Appl Environ Microbiol, 1999 Aug, 65(8), 3614 - 21 Regiospecificity of dioxygenation of di- to pentachlorobiphenyls and their degradation to chlorobenzoates by the bph-encoded catabolic pathway of Burkholderia sp . strain LB400; Seeger M et al.; Burkholderia sp . strain LB400 is one of the most potent aerobic polychlorobiphenyl (PCB)-degrading microorganisms that have been characterized . Its PCB-dioxygenating activity originates predominantly or exclusively from the biphenyl dioxygenase encoded by its bph gene cluster . Analysis of the dioxygenation products of several di- to pentachlorinated biphenyls formed by this enzyme revealed a complex dependence of the regiospecificity and the yield of dioxygenation on the substitution patterns of both the oxidized and the nonoxidized rings . No dioxygenolytic attack involving chlorinated meta or para carbons was observed . Therefore, the ability of the enzyme to hydroxylate chlorinated carbons appears to be limited to the ortho position . However, it is not limited to monochlorinated rings, as evidenced by dioxygenation of the 2, 4-disubstituted ring at carbons 2 and 3 . This site of attack is strikingly different from that of the 2,5-dichlorinated ring, which has been shown to be dihydroxylated at positions 3 and 4 (J . D . Haddock, J . R . Horton, and D . T . Gibson, J . Bacteriol . 177:20-26, 1995) . These results demonstrate that a second substituent of ortho-chlorinated rings crucially influences the site of dioxygenation at this ring and thereby determines whether or not the initial chlorobiphenyl oxidation product is further metabolized through the bph-encoded pathway . The 2,4-dichlorinated ring can alternatively be attacked at carbons 5 and 6 . The preferred site crucially depends on the substitution pattern of the other ring . The formation of more than a single dioxygenation product was found predominantly with congeners that contain two chlorinated rings, both of which are similarly prone to dioxygenation or one is substituted only at carbon 3. Antibiot Khimioter, 1999, 44(6), 21 - 6 {Burkholderia mallei and Burkholderia pseudomallei . Study of immuno- and pathogenesis of glanders and melioidosis . Heterologous vaccines}; Manzeniuk IN et al.; Parameters of the infectious activity of B.mallei and B.pseudomallei for animals of various species were determined . Pathomorphological characteristics of the process of malleus and melioidosis were studied on golden hamsters, mice, guinea pigs, rats and monkeys . Tularemia, plague and salmonellosis vaccines were shown to have protective effects in experimental malleus and melioidosis . An insignificant cross immune response between the malleus and melioidosis pathogens was observed. Vet Pathol, 1999 Jul, 36(4), 276 - 91 The hamster model of intraperitoneal Burkholderia mallei (glanders); Fritz DL et al.; Thirty-one female Syrian hamsters (Mesocricetus auratus) were inoculated intraperitoneally with a lethal dose of Burkholderia mallei (Budapest strain) . Hamsters were killed postinoculation on days 0 through 6 . Lesions were first noted in the spleens on postinoculation day 1, and in mediastinal and mesenteric lymph nodes, mediastinum, liver, and bone marrow on day 2 . Lesions were present in the lung and submandibular lymph nodes on day 3, and in the brain on day 5 . The characteristic histopathologic change was necrotizing pyogranulomatous inflammation, often with hemorrhage . Lesions indicative of impaired vascular perfusion, such as ischemia and infarction, were evident at the later time points . Pathologic changes generally increased in severity and distribution with time, and almost all tissues were ultimately affected . Our findings suggest that intraperitoneal bacteria were rapidly transported to mediastinal lymph nodes by transdiaphragmatic lymphatics and ultimately seeded other tissues hematogenously . The results of the study indicate that the Syrian hamster is a useful small animal model for glanders. J Bacteriol, 1999 Aug, 181(15), 4661 - 4 Molecular characterization of genetic loci required for secretion of exoproducts in Burkholderia pseudomallei; DeShazer D et al.; Previous studies have demonstrated that Burkholderia pseudomallei secretes protease, lipase, and phospholipase C (PLC) into the extracellular milieu, but their mechanisms of secretion and roles in pathogenesis have not been elucidated . In this study, we isolated and characterized 29 transposon mutants unable to secrete protease, lipase, and PLC. Infect Immun, 1999 Aug, 67(8), 4027 - 32 A murine model for infection with Burkholderia cepacia with sustained persistence in the spleen; Speert DP et al.; Burkholderia cepacia is an opportunistic pathogen that causes severe systemic infections in patients with chronic granulomatous disease (CGD) or with cystic fibrosis (CF), but its mechanisms of virulence are poorly understood . We developed a murine model of systemic infection in wild-type (WT) and gamma interferon knockout (GKO) BALB/c mice to facilitate dissection of components of pathogenicity and host defense . Both WT and GKO mice were susceptible to chronic splenic infection with B . cepacia, but not with Pseudomonas aeruginosa . B . cepacia strains from patients with CGD persisted longer than those from CF patients . C57BL/6 mice were the most susceptible murine strain; bacteria persisted in the spleen for 2 months . DBA/2, BALB/c, and A/J strains of mice were relatively resistant to infection . Certain strains of B . cepacia complex can persist in the murine spleen after systemic infection; this may provide clues to its virulence in compromised hosts, such as those with CGD and CF. Natl Med J India, 1999 Mar-Apr, 12(2), 59 - 61 Surgical presentation of melioidosis in India; Mathew S et al.; BACKGROUND: Melioidosis, the disease caused by Burkholderia pseudomallei, is common in Southeast Asia . It has also been reported from India, where some investigators feel it is under-diagnosed and under-reported . We report our experience with melioidosis presenting as abscesses at unusual sites . METHODS: All consecutive patients with culture proven B . pseudomallei, who presented to a single surgical unit between 1995 and 1998, were evaluated . RESULTS: Three patients presented with splenic abscesses and one with a soft tissue abscess in the neck . One patient developed septicaemia . All patients responded favourably to ceftazidime and/or co-trimoxazole which was started as soon as the diagnosis was confirmed . CONCLUSION: Melioidosis is under-diagnosed in India, probably due to a low index of suspicion of this disease among clinicians . It should be considered as a possibility when abscesses are encountered at unusual sites . The pus must then be cultured to identify the causative agent. Int J Biol Macromol, 1999 May, 24(4), 311 - 8 Biosynthesis and characterization of poly(3-hydroxybutyrate-co-3-hydroxyvalerate) produced by Burkholderia cepacia D1; Mitomo H et al.; Copolyesters of 3-hydroxybutyrate (3HB) and 3-hydroxyvalerate (3HV) were produced by Burkholderia cepacia D1 at 30 degrees C in nitrogen-free culture solutions containing n-butyric acid and/or n-valeric acid . When n-valeric acid was used as the sole carbon source, the 3HV fraction in copolyester increased from 36 to 90 mol% as the concentration of n-valeric acid in the culture solution increased from 1 to 20 g/l . The addition of n-butyric acid to the culture solution resulted in a decrease in the 3HV fraction in copolyester . The copolymers biosynthesized by this method were mixtures of random copolymers having a wide variety of composition of the 3HV component . The melting points of the fractionated copolymers show a concave curve with the minimum at the 3HV content of approximately 40 mol% . The alpha-parameter of lattice indices of the P(3HB) crystal for the fractionated copolymers largely increased as the 3HV composition increased . Biodegradability of the copolymer increased with the lower content of 3HV composition and/or the lower crystallinity. J Am Soc Nephrol, 1999 Jul, 10(7), 1566 - 74 Hepatitis C virus-associated glomerular disease in patients with human immunodeficiency virus coinfection; Cheng JT et al.; Chronic infection with hepatitis C virus (HCV) has been linked to the development of glomerular disease . HCV infection is highly prevalent among intravenous drug users, a population that is also at risk for HIV coinfection . This study reports the clinical-pathologic features and outcome of HCV-associated glomerular disease (HCV-GD) in 14 patients with HIV coinfection . All were intravenous drug users and all but one were African-Americans . Renal presentations included renal insufficiency, microscopic hematuria with active urine sediment, hypertension, and nephrotic syndrome or nephrotic-range proteinuria without hypercholesterolemia . Hypocomplementemia and cryoglobulinemia were present in 46 and 33% of patients, respectively . The predominant renal biopsy findings were membranoproliferative glomerulonephritis type 1 or type 3 (Burkholder subtype) in 79% of patients and membranous glomerulopathy with atypical features in 21% (including overlap with collapsing glomerulopathy in one patient) . The clinical course was characterized by rapid progression to renal failure requiring dialysis . The overall morbidity and mortality were high with median time of 5.8 mo to dialysis or death . Although most patients died in renal failure, cause of death was primarily attributable to long-term immunosuppression and advanced AIDS . Patients with AIDS had shorter survival than those without (median survival time of 6.1 mo versus 45.9 mo, log-rank test P = 0.02) . Only two patients were alive with stable renal function at follow-up of 28.5 mo . In patients with HCV-GD, coinfection with HIV leads to an aggressive form of renal disease that can be easily confused with HIV-associated nephropathy . Although hypocomplementemia, cryoglobulinemia, and more prominent hypertension and microscopic hematuria may provide clues to the presence of HCV-GD, renal biopsy is essential to differentiate HCV-GD from HIV-associated nephropathy. J Med Microbiol, 1999 Jul, 48(7), 657 - 62 Analysis of fliC variation among clinical isolates of Burkholderia cepacia; Winstanley C et al.; PCR and restriction fragment length polymorphism (RFLP) typing of flagellin genes (fliC) from 57 clinical isolates of Burkholderia cepacia indicated that only type 11 flagellins were present . Twenty-two isolates previously identified as the epidemic UK cystic fibrosis strain were indistinguishable by this method, as were 11 isolates from a pseudo-outbreak in Senegal . Other clinical isolates, including 19 from disparate sources in Malaysia, were separated into nine fliC RFLP groups, exhibiting a large degree of divergence . When isolates were indistinguishable by fliC genotyping, their similarity was confirmed by whole genome macro-restriction analysis with pulsed-field gel electrophoresis following XbaI digestion . The variation in fliC sequences of B . cepacia was far greater than that with B . pseudomallei, supporting the view that 'B . cepacia', as currently defined, may comprise several different genomic species. J Med Microbiol, 1999 Jul, 48(7), 649 - 56 Evidence for the presence in Burkholderia pseudomallei of a type III secretion system-associated gene cluster; Winstanley C et al.; Burkholderia pseudomallei, the causative agent of melioidosis, contains a cluster of putative genes homologous to those encoding HpaP, HrcQ, HrcR, HrcS and HrpV in the plant pathogen Ralstonia solanacearum . In R . solanacearum, these genes form part of a type III secretion-associated pathogenicity island . The order of the genes in B . pseudomallei is directly equivalent to that found in R . solanacearum . The B . pseudomallei proteins share 49.5% (HpaP), 52.6% (HrcQ), 80.0% (HrcR), 72.1% (HrcS) and 46.7% (HrpV) similarity, respectively, with their equivalent R . solanacearum proteins . The presence of type III secretion-associated genes in B . pseudomallei pathogens suggests a possible role for type III secretion systems in the pathogenicity of this organism. Biotechnol Bioeng, 1999 Jun 5, 63(5), 544 - 51 Tuning biphenyl dioxygenase for extended substrate specificity; Bruhlmann F et al.; Highly substituted polychlorinated biphenyls (PCBs) are known to be very resistant to aerobic biodegradation, particularly the initial attack by biphenyl dioxygenase . Functional evolution of the substrate specificity of biphenyl dioxygenase was demonstrated by DNA shuffling and staggered extension process (StEP) of the bphA gene coding for the large subunit of biphenyl dioxygenase . Several variants with an extended substrate range for PCBs were selected . In contrast to the parental biphenyl dioxygenases from Burkholderia cepacia LB400 and Pseudomonas pseudoalcaligenes KF707, which preferentially recognize either ortho- (LB400) or para- (KF707) substituted PCBs, several variants degraded both congeners to about the same extent . These variants also exhibited superior degradation capabilities toward several tetra- and pentachlorinated PCBs as well as commercial PCB mixtures, such as Aroclor 1242 or Aroclor 1254 . Sequence analysis confirmed that most variants contained at least four to six template switches . All desired variants contained the Thr335Ala and Phe336Ile substitutions confirming the importance of this critical region in substrate specificity . These results suggest that the block-exchange nature of gene shuffling between a diverse class of dioxygenases may be the most useful approach for breeding novel dioxygenases for PCB degradation in the desired direction . J Biochem (Tokyo), 1999 Jul, 126(1), 243 - 53 Short-chain acyl-CoA-dependent production of oxalate from oxaloacetate by Burkholderia glumae, a plant pathogen which causes grain rot and seedling rot of rice via the oxalate production; Li HQ et al.; In Burkholderia glumae (formerly named Pseudomonas glumae), isolated as the causal agent of grain rot and seedling rot of rice, oxalate was produced from oxaloacetate in the presence of short-chain acyl-CoA such as acetyl-CoA and propionyl-CoA . Upon purification, the enzyme responsible was separated into two fractions (tentatively named fractions II and III), both of which were required for the acyl-CoA-dependent production of oxalate . In conjugation with the oxalate production from oxaloacetate catalyzed by fractions II and III, acetyl-CoA used as the acyl-CoA substrate was consumed and equivalent amounts of CoASH and acetoacetate were formed . The isotope incorporation pattern indicated that the two carbon atoms of oxalate are both derived from oxaloacetate, and among the four carbon atoms of acetoacetate two are from oxaloacetate and two from acetyl-CoA . When the reaction was carried out with fraction II alone, a decrease in acetyl-CoA and an equivalent level of net utilization of oxaloacetate were observed without appreciable formation of CoASH, acetoacetate or oxalate . It appears that in the oxalate production from oxaloacetate and acetyl-CoA, fraction II catalyzes condensation of the two substrates to form an intermediate which is split into oxalate and acetoacetate by fraction III being accompanied by the release of CoASH. Antimicrob Agents Chemother, 1999 Jul, 43(7), 1644 - 50 Biochemical-genetic analysis and distribution of FAR-1, a class A beta-lactamase from Nocardia farcinica; Laurent F et al.; From genomic DNA of the clinical isolate Nocardia farcinica VIC, a 1 . 6-kb Sau3AI fragment was cloned and expressed in Escherichia coli JM109 . The recombinant strain expressed a beta-lactamase (pI, 4.6), FAR-1, which conferred high levels of resistance to amoxicillin, piperacillin, ticarcillin, and cephalothin . The hydrolysis constants (kcat, Km, Ki, and 50% inhibitory concentration) confirmed the MIC results and showed that FAR-1 activity is inhibited by clavulanic acid and at a low level by tazobactam and sulbactam . Moreover, FAR-1 beta-lactamase hydrolyzes aztreonam (at a low level) without significant activity against ceftazidime, cefotaxime and imipenem . FAR-1 mature protein of molecular mass ca 32 kDa, has less than 60% amino acid identity with any other class A beta-lactamases, being most closely related to PEN-A from Burkholderia cepacia (52%) . A blaFAR-1-like gene was found in all studied N . farcinica strains, underlining the constitutive origin of this gene. J Bacteriol, 1999 Jul, 181(13), 4106 - 9 Identification of a putative P-transporter operon in the genome of a Burkholderia strain living inside the arbuscular mycorrhizal fungus Gigaspora margarita; Ruiz-Lozano JM et al.; This article reports the identification of a putative P-transporter operon in the genome of a Burkholderia sp . living in the cytoplasm of the arbuscular mycorrhizal fungus Gigaspora margarita . Its presence suggests that Burkholderia sp . has the potential for P uptake from this environment . This finding raises new questions concerning the importance of intracellular bacteria for mycorrhizal symbiosis. Biochem Biophys Res Commun, 1999 Jun 24, 260(1), 181 - 7 cis-2,3-dihydro-2,3-dihydroxybiphenyl dehydrogenase and cis-1, 2-dihydro-1,2-dihydroxynaphathalene dehydrogenase catalyze dehydrogenation of the same range of substrates; Barriault D et al.; Pseudomonas putida strain G7 cis-1,2-dihydro-1, 2-dihydroxynaphthalene dehydrogenase (NahB) and Comamonas testosteroni strain B-356 cis-2,3-dihydro-2,3-dihydroxybiphenyl dehydrogenase (BphB) were found to be catalytically active towards cis-2,3-dihydro-2,3-dihydroxybiphenyl (specificity factors of 501 and 5850 s-1 mM-1 respectively), cis-1,2-dihydro-1, 2-dihydroxynaphthalene (specificity factors of 204 and 193 s-1 mM-1 respectively) and 3,4-dihydro-3,4-dihydroxy-2,2',5, 5'-tetrachlorobiphenyl (specificity factors of 1.6 and 4.9 s-1 mM-1 respectively) . A key finding in this work is the capacity of strain B-356 BphB as well as Burkholderia cepacia strain LB400 BphB to catalyze dehydrogenation of 3,4-dihydro-3,4-dihydroxy-2,2',5, 5'-tetrachlorobiphenyl which is the metabolite resulting from the catalytic meta-para hydroxylation of 2,2',5,5'-tetrachlorobiphenyl by LB400 biphenyl dioxygenase . Infect Immun, 1999 Jul, 67(7), 3593 - 600 Obligatory role of gamma interferon for host survival in a murine model of infection with Burkholderia pseudomallei; Santanirand P et al.; Burkholderia pseudomallei, the causative agent of melioidosis, is a gram-negative bacterium capable of causing either acute lethal sepsis or chronic but eventually fatal disease in infected individuals . However, despite the clinical importance of this infection in areas where it is endemic, there is essentially no information on the mechanisms of protective immunity to the bacterium . We describe here a murine model of either acute or chronic infection with B . pseudomallei in Taylor Outbred (TO) mice which mimics many features of the human pathology . Intraperitoneal infection of TO mice at doses of >10(6) CFU resulted in acute septic shock and death within 2 days . In contrast, at lower doses mice were able to clear the inoculum from the liver and spleen over a 3- to 4-week period, but persistence of the organism at other sites resulted in a chronic infection of between 2 and 16 months duration which was eventually lethal in all of the animals tested . Resistance to acute infection with B . pseudomallei was absolutely dependent upon the production of gamma interferon (IFN-gamma) in vivo . Administration of neutralizing monoclonal antibody against IFN-gamma lowered the 50% lethal dose from >5 x 10(5) to ca . 2 CFU and was associated with 8,500- and 4,400-fold increases in the bacterial burdens in the liver and spleen, respectively, together with extensive destruction of lymphoid architecture in the latter organ within 48 h . Neutralization of either tumor necrosis factor alpha or interleukin-12 but not granulocyte-macrophage colony-stimulating factor, also increased susceptibility to infection in vivo . Together, these results provide the first evidence of a host protective mechanism against B . pseudomallei . The rapid production of IFN-gamma within the first day of infection determines whether the infection proceeds to an acute lethal outcome or becomes chronic. Kaohsiung J Med Sci, 1999 May, 15(5), 292 - 6 An indigenous melioidosis: a case report; Chen YH et al.; Melioidosis is a rare but potentially fatal infectious disease in Taiwan, although it has been endemic in Southeast Asia, especially northeast Thailand, and northern Australia . In this article, we report a male diabetes with fulminant pneumonia, and septicemia caused by Burkholderia pseudomallei without traveling abroad before this episode . Productive cough and intermittent chills, high fever for one week, followed by progressively deteriorating dyspnea, shock, disturbed consciousness status were the major presentations . Blood culture grew B . pseudomallei on the fifth admission day . Unfortunately, the patient died on the 9th admission day, despite intensive care and the broad-spectrum antimicrobial regimen used. J Clin Microbiol, 1999 Jul, 37(7), 2201 - 8 Differentiation of Burkholderia species by PCR-restriction fragment length polymorphism analysis of the 16S rRNA gene and application to cystic fibrosis isolates; Segonds C et al.; Burkholderia cepacia, which is an important pathogen in cystic fibrosis (CF) owing to the potential severity of the infections and the high transmissibility of some clones, has been recently shown to be a complex of five genomic groups, i.e., genomovars I, II (B . multivorans), III, and IV and B . vietnamiensis . B . gladioli is also involved, though rarely, in CF . Since standard laboratory procedures fail to provide an accurate identification of these organisms, we assessed the ability of restriction fragment length polymorphism (RFLP) analysis of amplified 16S ribosomal DNA (rDNA), with the combination of the patterns obtained with six endonucleases, to differentiate Burkholderia species . This method was applied to 16 type and reference strains of the genus Burkholderia and to 51 presumed B . cepacia clinical isolates, each representative of one clone previously determined by PCR ribotyping . The 12 Burkholderia type strains tested were differentiated, including B . cepacia, B . multivorans, B . vietnamiensis, and B . gladioli, but neither the genomovar I and III reference strains nor the genomovar IV reference strain and B . pyrrociniaT were distinguishable . CF clinical isolates were mainly distributed in RFLP group 2 (which includes B . multivoransT) and RFLP group 1 (which includes B . cepacia genomovar I and III reference strains, as well as nosocomial clinical isolates) . Two of the five highly transmissible clones in French CF centers belonged to RFLP group 2, and three belonged to RFLP group 1 . The remaining isolates either clustered with other Burkholderia species (B . cepacia genomovar IV or B . pyrrocinia, B . vietnamiensis, and B . gladioli) or harbored unique combinations of patterns . Thus, if further validated by hybridization studies, PCR-RFLP of 16S rDNA could be an interesting identification tool and contribute to a better evaluation of the respective clinical risks associated with each Burkholderia species or genomovar in patients with CF. J Clin Microbiol, 1999 Jul, 37(7), 2158 - 64 Identification of Burkholderia spp . in the clinical microbiology laboratory: comparison of conventional and molecular methods; van Pelt C et al.; Cystic fibrosis (CF) predisposes patients to bacterial colonization and infection of the lower airways . Several species belonging to the genus Burkholderia are potential CF-related pathogens, but microbiological identification may be complicated . This situation is not in the least due to the poorly defined taxonomic status of these bacteria, and further validation of the available diagnostic assays is required . A total of 114 geographically diverse bacterial isolates, previously identified in reference laboratories as Burkholderia cepacia (n = 51), B . gladioli (n = 14), Ralstonia pickettii (n = 6), B . multivorans (n = 2), Stenotrophomonas maltophilia (n = 3), and Pseudomonas aeruginosa (n = 11), were collected from environmental, clinical, and reference sources . In addition, 27 clinical isolates putatively identified as Burkholderia spp . were recovered from the sputum of Dutch CF patients . All isolates were used to evaluate the accuracy of two selective growth media, four systems for biochemical identification (API 20NE, Vitek GNI, Vitek NFC, and MicroScan), and three different PCR-based assays . The PCR assays amplify different parts of the ribosomal DNA operon, either alone or in combination with cleavage by various restriction enzymes (PCR-restriction fragment length polymorphism {RFLP} analysis) . The best system for the biochemical identification of B . cepacia appeared to be the API 20NE test . None of the biochemical assays successfully grouped the B . gladioli strains . The PCR-RFLP method appeared to be the optimal method for accurate nucleic acid-mediated identification of the different Burkholderia spp . With this method, B . gladioli was also reliably classified in a separate group . For the laboratory diagnosis of B . cepacia, we recommend parallel cultures on blood agar medium and selective agar plates . Further identification of colonies with a Burkholderia phenotype should be performed with the API 20NE test . For final confirmation of species identities, PCR amplification of the small-subunit rRNA gene followed by RFLP analysis with various enzymes is recommended. J Med Microbiol, 1999 Jun, 48(6), 551 - 7 A novel mucin-sulphatase activity found in Burkholderia cepacia and Pseudomonas aeruginosa; Jansen HJ et al.; Lung infections due to Burkholderia cepacia and Pseudomonas aeruginosa in patients with cystic fibrosis (CF) are common, are associated with respiratory morbidity and are a cause of mortality . Respiratory mucin in CF patients is highly sulphated, which increases its resistance to bacterial degradation . Desulphation increases the susceptibility of mucin to degradation by bacterial glycosidases and proteinases, and subsequent deglycosylation may facilitate bacterial colonisation by increasing available substrates and binding sites . This study determined whether clinical and environmental strains of B . cepacia and P . aeruginosa had the ability to desulphate mucin . Mucin-sulphatase activity was tested by incubating bacterial cell suspensions with 35S-sulphated mucins purified from LS174T and HT29-MTX human colon carcinoma cell lines . These mucins were also used to test for differences in substrate specificities . Mucin-sulphatase activity was detected in all nine B . cepacia strains and in four of six P . aeruginosa strains . There was strain variability in the level of mucin-sulphatase activity . Aryl-sulphatase activities of Pseudomonas isolates (determined with methylumbelliferyl sulphate) were c . 20-fold higher than those of B . cepacia strains, and were independent of mucin-sulphatase activity . This is the first report to demonstrate desulphation of mucin by B . cepacia and P . aeruginosa . It is concluded that B . cepacia and P . aeruginosa produce one or more cell-bound glycosulphatase(s), in addition to aryl-sulphatase activity . Mucin-sulphatase activity of B . cepacia and P . aeruginosa may contribute to their association with airway infections in patients with cystic fibrosis. Zh Mikrobiol Epidemiol Immunobiol, 1999 Mar-Apr, (2), 49 - 51 {The efficacy and outlook for the study of live vaccines for the prevention of melioidosis}; Iliukhin VI et al.; The effect of immunization with Burkholderia pseudomallei, (Pur- and Ts), heterologous vaccines and the recombinant culture of Francisella tularensis RM2, carrying a plasmid with fragments of B . pseudomallei chromosome, was studied on four species of experimental animals, essentially differing by their sensitivity to melioidosis . B . pseudomallei mutants formed the statistically significant level of protection in subcutaneously challenged animals, moderately sensitive to melioidosis, but were not effective when tested, under the same conditions, in animals, highly sensitive to melioidosis . The effect produced by the experimental vaccines under study in animals of all species, subjected to aerogenic challenge, was leveled . The study showed good prospects for the use of tularemia vaccine with a view to create heterologous immunity to melioidosis and the possibility of its use as the basis of bivalent gene engineering vaccine. Zh Mikrobiol Epidemiol Immunobiol, 1999 Mar-Apr, (2), 22 - 5 {The role of the structural parts of bacterial lipopolysaccharide in its direct immunosuppressive activity}; Borisova EV; The direct immunosuppressive activity of lipopolysaccharides (LPS) and their structural parts (O-chains, R-core, lipid A), obtained from Salmonella, Pseudomonas and Burkholderia, was studied . LPS preparations were extracted by the phenol-water method . Structural parts of LPS were obtained by acetic acid hydrolysis and gel filtration . The study demonstrated that all these preparations, when injected intraperitoneally into mice, did not affect the level of delayed-type hypersensitivity (DTH) to the test antigen in the animals . After redox treatment all LPS preparations became capable of suppressing DTH . After redox treatment such immunosuppressive activity could be observed in lipid A, while O-specific chains and R-core remained inactive . After phenol treatment immunosuppressive activity disappeared . Chemical groups capable of activation were likely to be located in lipid A or in lipid A-associated protein. Antimicrob Agents Chemother, 1999 Jun, 43(6), 1435 - 40 In vitro activities of designed antimicrobial peptides against multidrug-resistant cystic fibrosis pathogens; Schwab U et al.; The emergence of multidrug-resistant pathogens renders antibiotics ineffective in the treatment of lung infections in patients with cystic fibrosis (CF) . Designed antimicrobial peptides (DAPs) are laboratory-synthesized peptide antibiotics that demonstrate a wide spectrum of antibacterial activity . Optimal conditions for susceptibility testing of these peptides have not yet been established . Medium composition is clearly a major factor influencing the results and reproducibilities of susceptibility tests . Using time-kill assays, we tested the effects of different media and buffers on the bactericidal activities of the peptides D2A21 and D4E1 on Staphylococcus aureus ATCC 29213 and Pseudomonas aeruginosa ATCC 27853 . Each peptide at 1 and 5 microM was incubated with bacteria in the different media and buffers . Both peptides were most active in Tris-HCl buffer against S . aureus and P . aeruginosa . Among the more complex media tested, modified RPMI medium was the medium in which the peptides demonstrated the highest activity, while it supported the growth of the bacteria . The broth microdilution technique was used to test the activities of D2A21 and D4E1 in modified RPMI medium against multidrug-resistant pathogens from patients with CF . The MICs of DAPs for methicillin-resistant S . aureus ranged from 0.25 to 4 microg/ml, those for multidrug-resistant P . aeruginosa ranged from 0.125 to 4 microg/ml, those for Stenotrophomonas maltophilia ranged from 0.5 to 32 microg/ml, and those for Burkholderia cepacia ranged from 32 to >/=64 microg/ml . When the activity of peptide D2A21 was compared with that of the tracheal antimicrobial peptide (TAP), D2A21 had greater potency than TAP against P . aeruginosa . In addition, no difference in the MICs of D2A21 was seen when it was tested in nutrient broth supplemented with NaCl at different concentrations . Thus, DAPs are a class of salt-insensitive antibiotics potentially useful in the treatment of CF patients harboring multidrug-resistant P . aeruginosa. Appl Environ Microbiol, 1999 Jun, 65(6), 2547 - 52 Degradation of chlorobenzenes at nanomolar concentrations by Burkholderia sp . strain PS14 in liquid cultures and in soil; Rapp P et al.; The utilization of 1,2,4,5-tetrachloro-, 1,2,4-trichloro-, the three isomeric dichlorobenzenes and fructose as the sole carbon and energy sources at nanomolar concentrations was studied in batch experiments with Burkholderia sp . strain PS14 . In liquid culture, all chlorobenzenes were metabolized within 1 h from their initial concentration of 500 nM to below their detection limits of 0.5 nM for 1,2,4,5-tetrachloro- and 1,2,4-trichlorobenzene and 7.5 nM for the three dichlorobenzene isomers, with 63% mineralization of the tetra- and trichloroisomers . Fructose at the same initial concentration was, in contrast, metabolized over a 4-h incubation period down to a residual concentration of approximately 125 nM with 38% mineralization during this time . In soil microcosms, Burkholderia sp . strain PS14 metabolized tetrachlorobenzene present at 64.8 ppb and trichlorobenzene present at 54.4 ppb over a 72-h incubation period to below the detection limits of 0.108 and 0.09 ppb, respectively, with approximately 80% mineralization . A high sorptive capacity of Burkholderia sp . strain PS14 for 1,2,4, 5-tetrachlorobenzene was found at very low cell density . The results demonstrate that Burkholderia sp . strain PS14 exhibits a very high affinity for chlorobenzenes at nanomolar concentrations. Infect Immun, 1999 Jun, 67(6), 2891 - 900 Characterization of a murine model of melioidosis: comparison of different strains of mice; Hoppe I et al.; Melioidosis is an infectious disease caused by the saprophytic gram-negative rod Burkholderia pseudomallei . The aim of this study was to establish and characterize a murine model of melioidosis to provide a basis for further investigations on the pathogenesis of the disease . After intravenous infection with B . pseudomallei, C57BL/6 mice were found to be significantly more resistant than BALB/c mice . There was a marked organotropism of B . pseudomallei for the spleen and liver in both strains of mice, with the highest bacterial load in the spleen . Electron microscopic investigations of the spleen clearly demonstrated intracellular replication within membrane-bound phagosomes . Electron micrographs of the liver provided evidence that B . pseudomallei-containing phagosomes in hepatocytes fuse with lysosomes, leading to degradation of bacteria . In both strains of mice, the course of infection was highly dependent on the infective dose and the bacterial strain used, ranging from death within a few days to death after several weeks . In comparison with BALB/c mice, the bacterial counts in C57BL/6 mice were decreased 12 h after infection, which is suggestive of an innate immune mechanism against B . pseudomallei in this early phase of infection contributing to the lower susceptibility of C57BL/6 mice . BALB/c mice developed a more pronounced lymphopenia, granulocytosis, and splenomegaly at a lower infective dose compared to C57BL/6 mice . Analysis of the antibody response against B . pseudomallei 11 days after infection revealed a significantly higher immunoglobulin G2A (IgG2a)/IgG1 ratio in C57BL/6 mice than in BALB/c mice, indicating that a T helper type 1 immune response is associated with resistance to infection with B . pseudomallei. J Clin Microbiol, 1999 Jun, 37(6), 1906 - 12 Phylogenetic analysis of Ara+ and Ara- Burkholderia pseudomallei isolates and development of a multiplex PCR procedure for rapid discrimination between the two biotypes; Dharakul T et al.; A Burkholderia pseudomallei-like organism has recently been identified among some soil isolates of B . pseudomallei in an area with endemic melioidosis . This organism is almost identical to B . pseudomallei in terms of morphological and biochemical profiles, except that it differs in ability to assimilate L-arabinose . These Ara+ isolates are also less virulent than the Ara- isolates in animal models . In addition, clinical isolates of B . pseudomallei available to date are almost exclusively Ara- . These features suggested that these two organisms may belong to distinctive species . In this study, the 16S rRNA-encoding genes from five clinical (four Ara- and one Ara+) and nine soil isolates (five Ara- and four Ara+) of B . pseudomallei were sequenced . The nucleotide sequences and phylogenetic analysis indicated that the 16S rRNA-encoding gene of the Ara+ biotype was similar to but distinctively different from that of the Ara- soil isolates, which were identical to the classical clinical isolates of B . pseudomallei . The nucleotide sequence differences in the 16S rRNA-encoding gene appeared to be specific for the Ara+ or Ara- biotypes . The differences were, however, not sufficient for classification into a new species within the genus Burkholderia . A simple and rapid multiplex PCR procedure was developed to discriminate between Ara- and Ara+ B . pseudomallei isolates . This new method could also be incorporated into our previously reported nested PCR system for detecting B . pseudomallei in clinical specimens. J Bacteriol, 1999 May, 181(10), 3069 - 75 Role of quinolinate phosphoribosyl transferase in degradation of phthalate by Burkholderia cepacia DBO1; Chang HK et al.; Two distinct regions of DNA encode the enzymes needed for phthalate degradation by Burkholderia cepacia DBO1 . A gene coding for an enzyme (quinolinate phosphoribosyl transferase) involved in the biosynthesis of NAD+ was identified between these two regions by sequence analysis and functional assays . Southern hybridization experiments indicate that DBO1 and other phthalate-degrading B . cepacia strains have two dissimilar genes for this enzyme, while non-phthalate-degrading B . cepacia strains have only a single gene . The sequenced gene was labeled ophE, due to the fact that it is specifically induced by phthalate as shown by lacZ gene fusions . Insertional knockout mutants lacking ophE grow noticeably slower on phthalate while exhibiting normal rates of growth on other substrates . The fact that elevated levels of quinolinate phosphoribosyl transferase enhance growth on phthalate stems from the structural similarities between phthalate and quinolinate: phthalate is a competitive inhibitor of this enzyme and the phthalate catabolic pathway cometabolizes quinolinate . The recruitment of this gene for growth on phthalate thus gives B . cepacia an advantage over other phthalate-degrading bacteria in the environment. Int J Syst Bacteriol, 1999 Apr, 49 Pt 2, 787 - 94 Burkholderia caribensis sp . nov., an exopolysaccharide-producing bacterium isolated from vertisol microaggregates in Martinique; Achouak W et al.; Twenty-one exopolysaccharide-producing strains were isolated from the 5-20 microns fraction of a vertisol in the south-east of the island of Martinique in the French West Indies . Although these strains were phenotypically identified as Burkholderia cepacia or as Burkholderia glathei using BIOLOG microplates, they did not cluster genotypically by amplified rDNA restriction analysis (ARDRA) with any described Burkholderia species . A phylogenetic analysis revealed that the rrs (16S rDNA) sequences of three representative strains clustered in a single branch within the genus Burkholderia and distantly from all of the previously described species of Burkholderia for which rrs sequences were available . DNA-DNA hybridization data as well as phenotypic analyses indicated that the 21 isolates represented a single and new species for which the name Burkholderia caribensis sp . nov . is proposed (type strain MWAP64T = LMG 18531T). J Med Microbiol, 1999 May, 48(5), 419 - 23 Nitric oxide-induced potentiation of the killing of Burkholderia cepacia by reactive oxygen species: implications for cystic fibrosis; Smith AW et al.; Burkholderia (formerly Pseudomonas) cepacia has emerged as an important pulmonary pathogen in cystic fibrosis, and survives within the lung despite a vigorous neutrophil-dominated immune response . Nitric oxide (NO) contributes to the antimicrobial activity of reactive oxygen species in the normal lung, but recent evidence suggests that inducible NO synthase is not expressed in the airway epithelial cells of cystic fibrosis (CF) patients . This may explain the failure of the neutrophil response to eliminate B . cepacia . To test this hypothesis, the present study examined the combined effect of NO, superoxide and H2O2 against B . cepacia . There was no killing of a highly transmissible strain by either superoxide or NO alone, but their combination reduced the bacterial count by >1000-fold over 75 min . This bactericidal activity was not sensitive to addition of superoxide dismutase, but was abrogated completely by catalase, suggesting that NO and hydrogen peroxide were the bactericidal mediators . Increased killing by NO in combination with H2O2 was seen for seven of a further 11 strains examined . The lack of NO in the lungs of CF patients may contribute to the survival of B . cepacia. Curr Opin Pulm Med, 1999 May, 5(3), 151 - 6 Pyogenic lung infections; Mwandumba HC et al.; Pyogenic lung infections still occur despite the availability of effective antibiotics for the treatment of patients with acute bacterial pneumonia . Our understanding of the pathogenesis and management of these conditions has steadily improved over the past few decades, although some areas remain obscure . The effect of HIV infection on the incidence of pyogenic lung infections remains largely unknown, and large studies are required to evaluate this . Burkholderia (formerly Pseudomonas) cepacia strains are now recognized as important respiratory pathogens in patients with cystic fibrosis, and the high transmissibility of some strains, combined with their inherent multiple antibiotic resistance, are continuing causes for concern. Am J Respir Cell Mol Biol, 1999 May, 20(5), 872 - 9 Activity of abundant antimicrobials of the human airway; Travis SM et al.; Human airways produce several antimicrobial factors; the most abundant are lysozyme and lactoferrin . Despite their likely importance in preventing infection, and their possible key role in the pathogenesis of cystic fibrosis (CF), we know little about their antibacterial activity in the context of the CF airway . We found that abundant airway antimicrobial factors kill common CF pathogens, although Burkholderia was relatively resistant . To study the antibacterial activity, we developed a rapid, sensitive, and quantitative in vitro luminescence assay . Because NaCl concentrations may be elevated in CF airway surface liquid, we tested the effect of salt on antibacterial activity . Activity of individual factors and of airway lavage fluid was inhibited by high ionic strength, and it was particularly sensitive to divalent cations . However, it was not inhibited by nonionic osmolytes and thus did not require hypotonic liquid . The inhibition by ionic strength could be partially compensated by increased concentrations of antibacterial factors, thus there was no one unique salt concentration for inhibition . CF airway secretions also contain abundant mucin and elastase; however, these had no effect on antibacterial activity of lysozyme, lactoferrin, or airway lavage fluids . When studied at low NaCl concentrations, CF and non-CF airway lavage fluids contained similar levels of antibacterial activity . These results suggest approaches toward developing treatments aimed at preventing or reducing airway infections in individuals with CF. Eur J Biochem, 1999 Apr, 261(2), 533 - 9 Hydroxylation reaction catalyzed by the Burkholderia cepacia AC1100 bacterial strain . Involvement of the chlorophenol-4-monooxygenase; Martin G et al.; The Burkholderia cepacia AC1100 strain, known to degrade the herbicide, 2,4,5-Trichlorophenoxyacetic acid (2,4,5-T), is able to metabolize 4-hydroxyarylaldehyde, not only into the corresponding acid, but also into a new hydroquinone, 2,5-dihydroxyarylaldehyde . When incubated with resting AC1100 cells or cell-free extracts, syringaldehyde and 3,5-dimethoxy-4-hydroxybenzaldehyde were converted into such metabolites, identified by comparison of their mass and 1H-NMR spectra with those of authentic chemically synthesized samples . With 5-bromovanillin, only one metabolite was formed, the structure of which was identified as 2, 5-dihydroxy-4-methoxy-6-bromobenzaldehyde through 1H-NMR two-dimensional NOESY experiments . All these products result formally from a para hydroxylation of the phenol followed by the cis migration of the aldehyde . This reaction is the only one to be associated with the 2,4,5-T degradation pathway, as the acid formation was retained when the AC1100 strain had lost its degradation ability . Through competitive experiments with halophenols and methimazole, an alternative substrate of flavin monooxygenase, the chlorophenol-4-monooxygenase was recognized to be the enzyme involved in the hydroxylation of 4-hydroxyarylaldehyde . The purified enzyme, previously reported to catalyze the para hydroxylation or dehalogenating hydroxylation of chlorophenols, also promotes this hydroxylation reaction in the presence of NADH and FAD . The kcat value determined for the best substrate, syringaldehyde, 0 . 08 s-1, was about 20% of that obtained for 2,6-dichlorophenol hydroxylation (0.38 s-1). Vet Rec, 1999 Mar 6, 144(10), 255 - 8 Equine glanders in Turkey; Arun S et al.; In the course of an epidemiological study of glanders on a number of Turkish islands in the Sea of Marmara, 1128 horses were examined by using the intracutaneous mallein test . Thirty-five (3-1 per cent) developed an increase in rectal temperature and a swelling at the point of injection . Ten of these horses were killed and glanders was confirmed in five cases by the presence of lesions and by the immunohistological demonstration of the causative agent, Burkholderia mallei . Clinical and pathological findings indicated that in all cases the infection was restricted to the mucous membrane of the nasal cavity with its parasinus, the nostrils and the upper lips . It was confirmed that equine glanders is endemic in Turkey. Mol Cell Probes, 1999 Apr, 13(2), 99 - 105 PCR-RFLP analysis of the flagellin sequences for identification of Burkholderia pseudomallei and Burkholderia cepacia from clinical isolates; Tungpradabkul S et al.; The flagellin genes of four Burkholderia pseudomallei and two Burkholderia cepacia clinical isolates were studied by a polymerase chain reaction (PCR)-based isolation method using the same pair of primers . The PCR-amplification products of the isolates showed a single band of about 1.1 kb, which is similar to a type II B . cepacia flagellin reported previously . In order to distinguish these two species based on the flagellin PCR-amplified products, Pst I and Xho I restriction endonuclease analysis was performed . The results suggest that there is sufficient diversity within the flagellin sequences of the closely related Burkholderia species, B . pseudomallei and B . cepacia, to enable flagellin-type identification on the basis of the pattern of restriction fragments . In addition, the flagellin gene should be of considerable use as a genetic marker for clinical identification of these organisms . J Clin Microbiol, 1999 May, 37(5), 1335 - 9 Discrimination of Burkholderia multivorans and Burkholderia vietnamiensis from Burkholderia cepacia genomovars I, III, and IV by PCR; Bauernfeind A et al.; We present a PCR procedure for identification of Burkholderia cepacia, Burkholderia multivorans, and Burkholderia vietnamiensis . 16S and 23S ribosomal DNAs (rDNAs) of B . multivorans and B . vietnamiensis were sequenced and aligned with published sequences for definition of species-specific 18-mer oligonucleotide primers . Specific antisense 16S rDNA primers (for B . cepacia, 5'-AGC ACT CCC RCC TCT CAG-3'; for B . multivorans, 5'-AGC ACT CCC GAA TCT CTT-3') and 23S rDNA primers (for B . vietnamiensis, 5'-TCC TAC CAT GCG TGC AA-3') were paired with a general sense primer of 16S rDNAs (5'-AGR GTT YGA TYM TGG CTC AG-3') or with a sense primer of 23S rDNA (5'-CCT TTG GGT CAT CCT GGA-3') . PCR with these primers under optimized conditions is appropriate to specifically and rapidly identify B . multivorans, B . vietnamiensis, and B . cepacia (genomovars I, III, and IV are not discriminated) . In comparison with the polyphasic taxonomic analyses presently necessary for species and genomovar identification within the B . cepacia complex, our procedure is more rapid and easier to perform and may contribute to clarifying the clinical significance of individual members of the complex in cystic fibrosis. J Appl Microbiol, 1999 Mar, 86(3), 460 - 8 Genotypic and phenotypic relationships in Burkholderia cepacia isolated from cystic fibrosis patients and the environment; Wigley P et al.; Twenty-one strains of Burkholderia cepacia isolated from the environment, and 21 clinical strains isolated principally from sputum of cystic fibrosis (CF) patients, were characterized genotypically by macrorestriction analysis (genome fingerprinting) and PCR ribotyping, and phenotypically by susceptibility to antibiotics and the ability to macerate onion tissue . The plasmid content of the strains was also investigated . Environmental isolates showed a high degree of genetic variability, all strains differing from both one another and the CF isolates . The CF isolates were less variable, with common strains found in patients attending three geographically distinct CF centres . Phenotypic variation was found both within and between CF and environmental strains . Generally, CF isolates displayed higher levels of antibiotic resistance, while the ability to macerate onion tissue was more prevalent amongst environmental isolates . Plasmids were more frequently found in CF isolates, but were of similar size in both groups of strains . Such variability is not surprising in view of the existence of multiple genomovars within the B . cepacia complex. J Infect Dis, 1999 May, 179(5), 1197 - 205 An epidemic of burkholderia cepacia transmitted between patients with and without cystic fibrosis; Holmes A et al.; Burkholderia cepacia is an important pathogen in cystic fibrosis (CF) and an infrequent cause of nosocomial infection in non-CF patients . This report describes a large hospital outbreak that appeared to involve both patient groups, a previously unrecognized phenomenon . Ribotype restriction fragment length polymorphism (RFLP) profiles and pulsed-field gel electrophoresis-resolved macrochromosomal RFLPs were analyzed, a ribotype-based phylogenic tree was constructed, and case-control and cohort studies were performed . A single dominant clone was found in both CF and non-CF groups . Phylogenic analysis suggests that it has evolved independently and that such highly transmissible strains can emerge rapidly and randomly . Acquisition risk in the CF patients was linked to hospitalization (odds ratio=5.47, P=.0158, confidence interval=1 . 28-26.86) and was associated with significantly increased mortality rates . Infection control policies must now consider this threat of transmission between non-CF and CF patients. Syst Appl Microbiol, 1999 Feb, 22(1), 68 - 78 Physiological and phylogenetic diversity of bacteria growing on resin acids; Mohn WW et al.; Resin acids are tricyclic diterpenes which are synthesized by trees and are a major cause of toxicity of pulp mill effluents . Bacterial strains isolated from three different sources and which grow on resin acids were physiologically characterized . Eleven strains, representating distinct groups, were further characterized physiologically and phylogenetically . The isolates had distinct specificities for use, as growth substrates, of the different resin acids tested . The isolates also used fatty acids but were generally limited in use of other diverse substrates tested . According to their 16S rDNA sequences, the representative isolates are related to members of the genera, Sphingomonas, Zoogloea, Ralstonia, Burkholderia, Pseudomonas and Mycobacterium . Analysis of whole-cell fatty acid profiles generally supported those phylogenetic relationships . However, most of the isolated did not have high similarities to reference strains in the Microbial Identification System database of fatty acid profiles or in the Biolog database of substrate oxidation patterns . Described species of Sphingomonas, Zoolgoea, Burkholderia Pseudomonas, most closely related to the isolates we characterized, failed to grow on, or degrade, resin acids . We propose recognition of Zoogloea resiniphila sp . nov., Pseudomonas vancouverensis sp . nov., P . abietaniphila sp . nov . and P . multiresinivorans sp . nov. Biotechnol Bioeng, 1998 Feb 5, 57(3), 287 - 96 Activity and stability of a recombinant plasmid-borne TCE degradative pathway in suspended cultures; Sharp RR et al.; The retention and expression of the plasmid-borne, TCE degradative toluene-ortho-monooxygenase (TOM) pathway in suspended continuous cultures of transconjugant Burkholderia cepacia 17616 (TOM31c) were studied . Acetate growth and TCE degradation kinetics for the transconjugant host are described and utilized in a plasmid loss model . Plasmid maintenance did not have a significant effect on the growth rate of the transconjugant . Both plasmid-bearing and plasmid-free strains followed Andrews inhibition growth kinetics when grown on acetate and had maximum growth rates of 0.22 h-1 . The transconjugant was capable of degrading TCE at a maximum rate of 9.7 nmol TCE/min . mg protein, which is comparable to the rates found for the original plasmid host, Burkholderia cepacia PR131 (TOM31c) . The specific activity of the TOM pathway was found to be a linear function of growth rate . Plasmid maintenance was studied at three different growth rates: 0.17/h, 0.1/h, and 0.065/h . Plasmid maintenance was found to be a function of growth rate, with the probability of loss ranging from 0.027 at a growth rate of 0.065/h to 0.034 at a growth rate 0.17/h . Rev Pneumol Clin, 1998 Dec, 54(6), 365 - 72 {Pulmonary melioidosis}; Perret JL et al.; Melioidosis is most frequently encountered in pulmonary localization . Melioidosis is an infectious disease caused by Burkholderia pseudomallei first described by Whitmore in 1912 in Burma . B . pseudomallei is a Gram negative rod belonging to the Pseudomonadaceae family . Soil and water are the natural reservoirs for the germ which is a specific pathogen for several mammal species . Long endemic in Southeast Asia and several tropical zones, B . pseudomallei has recently been found in temperate zones, including France . Human contamination occurs via the transcutaneous route and often leads to dormant inapparent infection . Many conditions, such as diabetes, renal lithiasis, various circumstances of immunodepression or stress, facilitate clinical manifestations which vary greatly . Pulmonary manifestations may be acute and extensive, producing a torpid pseudo-tuberculous condition or a variety of clinical and radiological features mimicking other diseases . Bacteriological and serological tests may be negative . Exposure in an endemic zone, the notion of a favorable context, weight loss, cavitary images on successive chest x-rays and the presence of extra-pulmonary localizations may be suggestive . Ceftazidime or the amoxicillin-clavulanic acid combination are indicated, but mortality in acute forms still reaches 40% . Relapse can be expected if the treatment duration is too short. J Toxicol Environ Health A, 1999 Mar 26, 56(6), 419 - 31 Colonization and clearance of environmental microbial agents upon intranasal exposure of strain C3H/HeJ mice; George SE et al.; Environmental dissemination of biotechnology agents is becoming a common practice . Most applications use historically innocuous species; however, potential health effects of individual products are not scrutinized unless they contain genetically engineered microorganisms . In order to investigate possible health concerns, four surrogate microbial agents were studied in vivo . Male C3H/HeJ (endotoxin-resistant) mice were administered intranasally (i.n.) with approximately 10(7) Pseudomonas aureofaciens, Burkholderia cepacia, P . fluorescens, or P . putida . To determine clearance of the dosed bacterial strains, lungs, small intestine, large intestine, cecum, mesenteric lymph nodes (MLN), spleen, and liver were homogenized individually, plated, and dilutions inoculated onto selective media . Pseudomonas fluorescens and P . putida were eliminated from the lungs by 2 d posttreatment, and P . aureofaciens was not detected in the lungs by 5 d posttreatment . Burkholderia cepacia was reisolated from the lungs and cecum for the experimental duration (14 d) . Translocation to extraintestinal sites (MLN, spleen, and liver) also occurred . Burkholderia cepacia was recovered from the MLN for 10 d after treatment of mice . Pulmonary exposure to several bacterial strains resulted in unexpected mortality . Pseudomonas aureofaciens was lethal at the lowest dose (8.26 x 10(6) CFU/ mouse), while P . fluorescens and B . cepacia were fatal at higher doses (6.15 x 10(8) CFU/mouse and 1.34 x 10(8) CFU/mouse, respectively) . By using the model described in this study, human safety issues can be more easily addressed and evaluated. Res Microbiol, 1999 Jan-Feb, 150(1), 45 - 59 Isolation and characterisation of a new antagonistic Burkholderia strain from the rhizosphere of healthy tomato plants; Sfalanga A et al.; A new Burkholderia strain (PVFi5A) which exhibits antagonism towards many bacterial and fungal plant pathogens has been partially characterised . This strain was isolated from the rhizosphere of tomato plants and was referred to the Burkholderia cepacia complex on the basis of cultural, morphological and biochemical characteristics, including determination of the 16S ribosomal DNA sequence and fatty acid profile . Strain PVFi5A is a Gram-negative, aerobic bacterium, oxidase- and catalase-positive, motile with a polar tuft of flagella, able to grow on a variety of media without producing diffusible pigments; it is avirulent to onion, able to grow at 41 degrees C and resistant to several antibiotic substances . Its fatty acid profile contains the hydroxy acids 18:1 20H, 14:0 3OH and 16:0 3OH, but not the hydroxy acids 16:0 2OH . The antagonistic activity of strain PVFi5A is due to its production of various, as yet unidentified, antimicrobial compounds, one or more of which may differ from those reported previously for certain 'B . cepacia' strains . The ability of PVFi5A to suppress the growth of important bacterial and fungal phytopathogens makes this strain a potential biocontrol agent. Eur J Biochem, 1999 Mar, 260(2), 373 - 83 Structural elucidation of a novel exopolysaccharide produced by a mucoid clinical isolate of Burkholderia cepacia . Characterization of a trisubstituted glucuronic acid residue in a heptasaccharide repeating unit; Cerantola S et al.; The structure of the exopolysaccharide (EPS) produced by a clinical isolate of Burkholderia cepacia isolated from a patient with fibrocystic lung disease has been investigated . By means of methylation analyses, carboxyl reduction, partial depolymerization by fuming HCl and chemical degradations such as Smith degradation, lithiumethylenediamine degradation and beta-elimination, supported by GC/MS and NMR spectroscopic analyses, the repeat unit of the EPS has been identified and was shown to correspond to the acidic branched heptasaccharide with the following structure: {formula: see text} . This partially acetylated acidic polymer, distinguished by the presence of the less usual D-isomer of rhamnose and of a trisubstituted glucuronic acid residue, could represent the main EPS produced by this bacterial species. Microbios, 1998, 96(384), 71 - 93 Effects of Burkholderia pseudomallei and other Burkholderia species on eukaryotic cells in tissue culture; Harley VS et al.; Burkholderia pseudomallei causes melioidosis, a serious and often fatal bacterial infection . B . pseudomallei can behave as a facultatively intracellular organism and this ability may be important in the pathogenesis of both acute and chronic infection . The uptake of B . pseudomallei and other Burkholderia spp . by cells in tissue culture was examined by electron microscopy . B . pseudomallei can invade cultured cell lines including phagocytic lines such as RAW264, J774 and U937, and non-phagocytic lines such as CaCO-2, Hep2, HeLa, L929, McCoy, Vero and CHO . Uptake was followed by the intracellular multiplication of B . pseudomallei and the induction of cell fusion and multinucleate giant cell formation . Similar effects were produced by B . mallei and B . thailandensis. Eur J Biochem, 1999 Feb, 259(3), 887 - 91 Structural analysis of a novel putative capsular polysaccharide from Pseudomonas (Burkholderia) caryophylli strain 2151; Molinaro A et al.; A novel putative capsular polysaccharide consisting of D-Glcp and D-Fruf in the molar ratio of 1:1 was isolated as minor constituent from the lipopolysaccharide (LPS) fraction of Pseudomonas (Burkholderia) caryophylli . Its structure was determined, using mainly one- and two-dimensional NMR spectroscopy, as: -->6)-alpha-D-Glcp-(1-->1)-beta-D-Fruf-(2-->. Diagn Microbiol Infect Dis, 1999 Feb, 33(2), 81 - 6 In vitro activity of ciprofloxacin, levofloxacin, and trovafloxacin, alone and in combination with beta-lactams, against clinical isolates of Pseudomonas aeruginosa, Stenotrophomonas maltophilia, and Burkholderia cepacia; Isenberg HD et al.; We tested three fluoroquinolones (ciprofloxacin, levofloxacin, and trovafloxacin), each combined with each of four beta-lactams (cefoperazone, ceftriaxone, imipenem, and meropenem) for synergy against clinical isolates of nosocomial strains of Pseudomonas aeruginosa, Stenotrophomonas maltophilia, and Burkholderia cepacia . The ciprofloxacin-beta-lactam combinations showed synergy against none or only a small fraction (7 to 10%) of the P . aeruginosa and B . cepacia isolates . Ciprofloxacin-cefoperazone, -ceftriaxone, and -meropenem were synergic against 50%, 25%, and 30% of the S . maltophilia isolates, respectively . Among the levofloxacin combinations, only those with cefoperazone and imipenem showed significant synergy, and this only against B . cepacia (50% and 30%, respectively) . Trovafloxacin-cefoperazone and -imipenem showed modest synergy against P . aeruginosa (23% and 27%, respectively), as did trovafloxacin-cefoperazone and -ceftriaxone against B . cepacia (30%) . The trovafloxacin-imipenem combination was synergic against all isolates of B . cepacia . Because of their synergy, the following combinations may be useful in the nosocomial setting: trovafloxacin-cefoperazone or -imipenem against P . aeruginosa; ciprofloxacin-cefoperazone, -ceftriaxone, or -meropenem against S . maltophilia; levofloxacin-cefoperazone and trovafloxacin-imipenem against B . cepacia. Chest, 1999 Mar, 115(3), 782 - 7 Timing of referral for lung transplantation for cystic fibrosis: overemphasis on FEV1 may adversely affect overall survival; Doershuk CF et al.; STUDY OBJECTIVES: (1) Report our experience with referral for lung transplantation . (2) Review survival in cvstic fibrosis (CF) patients without lung transplantation after FEV1 remains < 30% predicted for 1 years . DESIGN: Retrospective review . SETTING: A university hospital CF center . PATIENTS: (1) Forty-five patients referred for lung transplantation evaluation, and (2) 178 patients without Burkholderia sp infection, with the above FEVl criterion . MAIN OUTCOME MEASURE: Survival . MEASUREMENTS AND RESULTS: (1) One- and 2-year survival after transplantation was 55% and 45%, respectively . However, among patients without transplants with FEVl < 30% predicted, median survival, 1986 to 1990, ie, before the transplant era, was 4.6 years with 25% living > 9 years (before 1986, 25% lived > 6 vears) . (2) Survival after transplantation was not correlated to any of the following: age, sex, genotype, FEVI percent predicted, insulin-dependent diabetes mellitus, or with waiting time before transplantation, and did not seem to be correlated to serum bicarbonate or percent ideal body weight . Four of five patients already infected with Burkholderia species died within 5 months of transplantation; the fifth died at 17 months . All five died of pulmonary or extrapulmonarv infection with Burkholderia species CONCLUSIONS: Use of FEV! < 30% predicted to automatically establish transplantation eligibility could lead to decreased overall survival for CF patients . Referral for evaluation and transplantation should also be based on oxygen requirement, rate of deterioration, respiratory microbiology, quality of life, frequency of IV antibiotic therapy, and other considerations . If pulmonary status has unexpectedly improved when the patient is at or near the top of the waiting list, total survival may be improved by "inactivating the patient" until progression is again evident. J Clin Microbiol, 1999 Apr, 37(4), 1018 - 23 Identification and characterization of IS2404 and IS2606: two distinct repeated sequences for detection of Mycobacterium ulcerans by PCR; Stinear T et al.; Molecular analysis of Mycobacterium ulcerans has revealed two new insertion sequences (ISs), IS2404 and IS2606 . IS2404 was identified by complete sequencing of a previously described repetitive DNA segment from M . ulcerans . This element is 1,274 bp long, contains 12-bp inverted repeats and a single open reading frame (ORF) potentially encoding a protein of 327 amino acids (aa), and apparently generates 7-bp direct repeats upon transposition . Amino acid similarity was found between the putative transposase and those encoded by ISs in other bacterial sequences from Aeromonas salmonicida (AsIs1), Escherichia coli (H repeat element), Vibrio cholerae (VcIS1), and Porphyromonas gingivalis (PGIS2) . The second IS, IS2606, was discovered by sequence analysis of a HaeIII fragment of M . ulcerans genomic DNA containing a repetitive sequence . This element is 1,404 bp long, with 12-bp inverted repeats and a single ORF potentially encoding a protein of 445 aa . Database searches revealed a high degree of amino acid identity (70%) with the putative transposase of IS1554 from M . tuberculosis . Significant amino acid identity (40%) was also observed with transposases from several other microorganisms, including Rhizobium meliloti (ISRm3), Burkholderia cepacia (IS1356), Corynebacterium diphtheriae, and Yersinia pestis . PCR screening of DNA from 45 other species of mycobacteria with primers for IS2404 confirm that this element is found only in M . ulcerans . However, by PCR, IS2606 was also found in Mycobacterium lentiflavum, another slow-growing member of the genus Mycobacterium that is apparently genetically distinct from M . ulcerans . Testing the sensitivity of PCR based on IS2404 and IS2606 primers demonstrated the ability to detect 0.1 and 1 M . ulcerans genome equivalents, respectively . The ability to detect small numbers of cells by using two gene targets will be particularly useful for analyzing environmental samples, where there may be low concentrations of M . ulcerans among large numbers of other environmental mycobacteria. J Clin Microbiol, 1999 Apr, 37(4), 1004 - 7 Comparison of isolation media for recovery of Burkholderia cepacia complex from respiratory secretions of patients with cystic fibrosis; Henry D et al.; Burkholderia cepacia selective agar (BCSA) has previously been devised for isolation of B . cepacia from respiratory secretions of patients with cystic fibrosis and tested under research laboratory conditions . Here we describe a study in which BCSA, oxidation-fermentation polymyxin bacitracin lactose agar (OFPBL), and Pseudomonas cepacia agar (PCA) were compared in routine culture procedures for the ability to grow B . cepacia and inhibit other organisms . Three hundred twenty-eight specimens from 209 patients at two pediatric centers and 328 specimens from 109 adults were tested . Plates were inoculated, incubated, and read for quality and quantity of growth at 24, 48, and 72 h . Five (1.5%) specimens from 4 (1.9%) children and 75 (22.9%) specimens from 16 (14.7%) adults grew B . cepacia complex . At 24, 48, and 72 h, BCSA achieved 43, 93, and 100% detection, respectively; OFPBL achieved 26, 84, and 96%, respectively; and PCA achieved 33, 74, and 84% detection, respectively . Quality was assessed as pinpoint or good growth . At 24 h, most cultures growing B . cepacia complex had pinpoint colonies . By 48 and 72 h, 48 and 69% of B . cepacia complex cultures, respectively, had good growth on BCSA, while on OFPBL 19 and 30%, respectively, had good growth and on PCA 11 and 18%, respectively, had good growth . BCSA was superior to OFPBL and PCA in suppressing organisms other than B . cepacia complex; 40 non-B . cepacia complex organisms were isolated from BCSA, 263 were isolated from OFPBL, and 116 were isolated from PCA . We conclude that BCSA is superior to OFPBL and PCA in its ability to support the growth of B . cepacia complex and to suppress other respiratory organisms. J Basic Microbiol, 1999, 39(1), 17 - 24 Effect of urokinase on the extracellular virulence properties of Pseudomonas aeruginosa and Burkholderia cepacia; Heys SJ et al.; The influence of urokinase and oxygen availability on growth, siderophore, protease and lipase production in Burkholderia cepacia and non-mucoid (PA01) and mucoid (PaWH) strains of Pseudomonas aeruginosa was assessed for cells grown in batch culture under iron-restriction . Siderophore production decreased with increasing concentration of urokinase in B . cepacia independent of oxygen availability but decreased in both strains of P . aeruginosa only under oxygen-depleted conditions . Protease activity was enhanced for all three strains irrespective of oxygen content whereas lipase production increased in B . cepacia and decreased in PA01 under both sets of growth conditions and varied with oxygen availability in PaWH . The evidence presented suggests that urokinase could contribute to the pathophysiology of pulmonary infections. Curr Microbiol, 1999 Apr, 38(4), 233 - 8 A nonhemolytic phospholipase C from Burkholderia cepacia; Weingart CL et al.; Burkholderia cepacia is an opportunistic pathogen that causes serious pulmonary infections in cystic fibrosis patients . Although several potential virulence factors-a protease, lipase, and two phospholipases C (one hemolytic and one nonhemolytic)-have been identified, only two, the protease and the lipase, have been described in detail . The goal of this study was to purify and characterize a nonhemolytic phospholipase C secreted by B . cepacia strain Pc224c . The enzyme was concentrated from culture supernatants and purified by polyacrylamide gel electrophoresis . The 54-kDa protein was stable in the presence of sodium dodecyl sulfate (up to 10%) and at 4 degrees, 22 degrees, and 37 degrees C; it was, however, inactivated at 100 degrees C . The enzyme bound to glass, chromatography matrices, and polyvinylidene difluoride and cellulose membranes, suggesting that it is hydrophobic . In a genetic approach, primers based on conserved sequences of a B . cepacia Pc69 hemolytic phospholipase C and both the Pseudomonas aeruginosa hemolytic and nonhemolytic proteins were designed to identify the Pc224c nonhemolytic phospholipase C gene . One polymerase chain reaction product was identified; it was sequenced and the sequence compared with sequences in the BLAST database . The best match was the Pseudomonas aeruginosa hemolytic phospholipase C . Ten additional B . cepacia strains were screened for the gene by Southern hybridization; five had the 4-kb band, suggesting that these strains have a similar form of the PLC gene . Nine of the ten strains reacted with the probe, suggesting that similar sequences were present, but in another form. Antimicrob Agents Chemother, 1999 Mar, 43(3), 465 - 70 Efflux-mediated aminoglycoside and macrolide resistance in Burkholderia pseudomallei; Moore RA et al.; Burkholderia pseudomallei, the causative agent of melioidosis, is intrinsically resistant to a wide range of antimicrobial agents including beta-lactams, aminoglycosides, macrolides, and polymyxins . We used Tn5-OT182 to mutagenize B . pseudomallei to identify the genes involved in aminoglycoside resistance . We report here on the identification of AmrAB-OprA, a multidrug efflux system in B . pseudomallei which is specific for both aminoglycoside and macrolide antibiotics . We isolated two transposon mutants, RM101 and RM102, which had 8- to 128-fold increases in their susceptibilities to the aminoglycosides streptomycin, gentamicin, neomycin, tobramycin, kanamycin, and spectinomycin . In addition, both mutants, in contrast to the parent, were susceptible to the macrolides erythromycin and clarithromycin but not to the lincosamide clindamycin . Sequencing of the DNA flanking the transposon insertions revealed a putative operon consisting of a resistance, nodulation, division-type transporter, a membrane fusion protein, an outer membrane protein, and a divergently transcribed regulatorprotein . Consistent with the presence of an efflux system, both mutants accumulated {3H} dihydro streptomycin, whereas the parent strain did not . We constructed an amr deletion strain, B . pseudomallei DD503, which was hypersusceptible to aminoglycosides and macrolides and which was used successfully in allelic exchange experiments . These results suggest that an efflux system is a major contributor to the inherent high-level aminoglycoside and macrolide resistance found in B . pseudomallei. Int J Syst Bacteriol, 1999 Jan, 49 Pt 1, 37 - 42 Burkholderia cocovenenans (van Damme et al . 1960) Gillis et al . 1995 and Burkholderia vandii Urakami et al . 1994 are junior synonyms of Burkholderia gladioli (Severini 1913) Yabuuchi et al . 1993 and Burkholderia plantarii (Azegami et al . 1987) Urakami et al . 1994, respectively; Coenye T et al.; Reference strains of Burkholderia cocovenenans and Burkholderia vandii were compared with strains of other Burkholderia species using SDS-PAGE of whole-cell proteins, DNA-DNA hybridization and extensive biochemical characterization . Burkholderia gladioli and B . cocovenenans were indistinguishable in the chemotaxonomic and biochemical analyses . Burkholderia plantarii and B . vandii had indistinguishable whole-cell protein patterns but the B . vandii type strain differed from B . plantarii strains in several biochemical tests . The DNA-DNA binding levels (higher than 70%) indicated that (i) B . gladioli and B . cocovenenans, and (ii) B . plantarii and B . vandii each represent a single species . It is concluded that B . cocovenenans and B . vandii are junior synonyms of B . gladioli and B . plantarii, respectively. Infect Immun, 1999 Mar, 67(3), 1505 - 7 Lipopolysaccharide (LPS) from Burkholderia cepacia is more active than LPS from Pseudomonas aeruginosa and Stenotrophomonas maltophilia in stimulating tumor necrosis factor alpha from human monocytes; Zughaier SM et al.; Whole cells and lipopolysaccharides (LPSs) extracted from Burkholderia cepacia, Pseudomonas aeruginosa, Stenotrophomonas maltophilia, and Escherichia coli were compared in their ability to stimulate tumor necrosis factor alpha (TNF-alpha) from the human monocyte cell line MonoMac-6 . B . cepacia LPS, on a weight-for-weight basis, was found to have TNF-alpha-inducing activity similar to that of LPS from E . coli, which was approximately four- and eightfold greater than the activity of LPSs from P . aeruginosa and S . maltophilia, respectively . The LPS-stimulated TNF-alpha production from monocytes was found to be CD14 dependent . These results suggest that B . cepacia LPS might play a role in the pathogenesis of inflammatory lung disease in cystic fibrosis, and in some patients it might be responsible, at least in part, for the sepsis-like cepacia syndrome. Mol Microbiol, 1998 Dec, 30(5), 1081 - 100 The type II O-antigenic polysaccharide moiety of Burkholderia pseudomallei lipopolysaccharide is required for serum resistance and virulence; DeShazer D et al.; Melioidosis, an infection caused by the gram-negative bacterial pathogen Burkholderia pseudomallei, is endemic in south-east Asia and northern Australia . Acute septicaemic melioidosis is a major cause of morbidity and mortality, especially in north-east Thailand . B . pseudomallei is highly resistant to the bactericidal activity of normal human serum (NHS), and we have found that B . pseudomallei 1026b multiplies in 10-30% NHS . We developed a simple screen for the identification of serum-sensitive mutants based on this novel phenotype . Approximately 1200 Tn5-OT182 mutants were screened, and three serum-sensitive mutants were identified . The type II O-antigenic polysaccharide (O-PS) moiety of lipopolysaccharide was not present in the serum-sensitive mutants . A representative serum-sensitive mutant, SRM117, was killed by the alternative pathway of complement and was less virulent than 1026b in three animal models of melioidosis . The Tn5-OT182 integrations in the serum-sensitive mutants were physically linked on the B . pseudomallei chromosome, and further genetic analysis of this locus revealed a cluster of 15 genes required for type II O-PS production . The proteins encoded by these genes were similar to proteins involved in bacterial polysaccharide biosynthesis . The results presented here demonstrate that type II O-PS is essential for B . pseudomallei serum resistance and virulence. Am J Trop Med Hyg, 1999 Jan, 60(1), 90 - 3 Attachment of Burkholderia pseudomallei to pharyngeal epithelial cells: a highly pathogenic bacteria with low attachment ability; Ahmed K et al.; Respiratory infections are initiated by the attachment of bacteria to pharyngeal epithelial cells . We studied the attachment of Burkholderia pseudomallei to pharyngeal epithelial cells . After one, two, three, and four washes, there were 22.6+/-8.9, 15.7+/-7.0, 6.8+/-3.1, and 4.6+/-1.1 (mean+/-SD) attached bacteria/cell, respectively . If the bacterial concentration was maintained at 1 X 10(8) colony-forming units (cfu)/ml and three washes were done, at concentrations of 2.5 x 10(4), 5 X 10(4), and 1 x 10(5) cells/ml there were 9.9+/-3.6, 3.3+/-0.8, and 2.5+/-1.1 attached bacteria/cell, respectively . If the cell concentration was kept at 2.5 x 10(4) cells/ml and three washes were done, at bacterial concentrations of 1 x 10(5), 1 X 10(6), 1 X 10(7), 1 x 10(8), and 1 x 10(9) cfu/ml, there were 0.3+/-0.3, 0.6+/-0.6, 1.0+/-0.2, 5.1+/-2.3, and 9.6+/-1.9 attached bacteria/cell, respectively . There were 4.8+/-1.9, 5.5+/-2.5, 5.6+/-1.9, and 6.4+/-2.6 attached bacteria/cell at 0, 30, 120, and 240 min of incubation, respectively . Pharyngeal cells from 10 persons (seven men and three women, mean+/-SD age = 30.7+/-8.1 years, 12 experiments with a single isolate) showed that there were 7.8+/-4.3 attached bacteria/cell . It was found that the efficiency of attachment of this bacteria was very low (7.0+/-3.3 bacteria/cell) . Electron microscopy revealed that there were no fimbriae but a thin capsular polysaccharide layer on the surface of B . pseudomallei . Attachment to pharyngeal epithelial cells appeared to be mediated by this structure. Mol Gen Mikrobiol Virusol, 1998, (4), 28 - 33 {Isolation and primary characteristics of a Pseudomonas (Burkholderia) pseudomallei plasmid}; Zamaraev VS et al.; Plasmid screening of reference Pseudomonas pseudomallei strains isolated from patients and animals revealed cryptic plasmids with different molecular weights in 30 strains . Plasmids were investigated by restriction analysis and DNA-DNA hybridization . Cryptic plasmids were denoted as pPM1, pCM2, pCM3, and pCM4 . The presence of plasmids in melioidosis agent permits their use for intraspecies typing of strains and for genetic studies. J Clin Microbiol, 1999 Mar, 37(3), 753 - 7 Impact of microbiology practice on cumulative prevalence of respiratory tract bacteria in patients with cystic fibrosis; Shreve MR et al.; Investigators participating in the Epidemiologic Study of Cystic Fibrosis project began to collect microbiological, pulmonary, and nutritional data on cystic fibrosis (CF) patients at 180 North American sites in 1994 . Part of this study was a survey undertaken in August 1995 to determine microbiology laboratory practices with regard to pulmonary specimens from CF patients . The survey included a section on test ordering, completed by a site clinician, and a section on test performance and reporting, completed by each site's clinical microbiology laboratory staff . Seventy-nine percent of the surveys were returned . There was intersite consistency of microbiology laboratory practices in most cases . The majority of sites follow most of the CF Foundation consensus conference recommendations . There were differences in the frequency at which specimens for culture were obtained, in the use of selective media for Staphylococcus aureus and Haemophilus influenzae, and in the use of a prolonged incubation for Burkholderia cepacia . These variations in practice contribute to prevalence differences among sites and may result in differences in clinical care. Biosci Biotechnol Biochem, 1998 Dec, 62(12), 2312 - 7 Purification and characterization of tert-butyl ester-hydrolyzing lipase from Burkholderia sp . YY62; Yeo SH et al.; An intracellular novel lipase which can hydrolyze t-butyl octanoate (TBO) was purified to homogeneity from crude cell-free extracts of Burkholderia (formerly Pseudomonas) sp . YY62 with 9% overall yield . Seventy-four-fold purification was achieved by ammonium-sulfate precipitation, three consecutive open-column chromatographies (DEAE anion-exchange, Sepharose CL-6B gel-filtration, and the second DEAE anion-exchange columns), and two HPLCs (TSK G2000SWXL gel-filtration and phenyl 5PW hydrophobic interaction columns) . Enzymes hydrolyzing p-nitrophenyl acetate were separated into two peaks (peak I and II) on the hydrophobic HPLC, and only peak II was found to have TBO-hydrolyzing activity . The peak preparation showed a single band of 40 kDa on SDS-PAGE and a molecular mass of 39 kDa on gel-filtration under non-denatured conditions, indicating the monomeric nature of the TBO-hydrolyzing lipase . The lipase showed maximum activity at pH 7.0 and 28 degrees C . The N-terminal 15 amino acid residues were determined as Met-Asp-Phe-Tyr-Asp-Ala-Asn-Glu-Thr-Arg-His-Pro-Glu-Gln-Arg, which showed no homology to known proteins, suggesting that the purified enzyme may belong to a novel class of hydrolase. Appl Environ Microbiol, 1999 Feb, 65(2), 849 - 52 New assay for rhizobitoxine based on inhibition of 1-aminocyclopropane-1-carboxylate synthase; Yasuta T et al.; Rhizobitoxine is synthesized by the legume symbiont Bradyrhizobium elkanii and the plant pathogen Burkholderia andropogonis . Rhizobitoxine competitively inhibited 1-aminocyclopropane-1-carboxylate (ACC) synthase bLE-ACS2 from the tomato, a key enzyme in the pathway of ethylene biosynthesis . Based on this inhibition of ACC synthase, we have developed a new assay for rhizobitoxine. Appl Environ Microbiol, 1999 Feb, 65(2), 759 - 65 Alterations in adhesion, transport, and membrane characteristics in an adhesion-deficient pseudomonad; DeFlaun MF et al.; A stable adhesion-deficient mutant of Burkholderia cepacia G4, a soil pseudomonad, was selected in a sand column assay . This mutant (ENV435) was compared to the wild-type strain by examining the adhesion of the organisms to silica sand and their transport through two aquifer sediments that differed in their sand, silt, and clay contents . We compared the longitudinal transport of the wild type and the adhesion mutant to the transport of a conservative chloride tracer in 25-cm-long glass columns . The transport of the wild-type strain was severely retarded compared to the transport of the conservative tracer in a variety of aquifer sediments, while the adhesion mutant and the conservative tracer traveled at similar rates . An intact sediment core study produced similar results; ENV435 was transported at a faster rate and in much greater numbers than G4 . The results of hydrophobic interaction chromatography revealed that G4 was significantly more hydrophobic than ENV435, and polyacrylamide gel electrophoresis revealed significant differences in the lipopolysaccharide O-antigens of the adhesion mutant and the wild type . Differences in this cell surface polymer may explain the decreased adhesion of strain ENV435. Appl Environ Microbiol, 1999 Feb, 65(2), 787 - 94 High-level formation of active Pseudomonas cepacia lipase after heterologous expression of the encoding gene and its modified chaperone in Escherichia coli and rapid in vitro refolding; Quyen DT et al.; The lipase from Pseudomonas cepacia ATCC 21808 (recently reclassified as Burkholderia cepacia) is widely used by organic chemists for enantioselective synthesis and is manufactured from recombinant P . cepacia harboring on a plasmid the clustered genes for lipase and its chaperone . High levels of expression of inactive lipase (40%) in Escherichia coli were achieved with pCYTEXP1 under the control of the strong, temperature-inducible lambdaPRL promoter . However, no overexpression of the lipase chaperone was achieved in E . coli . Thus, chemical refolding of inactive lipase in the absence of its chaperone yielded only 25 U/mg, compared to 3,470 U of the purified lipase secreted by recombinant P . cepacia per mg . Sequence analysis of the chaperone revealed a high GC content (>90%) in the 5' region of the gene and the presence of a putative membrane anchor at the N terminus . Hence, the 5' region of the gene was replaced by a synthetic fragment, and the putative membrane anchor was removed by deletion of the first 34 or 70 N-terminal amino acids . Only truncation of the gene led to overexpression of the chaperone (up to 60%) in E . coli . With this chaperone, it was possible to obtain for the first time in a simple refolding procedure a highly active Pseudomonas lipase (classes I and II) expressed in E . coli with a specific activity of up to 4,850 U/mg and a yield of 314,000 U/g of E . coli wet cells. Appl Environ Microbiol, 1999 Feb, 65(2), 632 - 9 Inactivation of toluene 2-monooxygenase in Burkholderia cepacia G4 by alkynes; Yeager CM et al.; High concentrations of acetylene (10 to 50% {vol/vol} gas phase) were required to inhibit the growth of Burkholderia cepacia G4 on toluene, while 1% (vol/vol) (gas phase) propyne or 1-butyne completely inhibited growth . Low concentrations of longer-chain alkynes (C5 to C10) were also effective inhibitors of toluene-dependent growth, and 2- and 3-alkynes were more potent inhibitors than their 1-alkyne counterparts . Exposure of toluene-grown B . cepacia G4 to alkynes resulted in the irreversible loss of toluene- and o-cresol-dependent O2 uptake activities, while acetate- and 3-methylcatechol-dependent O2 uptake activities were unaffected . Toluene-dependent O2 uptake decreased upon the addition of 1-butyne in a concentration- and time-dependent manner . The loss of activity followed first-order kinetics, with apparent rate constants ranging from 0.25 min-1 to 2.45 min-1 . Increasing concentrations of toluene afforded protection from the inhibitory effects of 1-butyne . Furthermore, oxygen, supplied as H2O2, was required for inhibition by 1-butyne . These results suggest that alkynes are specific, mechanism-based inactivators of toluene 2-monooxygenase in B . cepacia G4, although the simplest alkyne, acetylene, was relatively ineffective compared to longer alkynes . Alkene analogs of acetylene and propyne-ethylene and propylene-were not inactivators of toluene 2-monooxygenase activity in B . cepacia G4 but were oxidized to their respective epoxides, with apparent Ks and Vmax values of 39.7 microM and 112.3 nmol min-1 mg of protein-1 for ethylene and 32.3 microM and 89.2 nmol min-1 mg of protein-1 for propylene. Antimicrob Agents Chemother, 1999 Feb, 43(2), 213 - 7 Comparative in vitro activities of meropenem, imipenem, temocillin, piperacillin, and ceftazidime in combination with tobramycin, rifampin, or ciprofloxacin against Burkholderia cepacia isolates from patients with cystic fibrosis; Bonacorsi S et al.; We evaluated the activities of meropenem, imipenem, temocillin, piperacillin, and ceftazidime by determination of the MICs for 66 genotypically characterized Burkholderia cepacia isolates obtained from the sputum of cystic fibrosis patients . In vitro synergy assays, as performed by the time-kill methodology, of two- and three-drug combinations of the beta-lactams with tobramycin, rifampin, and/or ciprofloxacin were also performed with 10 strains susceptible, intermediate, or resistant to fluoroquinolones . On the basis of the MICs, meropenem and temocillin were the most active beta-lactam agents, with MICs at which 90% of isolates are inhibited of 8 and 32 micrograms/ml, respectively . The addition of ciprofloxacin significantly enhanced the killing activities of piperacillin, imipenem, and meropenem against the 10 strains tested (P < 0.05) . The best killing activity was obtained with the combination of meropenem and ciprofloxacin, with bactericidal activity of 3.31 +/- 0.36 log10 CFU/ml (P < 0.05) . Compared to the activity of the two-drug beta-lactam-ciprofloxacin combination, the addition of rifampin or tobramycin did not significantly increase the killing activity (P > 0.05) . The three-drug combinations (with or without ciprofloxacin) significantly enhanced the killing activities of piperacillin, imipenem, and meropenem relative to the activities of the beta-lactams used alone (P < 0.05) . The combination beta-lactam-ciprofloxacin-tobramycin was the combination with the most consistently synergistic effect. Ann Trop Paediatr, 1998 Jun, 18(2), 161 - 4 Neonatal meningitis and septicaemia caused by Burkholderia pseudomallei; Halder D et al.; We report a case of meningitis and one of fatal septicaemia in neonates due to Burkholderia pseudomallei and review the literature on neonatal melioidosis . Pneumonia was the primary presentation and was complicated by shock in the latter case . The epidemiological findings suggest that the cases reported from Malaysia were community-acquired in contrast with those from the USA and Thailand. J Bacteriol, 1999 Feb, 181(3), 965 - 72 Biochemical and genetic evidence for meta-ring cleavage of 2,4, 5-trihydroxytoluene in Burkholderia sp . strain DNT; Haigler BE et al.; 2,4,5-Trihydroxytoluene (THT) oxygenase from Burkholderia sp . strain DNT catalyzes the conversion of THT to an unstable ring fission product . Biochemical and genetic studies of THT oxygenase were undertaken to elucidate the mechanism of the ring fission reaction . The THT oxygenase gene (dntD) was previously localized to the 1.2-kb DNA insert subcloned in the recombinant plasmid designated pJS76 (W . C . Suen and J . C . Spain, J . Bacteriol . 175:1831-1837, 1993) . Analysis of the deduced amino acid sequence of DntD revealed the presence of the highly conserved residues characteristic of the catechol 2,3-dioxygenase gene family I . The deduced amino acid sequence of DntD corresponded to a molecular mass of 35 kDa . The native molecular masses for the THT oxygenase estimated by using gel filtration chromatography and nondenaturing gel electrophoresis were 67.4 and 77.8 kDa, respectively . The results suggested that the native protein consists of two identical subunits . The colorless protein contained 2 mol of iron per mol of protein . Stimulation of activity in the presence of ferrous iron and ascorbate suggested a requirement for ferrous iron in the active site . The properties of the enzyme are similar to those of the catechol 2,3-dioxygenases (meta-cleavage dioxygenases) . In addition to THT, the enzyme exhibited activity towards 1,2,4-benzenetriol, catechol, 3- and 4-methylcatechol, and 3- and 4-chlorocatechol . The chemical analysis of the THT ring cleavage product showed that the product was 2, 4-dihydroxy-5-methyl-6-oxo-2,4-hexadienoic acid, consistent with extradiol ring fission of THT. J Bacteriol, 1999 Feb, 181(3), 748 - 56 Quorum sensing in Burkholderia cepacia: identification of the LuxRI homologs CepRI; Lewenza S et al.; Burkholderia cepacia has emerged as an important pathogen in patients with cystic fibrosis . Many gram-negative pathogens regulate the production of extracellular virulence factors by a cell density-dependent mechanism termed quorum sensing, which involves production of diffusible N-acylated homoserine lactone signal molecules, called autoinducers . Transposon insertion mutants of B . cepacia K56-2 which hyperproduced siderophores on chrome azurol S agar were identified . One mutant, K56-R2, contained an insertion in a luxR homolog that was designated cepR . The flanking DNA region was used to clone the wild-type copy of cepR . Sequence analysis revealed the presence of cepI, a luxI homolog, located 727 bp upstream and divergently transcribed from cepR . A lux box-like sequence was identified upstream of cepI . CepR was 36% identical to Pseudomonas aeruginosa RhlR and 67% identical to SolR of Ralstonia solanacearum . CepI was 38% identical to RhlI and 64% identical to SolI . K56-R2 demonstrated a 67% increase in the production of the siderophore ornibactin, was protease negative on dialyzed brain heart infusion milk agar, and produced 45% less lipase activity in comparison to the parental strain . Complementation of a cepR mutation restored parental levels of ornibactin and protease but not lipase . An N-acylhomoserine lactone was purified from culture fluids and identified as N-octanoylhomoserine lactone . K56-I2, a cepI mutant, was created and shown not to produce N-octanoylhomoserine lactone . K56-I2 hyperproduced ornibactin and did not produce protease . These data suggest both a positive and negative role for cepIR in the regulation of extracellular virulence factor production by B . cepacia. Infect Immun, 1999 Feb, 67(2), 908 - 13 A melanin pigment purified from an epidemic strain of Burkholderia cepacia attenuates monocyte respiratory burst activity by scavenging superoxide anion; Zughaier SM et al.; The acquisition of Burkholderia cepacia in some cystic fibrosis patients is associated with symptoms of acute pulmonary inflammation that may be life threatening . The ability of lipopolysaccharide (LPS) from B . cepacia to prime a monocyte cell line for enhanced superoxide anion generation was investigated and compared with the priming activities of LPSs from Pseudomonas aeruginosa, Stenotrophomonas maltophilia, and Escherichia coli . The human monocyte cell line MonoMac-6 (MM6) was primed overnight with different LPSs (100 ng/ml), and the respiratory burst was triggered by exposure to opsonized zymosan (125 micrograms/ml) . Superoxide generation was detected by enhanced chemiluminescence with Lucigenin . B . cepacia LPS was found to prime MM6 cells to produce more superoxide anion than P . aeruginosa or S . maltophilia LPS, and this priming response was CD14 dependent . In addition, the inhibition of respiratory burst responses in monocytes by a bacterial melanin-like pigment purified from an epidemic B . cepacia strain was investigated . The melanin-like pigment was isolated from tyrosine-enriched media on which B . cepacia had been grown and was purified by gel filtration, anion ion-exchange chromatography, and ethanol precipitation . The scavenging potential of the melanin-like pigment for superoxide anion radical (*O2-) generated during the respiratory burst was confirmed with superoxide produced from a cell-free system with xanthine-xanthine oxidase and detected by electron paramagnetic resonance spectroscopy with the spin trap 5-diethoxyphosphoryl-5-methyl-1-pyrroline-n-oxide . The addition of melanin during the LPS priming stage had no effect on the subsequent triggering of the respiratory burst, but melanin inhibited *O2- detection when added at the triggering stage of the respiratory burst . We conclude that melanin-producing B . cepacia may derive protection from the free-radical-scavenging properties of this pigment. Microbiol Immunol, 1998, 42(11), 731 - 7 Antigenic differences between clinical and environmental isolates of Burkholderia pseudomallei; Sirisinha S et al.; Burkholderia pseudomallei is a free-living organism that causes the potentially lethal tropical infection melioidosis . The disease is endemic in many parts of eastern Asia and northern Australia . The presence of two distinct biotypes in soil can be reliably distinguished by their ability to assimilate L-arabinose . Whereas some soil isolates could utilize this substrate (Ara+), the remaining soil isolates and all clinical isolates tested so far could not (Ara-) . Only the Ara- isolates were virulent in animal models . We have raised a murine monoclonal antibody (MAb) that can readily distinguish Ara- from Ara+ biotypes . The MAb reacted with a high molecular weight component present only on the Ara- biotype . With this MAb, clinical and soil Ara- isolates gave identical positive reactions in agglutination, immunofluorescence, ELISA and immunoblot assays . Using these same assay systems, the soil Ara+ biotype did not react with the MAb . Similar but distinct immunoblot patterns were also noted when these two Ara biotypes were probed with sera from patients with melioidosis or with polyclonal immune rabbit sera . These data showed that the Ara- biotype from both clinical and environmental isolates is antigenically different from its Ara+ environmental counterpart . The SDS-PAGE protein and lectin-binding profiles of both groups of Ara- isolates were also found to be different from those of the Ara+ biotype. Southeast Asian J Trop Med Public Health, 1998 Jun, 29(2), 410 - 5 Burkholderia pseudomallei: the unbeatable foe? Leelarasamee A. Although melioidosis has been recognized in Thailand for many years and considerable progress in term of diagnosis and treatment was achieved, B . pseudomallei is still "the unbeatable foe", for several reasons as outlined here: under-recognition, high case-fatality rate, unacceptable relapse rate and a "time-bomb" for sero-positive patients . Melioidosis is largely restricted to certain geographical areas . In Thailand, it had long been considered a rare disease in Thailand until ten cases with culture-proven melioidosis were reported by Sompone Punyagupta and his associates at a meeting of the Infectious Disease Group of Thailand . Since then awareness of young physicians and laboratory personnel for melioidosis has been increased . The most dramatic consequence was seen at Sappasitprasong Hospital in Ubon Ratchathani where over 100 strains of B . pseudomallei are isolated each year . But the frequent isolation of B . pseudomallei is surprisingly restricted to some provinces in the northeast, namely Khon Kaen and Ubon Ratchathani provinces and only 1-10 cases or none from adjacent provinces . The discrepancy was well illustrated by mapping the number of isolations by province . Thus many cases of septicemic melioidosis are certain to receive inappropriate chemotherapy and nearly half of them probably leave this world without proper diagnosis in area where under-recognition unfortunately still prevails . Mortality in disseminated septicemic melioidosis used to occur in 82-87% of the patients who were treated with doxycycline, chloramphenicol, cotrimoxazole and kanamycin and in non-disseminated septicemic melioidosis about 20% . With ceftazidime therapy, the mortality rate was cut by half to 35-40% . About 50% of the patients deteriorated rapidly and died within the first few days of fever . Fatalities are related to the speed of positive results of blood culture . Accordingly, awareness of the disease, familiarity of clinical syndrome compatible with septicemic melioidosis, gram-staining of exudate to include or exclude melioidosis, are all crucial factors to lead to proper empiric chemotherapy . Since the addition of anti-cytokine and platelet activating factor receptor antagonist to current antimicrobials failed to lower the mortality rate, we need to find a new antimicrobial such as protegrin-1 which exhibits rapid microbicidal activity, especially against stationary-phase cell . We need to optimize the bactericidal action of currently used antimicrobials by examining their pharmacokinetics . With prolonged maintenance treatment with cotrimoxazole plus doxycycline or co-amoxiclav, relapse occurs in 4 to 23% . Various explanations for the relapse are the ability of the organism to produce glycocalyx, form microcolonies in damaged tissues and survive within phagocytic cells . Again, the bactericidal antimicrobial which is concentration-dependent, may be used to shorten the duration of treatment and reduce relapse . Studies so far can not relate relapse to any defect of host defense mechanism . In endemic areas, seroepidemiological surveys showed that infection, mostly latent, occurred fairly commonly since childhood as 80% of children had antibodies by the age of four years . However, clinical melioidosis is more common in the elderly which in some cases are due to reactivation of primary latent infection . Since the incubation period of the reactivation can vary from weeks to many years, a vaccine or short-course secondary chemoprophylaxis may be possible interventions for the high risk group to get rid of the "time-bomb" reactivation . The vaccine may also be used to reduce the relapse rate . We need to discover the cellular determinants which is critical to awake the host defense to the sleeping bacteria and provoke local inflammatory response to newly born bacteria before dissemination takes place again . Basic research into the pathogenic mechanisms are key to understanding how to make an effective vaccine. J Bacteriol, 1999 Jan, 181(2), 531 - 40 The phn genes of Burkholderia sp . strain RP007 constitute a divergent gene cluster for polycyclic aromatic hydrocarbon catabolism; Laurie AD et al.; Cloning and molecular ecological studies have underestimated the diversity of polycyclic aromatic hydrocarbon (PAH) catabolic genes by emphasizing classical nah-like (nah, ndo, pah, and dox) sequences . Here we report the description of a divergent set of PAH catabolic genes, the phn genes, which although isofunctional to the classical nah-like genes, show very low homology . This phn locus, which contains nine open reading frames (ORFs), was isolated on an 11.5-kb HindIII fragment from phenanthrene-degrading Burkholderia sp . strain RP007 . The phn genes are significantly different in sequence and gene order from previously characterized genes for PAH degradation . They are transcribed by RP007 when grown at the expense of either naphthalene or phenanthrene, while in Escherichia coli the recombinant phn enzymes have been shown to be capable of oxidizing both naphthalene and phenanthrene to predicted metabolites . The locus encodes iron sulfur protein alpha and beta subunits of a PAH initial dioxygenase but lacks the ferredoxin and reductase components . The dihydrodiol dehydrogenase of the RP007 pathway, PhnB, shows greater similarity to analogous dehydrogenases from described biphenyl pathways than to those characterized from naphthalene/phenanthrene pathways . An unusual extradiol dioxygenase, PhnC, shows no similarity to other extradiol dioxygenases for naphthalene or biphenyl oxidation but is the first member of the recently proposed class III extradiol dioxygenases that is specific for polycyclic arene diols . Upstream of the phn catabolic genes are two putative regulatory genes, phnR and phnS . Sequence homology suggests that phnS is a LysR-type transcriptional activator and that phnR, which is divergently transcribed with respect to phnSFECDAcAdB, is a member of the sigma54-dependent family of positive transcriptional regulators . Reverse transcriptase PCR experiments suggest that this gene cluster is coordinately expressed and is under regulatory control which may involve PhnR and PhnS. J Med Microbiol, 1998 Aug, 47(8), 689 - 94 Flagellin gene variation between clinical and environmental isolates of Burkholderia pseudomallei contrasts with the invariance among clinical isolates; Winstanley C et al.; The flagellin gene sequence from a clinical isolate of Burkholderia pseudomallei was used to design oligonucleotide primers for PCR/RFLP analysis of flagellin gene variation among clinical and environmental isolates of B . pseudomallei . Genes from four clinical and six environmental isolates were amplified and compared by RFLP . The clinical isolates were indistinguishable, but variation was detected among some of the environmental isolates . Sequence analysis of flagellin gene amplified products demonstrated high levels of conservation amongst the flagellin genes of clinical isolates (>99% similarity), compared to the variation observed between the clinical isolates and one of the environmental isolates (<90% similarity) . Genomic comparisons with pulsed-field gel electrophoresis (PFGE) revealed differences between the relationships inferred by flagellin genotyping and PFGE, suggesting that a combination of molecular methods may be useful for the subtyping of B . pseudomallei strains. Eur J Biochem, 1998 Dec 1, 258(2), 696 - 701 Structural characterisation of a rhamnan and a fucorhamnan, both present in the lipopolysaccharide of Burkholderia vietnamiensis strain LMG 10926; Gaur D et al.; The polymeric fraction isolated after mild acid hydrolysis of the lipopolysaccharide (LPS) from Burkholderia vietnamiensis strain LMG 10926 contained L-rhamnose (Rha) and D-fucose (Fuc) . From NMR studies supported by the results of methylation analysis and Smith degradation, it could be inferred that the material was probably a mixture of two glycans . One component was a linear rhamnan with a trisaccharide repeating unit (1); the other was a branched fucorhamnan with a tetrasaccharide repeating unit (2) . The presence of two distinct polymeric fractions in LPS is a common feature for Burkholderia species . {structures: see text} Appl Environ Microbiol, 1999 Jan, 65(1), 251 - 9 Polycyclic aromatic hydrocarbon degradation by a new marine bacterium, Neptunomonas naphthovorans gen . nov., sp . nov; Hedlund BP et al.; Two strains of bacteria were isolated from creosote-contaminated Puget Sound sediment based on their ability to utilize naphthalene as a sole carbon and energy source . When incubated with a polycyclic aromatic hydrocarbon (PAH) compound in artificial seawater, each strain also degraded 2-methylnaphthalene and 1-methylnaphthalene; in addition, one strain, NAG-2N-113, degraded 2,6-dimethylnaphthalene and phenanthrene . Acenaphthene was not degraded when it was used as a sole carbon source but was degraded by both strains when it was incubated with a mixture of seven other PAHs . Degenerate primers and the PCR were used to isolate a portion of a naphthalene dioxygenase iron-sulfur protein (ISP) gene from each of the strains . A phylogenetic analysis of PAH dioxygenase ISP deduced amino acid sequences showed that the genes isolated in this study were distantly related to the genes encoding naphthalene dioxygenases of Pseudomonas and Burkholderia strains . Despite the differences in PAH degradation phenotype between the new strains, the dioxygenase ISP deduced amino acid fragments of these organisms were 97.6% identical . 16S ribosomal DNA-based phylogenetic analysis placed these bacteria in the gamma-3 subgroup of the Proteobacteria, most closely related to members of the genus Oceanospirillum . However, morphologic, physiologic, and genotypic differences between the new strains and the oceanospirilla justify the creation of a novel genus and species, Neptunomonas naphthovorans . The type strain of N . naphthovorans is strain NAG-2N-126. Appl Environ Microbiol, 1999 Jan, 65(1), 80 - 7 In situ, real-time catabolic gene expression: extraction and characterization of naphthalene dioxygenase mRNA transcripts from groundwater; Wilson MS et al.; We developed procedures for isolating and characterizing in situ-transcribed mRNA from groundwater microorganisms catabolizing naphthalene at a coal tar waste-contaminated site . Groundwater was pumped through 0.22-microm-pore-size filters, which were then frozen in dry ice-ethanol . RNA was extracted from the frozen filters by boiling sodium dodecyl sulfate lysis and acidic phenol-chloroform extraction . Transcript characterization was performed with a series of PCR primers designed to amplify nahAc homologs . Several primer pairs were found to amplify nahAc homologs representing the entire diversity of the naphthalene-degrading genes . The environmental RNA extract was reverse transcribed, and the resultant mixture of cDNAs was amplified by PCR . A digoxigenin-labeled probe mixture was produced by PCR amplification of groundwater cDNA . This probe mixture hybridized under stringent conditions with the corresponding PCR products from naphthalene-degrading bacteria carrying a variety of nahAc homologs, indicating that diverse dioxygenase transcripts had been retrieved from groundwater . Diluted and undiluted cDNA preparations were independently amplified, and 28 of the resulting PCR products were cloned and sequenced . Sequence comparisons revealed two major groups related to the dioxygenase genes ndoB and dntAc, previously cloned from Pseudomonas putida NCIB 9816-4 and Burkholderia sp . strain DNT, respectively . A distinctive subgroup of sequences was found only in experiments performed with the undiluted cDNA preparation . To our knowledge, these results are the first to directly document in situ transcription of genes encoding naphthalene catabolism at a contaminated site by indigenous microorganisms . The retrieved sequences represent greater diversity than has been detected at the study site by culture-based approaches. J Bacteriol, 1999 Jan, 181(1), 341 - 6 Genetic and biochemical analyses of the tec operon suggest a route for evolution of chlorobenzene degradation genes; Beil S et al.; The TecA broad-spectrum chlorobenzene dioxygenase of Burkholderia sp . strain PS12 catalyzes the first step in the mineralization of 1,2,4, 5-tetrachlorobenzene . The catabolic genes were localized on a small plasmid that belongs to the IncPbeta incompatibility group . PCR analysis of the genetic environment of the tec genes indicated high similarity to the transposon-organized catabolic tcb chlorobenzene degradation genes of Pseudomonas sp . strain P51 . Sequence analysis of the regions flanking the tecA genes revealed an upstream open reading frame (ORF) with high similarity to the todF 2-hydroxy-6-oxo-2,4-heptadienoate hydrolase g