of B . pseudomallei . Two monoclonal antibodies were found to be highly specific to B.pseudomallei as tested by indirect enzyme-linked immunosorbent assay and immunoblotting against a panel of crude whole cell extracts from B . pseudomallei, B . cepacia, Pseudomonas aeruginosa, P.putida, and Escherichia coli . One of the specific MAbs, clone SP-M, IgM subclass, could directly agglutinate all 42 B . pseudomallei, isolates . The study has shown that the agglutinating MAb has potential for rapid identification of B . pseudomallei in primary bacterial culture from clinical specimens . The antibody can be used in bacteriology laboratories to reduce time of biochemical methods, which require a few days. Clin Infect Dis, 1996 Jun, 22(6), 1092 - 5 Nosocomial outbreak of Burkholderia pickettii infection due to a manufactured intravenous product used in three hospitals; Fernandez C et al.; Forty-six cases of nosocomial infection caused by Burkholderia pickettii were reported between June and November 1993 in three metropolitan hospitals in Madrid . A case-control study of the outbreak was conducted to identify its cause . Seventy-four percent of the patients were males; the mean age +/- SD of the patients was 54 +/- 20 years . Sixty-five percent of the patients presented with some gastrointestinal disorder, and 80% had a peripheral catheter; 98% were treated with intravenous fluids, and 96% were treated with intravenous ranitidine . On the basis of results of a descriptive study and knowledge of the epidemiologic features of B . pickettii, a provisional causal hypothesis was formulated: intravenous ranitidine was the source of the outbreak . As a control measure, it was advised to stop treatment with this drug . On the basis of results of logistic regression and the microbiological isolation of B . pickettii in an ampule of the drug, we concluded that intravenous ranitidine was the cause of the outbreak. Epidemiol Infect, 1996 Jun, 116(3), 309 - 17 Burkholderia cepacia respiratory tract acquisition: epidemiology and molecular characterization of a large nosocomial outbreak; Pegues CF et al.; In 1994 we investigated a large outbreak of Burkholderia (formerly Pseudomonas) cepacia respiratory tract acquisition . A case patient was defined as any patient with at least one sputum culture from which B . cepacia was isolated from 1 January to 31 December 1994 . Seventy cases were identified . Most (40 {61%}) occurred between 1 February and 31 March 1994; of these, 35 (86%) were mechanically ventilated patients, 30 of whom were in an intensive-care unit (ICU) when B . cepacia was first isolated . Compared with control patients who were mechanically ventilated in an ICU, these 30 case-patients were significantly more likely to have been ventilated for 2 or more days (30/30 v . 15/30; P < 0.001) or to have been intubated more than once (12/30 v . 2/30; OR = 9.3, 95% CI 1.6-68.8; P = 0.002) before the first isolation of B . cepacia . By multivariate analysis, the 35 mechanically ventilated case-patients were significantly more likely to have received a nebulized medication (OR = 11.9, 95% CI = 1.6-553.1; P < 0.001) and a cephalosporin antimicrobial (OR = 11.9, 95% CI = 1.6-553.1) in the 10 days before the first isolation of B . cepacia, compared with B . cepacia-negative control-patients matched by date and duration of most recent mechanical ventilation . Although B . cepacia was not cultured from medications or the hospital environment, all outbreak strains tested had an identical DNA restriction endonuclease digestion pattern by pulsed-field gel electrophoresis . Review of respiratory therapy procedures revealed opportunities for contamination of nebulizer reservoirs . This investigation suggests that careful adherence to standard procedures for administration of nebulized medications is essential to prevent nosocomial respiratory infections. J Bacteriol, 1996 Jun, 178(12), 3462 - 9 HrpXv, an AraC-type regulator, activates expression of five of the six loci in the hrp cluster of Xanthomonas campestris pv . vesicatoria; Wengelnik K et al.; hrp genes, basic pathogenicity genes of the pepper and tomato pathogen Xanthomonas campestris pv . vesicatoria, are regulated dependent on environmental conditions . We isolated the hrpXv gene, which was found to be outside the large hrp cluster comprising the six loci hrpA to hrpF . The predicted HrpXv protein is 476 amino acids long and has a molecular mass of 52.5 kDa . HrpX is highly conserved among xanthomonads and is a member of the AraC family of regulatory proteins . An hrpXv insertion mutant has a typical hrp phenotype and no longer allows induction of the five hrp loci hrpB to hrpF in the new hrp induction medium XVM2, indicating that HrpXv is the positive regulator of these loci . An hrpXv mutant could be partially complemented by the related hrpB gene of Burkholderia solanacearum, the protein product of which shows 40 and 58% amino acid identity and similarity, respectively, to HrpXv . The hrpXv gene itself has a low basal level of expression that is enhanced in XVM2 . Expression of hrpXv as well as that of the hrpA locus is independent of the hrpXv gene . The transcription start site of hrpXv was mapped . Comparison between the hrpXv promoter and the corresponding region of the hrpXc gene from X . campestris pv . campestris revealed sequence conservation up to position -84 . A putative helix-turn-helix motif in the C-terminal region of HrpXv and its possible interaction with a conserved hrp promoter element, the plant-inducible promoter box, are discussed. Am J Epidemiol, 1996 May 15, 143(10), 1007 - 17 Determinants of mortality from cystic fibrosis in Canada, 1970-1989; Corey M et al.; The frequency, prevalence, and mortality patterns of cystic fibrosis were analyzed in 3,795 patients documented in the Canadian Patient Data Registry in 1970-1989 . Cystic fibrosis frequency in the 1970-1979 birth cohort was virtually identical to the commonly quoted 1 in 2,500 . In 1985-1989, median survival age was 36.7 years for males and 27.8 years for females, compared with 26.6 and 19.7, respectively, in 1970-1974 . However, there were significant regional differences when Canada was divided into the four regions, East, Quebec, Ontario, and West . In Quebec, patients were younger at diagnosis and until recently had greater mortality than patients in other regions, which suggests more severe disease; dramatically improved survival in the 1980s coincided with a change from a restricted fat diet to a high fat diet . Improved survival in Ontario in the 1970s accompanied this change in dietary therapy, which may also account for good survival throug