Int J Syst Evol Microbiol, 2001 Jan, 51(Pt 1), 183 - 5 Genotypic and chemotaxonomic evidence for the reclassification of Pseudomonas woodsii (Smith 1911) Stevens 1925 as Burkholderia andropogonis (Smith 1911) Gillis et al . 1995; Coenye T et al.; It was reported before that {Pseudomonas} woodsii and Burkholderia andropogonis are phenotypically indistinguishable and probably represent the same taxon, for which the name B . andropogonis has been proposed . In the present study, it was found that {P.} woodsii and B . andropogonis strains were indistinguishable by whole-cell protein electrophoresis and have a highly similar cellular fatty acid composition . A high DNA-DNA binding value of 95% was found between the type strains of both species . In addition, the 16S rDNA sequence of {P.} woodsii strain LMG 2362T was very similar to that of B . andropogonis LMG 2129T (99.0%) . The chemotaxonomic and genotypic data confirm that {P.} woodsii and B . andropogonis represent the same species, for which it is proposed to retain the name B . andropogonis. J Med Microbiol, 2001 Feb, 50(2), 116 - 26 Type III secretion systems and pathogenicity islands; Winstanley C et al.; Some bacterial pathogens have evolved by acquiring pathogenicity islands (PIs), which are clusters of genes encoding virulence traits . PIs encoding the secretion of effector molecules via type III secretion (TTS) systems have been discovered in several gram-negative pathogens . TTS systems are involved in contact-dependent secretion of virulence factors and can facilitate delivery of toxins directly into target cells . The expanding list of bacteria found to contain clusters of TTS genes includes members of the genera Yersinia, Salmonella, Shigella, Escherichia, Pseudomonas, Bordetella, Burkholderia, Chlamydia and a number of plant pathogens or symbionts . This review discusses the current knowledge of the role of TTS PIs in pathogenicity, the genetic organisation and evolution of such systems,and the potential for using TTS systems as targets for novel treatments. Zh Mikrobiol Epidemiol Immunobiol, 2000 Nov-Dec, (6), 94 - 9 {Pathogenicity of Burkholderia pseudomallei: extracellular and surface antigen functions}; Piven' NN et al.; The data of literature and the results of investigations carried out by the authors on the analysis of B . pseudomallei pathogenicity factors . They include mucoid, endotoxin, lecithinase, proteases, hemolysins, etc . In addition to fimbriae and pili, the adhesive properties of B . pseudomallei are supposed to be associated with surface capsule-like structures whose composition includes Ag8 . The characterization of exoproteases, hemolysins and lethal toxins is given . The virulence and immunogenicity of B . pseudomallei were shown to be linked with the components of surface glycoprotein Ag8 . The conclusion has been made that the analyzed pathogenicity factors are of importance for deciphering the pathogenesis of melioidosis. Ceylon Med J, 2000 Sep, 45(3), 116 - 8 The isolation of Burkholderia cepacia in a hospital setting in Sri Lanka; Perera N et al.; INTRODUCTION: Burkholderia cepacia is widely prevalent in nature . The natural habitat of this organism is soil, water and vegetation, but the hospital environment remains the main source of infection . It causes a wide variety of infections in hospitalised patients . Since there are no reports of its prevalence in Sri Lanka, a data retrieval and analysis was undertaken to determine its incidence among patients at Sri Jayawardenepura General Hospital (SJGH) . OBJECTIVE: To determine the prevalence, morphological characteristics, biochemical profile and antibiotic susceptibility pattern of B cepacia in a Sri Lankan tertiary care hospital . METHODS: Relevant clinical data were retrieved from all isolates of B cepacia from SJGH for 12 months from October 1996 . Standard procedures were used to isolate the organism from clinical specimens . API 20E was employed for biochemical identification . Antibiotic susceptibility tests was carried out using the modified Kirby Bauer method . RESULTS: B cepacia was isolated from 17 patients . 16 of them were hospitalised and were from the medical, surgical, and intensive care units . All were in a compromised state of health . The organism was isolated from a variety of specimens which included swabs of surgical wounds, chronic ulcers, sputum, bronchial lavage, endotracheal aspirate, urine, peritoneal fluid and blood . Blood agar, MacConkey agar and cystine lactose electrolyte deficient medium supported the growth of all strains . They were motile Gram negative rods . All strains were oxidase positive . Strains gave variable results with other biochemical tests . Antibiograms too were variable . All strains were sensitive to ceftazidime, and the majority of the strains were sensitive to the other third generation cephalosporines . All strains were resistant to four or more antimicrobial agents included in the study . Of the 17 patients from whom B cepacia was isolated, only 9 seem to have had actual infection; others were probably instances of colonisation or contamination . CONCLUSIONS: The present report confirms the prevalence and importance of B cepacia as a hospital pathogen in Sri Lanka . Hospital laboratories should be equipped to isolate, identify and perform antibiotic sensitivity test on these strains . Antibiotic susceptibility testing is necessary as the patterns seem to differ among strains . The multiple drug resistant nature of the organism warrants strict infection control measures to prevent spread in a hospital setting. J Med Microbiol, 2001 Jan, 50(1), 55 - 61 Distribution of immunoglobulin classes and IgG subclasses against a culture filtrate antigen of Burkholderia pseudomallei in melioidosis patients; Chenthamarakshan V et al.; The class and subclass distribution of antibody response to the culture filtrate antigen (CFA) of Burkholderia pseudomallei was examined in the sera of 45 septicaemic and 17 localised melioidosis cases and 40 cases clinically suspected of melioidosis and the results were compared with those from high-risk and healthy control groups . The geometric mean titre index (GMTI) values for all classes and subclasses of immunoglobulins examined were higher for sera from the proven and clinically suspected melioidosis cases than for the control groups . However, the highest response in the three patient groups was that of IgG with GMTIs ranging from 219.4 to 291.6 and the lowest was for IgM with GMTIs of 22.5, 24.3 and 28.7 . The IgA response was intermediate with GMTIs ranging from 119.2 to 170 . The GMTIs were highest for IgG in septicaemic and localised infections and for IgA and IgM in localised infections . As regards IgG subclass distribution, IgG1 and IgG2 were the predominant subclasses produced against the CFA in contrast to IgG3 and IgG4, which were produced in low amounts . None of the sera from the control groups had any significant titres of antibodies. Am J Respir Crit Care Med, 2001 Jan, 163(1), 43 - 8 Infection with Burkholderia cepacia in cystic fibrosis: outcome following lung transplantation; Chaparro C et al.; As a result of concern over excessive mortality after lung transplantation, many transplant programs refuse to accept cystic fibrosis (CF) patients infected with Burkholderia cepacia . As a significant proportion of patients with CF in our community are infected with this organism, we have continued to provide lung transplantation as an option . A retrospective review was conducted of medical records of all patients with CF transplanted between March 1988 and September 1996 . Fifty-six transplant procedures were performed in 53 recipients with CF between March 1988 and September 1996 . Twenty-eight had B . cepacia isolated pretransplant and 25 remaining positive post-transplant . Of the 53 recipients, 19 have died (15 of 28 {54%} B . cepacia positive and 4 of 25 {16%} B . cepacia negative) . B . cepacia was responsible for or involved in 14 deaths . Nine of the deaths occurred in the first 3 mo post-transplantation . One-year survival was 67% for B . cepacia positive patients and 92% for B . cepacia negative patients . Recent modifications in antimicrobial and immunosuppressive therapy since 1995 have resulted in no deaths early post-transplant in the last five patients transplanted . We conclude that early mortality in patients with CF infected with B . cepacia is significantly higher than in those not infected with B . cepacia . Modifications in post-transplant medical therapy may improve outcome. Environ Microbiol, 1999 Oct, 1(5), 393 - 9 Development and characterization of a lux-modified 2,4-dichlorophenol-degrading Burkholderia sp . RASC; Shaw LJ et al.; lux-marked biosensors for assessing the toxicity and bioremediation potential of polluted environments may complement traditional chemical techniques . luxCDABE genes were introduced into the chromosome of the 2,4-dichlorophenol (2,4-DCP)-mineralizing bacterium, Burkholderia sp . RASC c2, by biparental mating using the Tn4431 system . Experiments revealed that light output was constitutive and related to cell biomass concentration during exponential growth . The transposon insertion was stable and did not interrupt 2,4-DCP-degradative genes, and expression of luxCDABE did not constitute a metabolic burden to the cell . A bioluminescence response was detectable at sublethal 2,4-DCP concentrations: at < 10.26 microg ml(-1), bioluminescence was stimulated (e.g . 218% of control), but at concentrations >60 microg ml(-1) it declined to < 1% . Investigating the effect of {14C}-2,4-DCP concentration on the evolution of 14CO2 revealed that, for initial concentrations of 2.5-25 microg ml(-1), approximately equals 55% of the added 14C was mineralized after 24 h compared with <1% at 50 and 100 microg ml(-1) . Inhibition of 2,4-DCP mineralization between 25 and 50 microg ml(-1) corresponded well to the EC50 value (33.83 microg ml(-1)) obtained from bioluminescence inhibition studies . lux-marked RASC c2 may therefore be used as a functionally (i.e . 2,4-DCP degrader) and environmentally relevant biosensor of toxicity and biodegradation inhibition. Environ Microbiol, 1999 Jun, 1(3), 199 - 212 Identification of the metabolically active members of a bacterial community in a polychlorinated biphenyl-polluted moorland soil; Nogales B et al.; The presumptive metabolically active members of a bacterial community in a moorland soil in Germany, highly polluted with polychlorinated biphenyls (PCBs), were identified by sequencing of cloned reverse transcription-polymerase chain reaction (RT-PCR) amplification products of 16S rRNA generated from total RNA extracts . Analysis of the 16S rRNA clone library revealed a considerable diversity of metabolically active bacteria in the soil, despite the acidic pH and high concentrations of PCBs . Cloned sequence types clustered within the Proteobacteria (34% alpha-, 33% beta- and 7% gamma-subclasses), the Holophaga-Acidobacterium phylum (14%), the Actinobacteria (6.5%) and the Planctomycetales (2%) . Three cloned sequence types were not affiliated to any described phylogenetic group . An unusual feature of this soil was the abundance of sequence types within the beta-subclass of the Proteobacteria, most of which were similar to the 16S rRNA gene sequences of species from only two genera, Burkholderia and Variovorax . Three other numerous 16S rRNA sequence types were similar to the sequences of Sphingomonas species, members of the Rhodopila globiformis group and Acidobacterium capsulatum . Some of the sequence types retrieved were similar to the 16S rRNA sequences of bacterial isolates able to degrade a variety of organic pollutants, including PCBs . As the PCB contamination is the major source of measurable carbon in this soil, some of the 16S rRNA sequence types detected and presumed to represent the metabolically active members of the community indicate the organisms likely to be involved, directly or indirectly, in the utilization of the PCBs as carbon and energy sources. Phytother Res, 2001 Feb, 15(1), 39 - 43 Activity of plant flavonoids against antibiotic-resistant bacteria; Xu HX et al.; Thirty eight plant-derived flavonoids representing seven different structural groups were tested for activities against antibiotic-resistant bacteria using the disc-diffusion assay and broth dilution assay . Among the flavonoids examined, four flavonols (myricetin, datiscetin, kaempferol and quercetin) and two -flavones (flavone and luteolin) exhibited inhibitory activity against methicillin-resistant Staphylococcus aureus (MRSA) . Myricetin was also found to inhibit the growth of multidrug-resistant Burkholderia -cepacia, vancomycin-resistant enterococci (VRE) and other medically important organisms such as -Klebsiella pneumoniae and Staphylococcus epidermidis . Myricetin was bactericidal to B . cepacia . The results of the radiolabel incorporation assay showed that myricetin inhibited protein synthesis by -B . cepacia . The structure-activity relationship of these flavonoids is discussed . Gene, 2001 Jan 10, 262(1-2), 137 - 45 Expression of 2-halobenzoate dioxygenase genes (cbdSABC) involved in the degradation of benzoate and 2-halobenzoate in Burkholderia sp . TH2; Suzuki K et al.; Burkholderia sp . TH2, isolated from soil, utilizes 2-chlorobenzoate (2CB) and benzoate (BA) as its sole source of carbon and energy . The genes for 2-halobenzoate dioxygenase (cbdABC) from Burkholderia sp . TH2 were cloned and sequenced . The predicted amino acid sequences of all the gene products are highly similar to the cbd gene products of Pseudomonas sp . 2CBS . Disruption of the promoter region of cbdA resulted in loss of growth on 2CB and BA, indicating that these genes are involved in the growth of TH2 on these substrates . Expression of the cbd genes was analyzed by transcriptional fusion assay . The cbdS gene, a possible araC/xylS-type transcriptional regulatory gene, was shown to positively regulate the expression of cbdA . In addition, the effectors of CbdS were shown to be 2CB, 2-bromobenzoate, o-toluate (2-methylbenzoate), 2-iodobenzoate, and BA . Primer extension analysis showed that the cbdA mRNA started at two positions, 14 and 15 nucleotides upstream from the cbdA start codon, ATG . A pair of direct repeats, identical to that of the Pm promoter of the TOL plasmid, was found upstream of -35 hexamer of the cbdA promoter. FEMS Microbiol Lett, 2001 Feb 20, 195(2), 231 - 5 Screening and characterization of trehalose-oleate hydrolyzing lipase; Ishimoto R et al.; Various soil samples were collected to screen the presence of microorganisms which have ability to degrade TOE . One strain (AKU-883) with good TOE degrading activity was isolated and identified as Burkholderia cepacia and the extracellular enzyme was purified to homogeneity . The purification was achieved by ultrafiltration, Super Q anion-exchange chromatography and Superdex 200HR gel-filtration in the presence of Triton X . The enzyme was purified to 85-fold, and specific activity of 4.910 kU mg protein(-1) . The peak preparation on gel filtration showed a single band of 34 kDa on SDS-PAGE and native PAGE which indicate the monomeric nature of the enzyme . The pI of the enzyme was 6.3 . The enzyme showed the maximum activity at pH 9 and 65 degrees C, and was stable in the range of pH 5--10 and up to 60 degrees C . Almost all the activity (92%) was kept after incubation for more than 1 week at 50 degrees C (pH 7.3) . High activities remained even in water-miscible solvents such as ethanol, dimethyl formamide, diisopropyl ether, and dioxane . The N-terminal 16 amino acid residues were determined as A-N-G-Y-A-A-T-R-Y-P-I-I-L-V-G-G, which showed a consensus sequence for lipases from Burkholderia species . Thus the enzyme was concluded to be a kind of lipase. Curr Microbiol, 2001 Apr, 42(4), 269 - 75 Phenotypic and phylogenetic characterization of Burkholderia (Pseudomonas) sp . strain LB400; Fain MG et al.; The relevant phenotypic traits and phylogenetic relationships between Burkholderia (Pseudomonas) sp . strain LB400 and B . cepacia ATCC 25416(T) were compared to determine the degree to which these two strains might be related . Strain LB400 degrades chlorinated biphenyls and has been a model system for potential use in the bioremediation of polychlorinated biphenyls, while some strains of B . cepacia are plant and human pathogens . The fatty acid methyl ester profile, sole carbon source utilization, and biochemical tests confirmed that strain LB400 was a member of the genus Burkholderia . The 16S rRNA gene sequence showed that this strain was not as closely related to B . cepacia as previously suspected or to other known pathogens of this genus, but is closely related to B . phenazinium, B . caribensis, B . graminis, and three unnamed Burkholderia spp . not known to be pathogenic. Diagn Microbiol Infect Dis, 2001 Jan, 39(1), 1 - 7 Detection of immunoglobulins M and G using culture filtrate antigen of Burkholderia pseudomallei; Chenthamarakshan V et al.; IgM and IgG based ELISA systems were developed using the culture filtrate antigen (CFA) of Burkholderia pseudomallei . The assays were evaluated using 95 sera from 66 septicemic cases and 47 sera from 20 cases with localized melioidosis . In addition 65 sera from culture negative cases that were also serologically negative for other endemic infections clinically suspected of melioidosis were included . These were compared with sera from 260 non-melioidosis cases, 169 sera from individuals with high risk of acquiring the infection and 48 sera from healthy controls . The IgG-ELISA was 96% sensitive and 94% specific . All sera from cases with septicemic and localized infections and 61 of 63 sera from clinically suspected melioidosis cases were positive for IgG antibody . The geometric mean titre index (GMTI) values of IgG antibody in melioidosis cases were significantly higher (p < 0.0005) compared to that of healthy subjects, high risk group and subjects with non-melioidosis infections . The sensitivity and specificity of IgM ELISA was 74 and 99% respectively . The GMTI value of IgM antibody in the sera of melioidosis cases was significantly higher as compared to that of non-melioidosis disease controls (p < or = 0.001) . These results demonstrate that the detection of IgG is a better indicator of the disease in the diagnosis of melioidosis. FEMS Immunol Med Microbiol, 2001 Feb, 30(1), 53 - 63 Study on the pathophysiology of experimental Burkholderia pseudomallei infection in mice; Gauthier YP et al.; Burkholderia pseudomallei is the etiological agent of melioidosis, a potentially fatal disease occurring in man and animals . The aim of this study was to investigate the pathophysiological course of experimental melioidosis, and to identify the target organs, in an animal model . For this purpose SWISS mice were infected intraperitoneally with the virulent strain B . pseudomallei 6068 . The bacterial load of various organs was quantified daily by bacteriological analysis and by an enzyme-linked immunosorbent assay (ELISA) based on a monoclonal antibody specific to B . pseudomallei exopolysaccharide (EPS) . Electron microscopic investigation of the spleen was performed to locate the bacteria at the cellular level . In this model of acute melioidosis, B . pseudomallei had a marked organ tropism for liver and spleen, and showed evidence of in vivo growth with a bacterial burden of 1.6x10(9) colony forming units (CFU) per gram of spleen 5 days after infection with 200 CFU . The highest bacterial loads were detected in the spleen at all time points, in a range from 2x10(6) to 2x10(9) CFU g(-1) . They were still 50-80 times greater than the load of the liver at the time of peak burden . Other investigated organs such as lungs, kidneys, and bone marrow were 10(2)-10(4)-fold less infected than the spleen, with loads ranging from 3x10(2) to 3x10(6) CFU g(-1) . The heart and the brain were sites of a delayed infection, with counts in a range from 10(3) to 10(7) times lower than bacterial counts in the spleen . The EPS-specific ELISA proved to be highly sensitive, particularly at the level of those tissues in which colony counting on agar revealed low contamination . In the blood, EPS was detected at concentrations corresponding to bacterial loads ranging from 8x10(3) to 6x10(4) CFU ml(-1) . Electron microscopic examination of the spleen revealed figures of phagocytosis, and the presence of large numbers of intact bacteria, which occurred either as single cells or densely packed into vacuoles . Sparse figures suggesting bacterial replication were also observed . In addition, some bacteria could be seen in vacuoles that seemed to have lost their membrane . These observations provide a basis for further investigations on the pathogenesis of the disease. FEMS Microbiol Lett, 2001 Feb 5, 195(1), 91 - 6 New broad-host-range promoter probe vectors based on the plasmid RK2 replicon; Santos PM et al.; Broad-host-range plasmid RK2-based promoter probe vectors with a known nucleotide sequence were constructed . In the absence of an upstream promoter, the expression of two tested reporter genes (luc and lacZ) in Escherichia coli was virtually zero, while insertion of the Ptrc promoter resulted in strong inducer-dependent expression . The lacZ-based vectors were mobilized into Pseudomonas fluorescens ST, Pseudomonas putida KT2442, Sphingomonas spp . and Burkholderia spp . LB400, and expression analyses indicated that the properties observed in E . coli are maintained across the species barriers . In addition, the previously established knowledge of RK2 molecular biology allows easy manipulations of features such as plasmid copy number, further extending the application potential of the vectors. Int J Antimicrob Agents, 2001 Feb, 17(2), 109 - 13 Antibiotic susceptibility of Burkholderia pseudomallei from tropical northern Australia and implications for therapy of melioidosis; Jenney AW et al.; From a prospective melioidosis study commencing in 1989 at Royal Darwin Hospital, 170 initial isolates of Burkholderia pseudomallei were available for susceptibility testing . Of these 163 (96%) were susceptible to meropenem/imipenem, ceftazidime, trimethoprim-sulphamethoxazole (SMX/TMP) and doxycycline . Seven (4%) showed primary resistance; three had low-level resistance to SMX/TMP, one to ceftriaxone and amoxycillin/clavulanate (AMOX/CA) and three to doxycycline . Of 167 patients who survived their initial presentation, seven (4%) had culture positive infections which persisted for greater than 3 months after start of therapy . All ultimately cleared carriage of B . pseudomallei though three required changing to SMX/TMP after development of doxycycline resistance . Nineteen (11%) of the initial survivors clinically relapsed and 17 of these had repeat isolates available for testing . Four of these had acquired resistance: one to doxycycline, one to AMOX/CA and ceftazidime, one to SMX/TMP and one to both SMX/TMP and doxycycline . Molecular typing using randomly amplified polymorphic DNA and pulsed-field gel electrophoresis showed all but one relapse isolate to be the same as the original strain . These data are similar to published data from Thailand . As melioidosis has a high mortality (21% in this series) these results emphasize the need for prolonged eradication therapy and regular clinical and microbiological monitoring so that the emergence of resistance can be detected early and appropriate treatment modifications made. Microbiology, 2001 Jan, 147(Pt 1), 111 - 20 Identification of the acid phosphatase (acpA) gene homologues in pathogenic and non-pathogenic Burkholderia spp . facilitates TnphoA mutagenesis; Burtnick M et al.; Burkholderia pseudomallei and Burkholderia mallei are pathogens responsible for disease in both humans and animals . Burkholderia thailandensis, while phylogenetically similar, is considered avirulent in comparison . These three species exhibit phosphatase activity when grown on media containing chromogenic substrates such as 5-bromo-4-chloro-3-indolyl phosphate (XP) . Tn5-OT182 mutagenesis has been utilized to isolate mutants of B . pseudomallei and B . thailandensis unable to hydrolyse XP . Sequence analysis of these mutants revealed an ORF of 1734 nucleotides demonstrating a high degree of homology to the acpA gene product of Francisella tularensis . PCR primers were designed based on the B . pseudomallei acpA gene sequence and were used to amplify an acpA homologue from B . mallei . The predicted amino acid sequence of B . pseudomallei AcpA differed from those of the predicted B . thailandensis AcpA and B . mallei AcpA by 15 and 3 amino acids, respectively . Allelic exchange was used to construct DeltaacpA mutants in each of these Burkholderia spp . These mutants were shown to be devoid of phosphatase activity and have subsequently allowed for the implementation of phoA fusion transposon mutagenesis systems . Two such systems have been successfully utilized in Burkholderia spp . for the identification of several genes encoding exported proteins. Microbiology, 2001 Jan, 147(Pt 1), 97 - 109 In vitro resistance of Burkholderia cepacia complex isolates to reactive oxygen species in relation to catalase and superoxide dismutase production; Lefebre M et al.; The Burkholderia cepacia complex comprises groups of genomovars (genotypically distinct strains with very similar phenotypes) that have emerged as important opportunistic pathogens in cystic fibrosis (CF) patients . The inflammatory response against bacteria in the airways of CF individuals is dominated by polymorphonuclear cells and involves the generation of oxidative stress, which leads to further inflammation and tissue damage . Bacterial catalase, catalase-peroxidase and superoxide dismutase activities may contribute to the survival of B . cepacia following exposure to reactive oxygen metabolites generated by host cells in response to infection . In the present study the authors investigated the production of catalase, peroxidase and SOD by isolates belonging to various genomovars of the B . cepacia complex . Production of both catalase and SOD was maximal during late stationary phase in almost all isolates examined . Native PAGE identified 13 catalase electrophoretotypes and two SOD electrophoretotypes (corresponding to an Fe-SOD class) in strains belonging to the six genomovars of the B . cepacia complex . Seven out of 11 strains displaying high-level survival after H(2)O(2) treatment in vitro had a bifunctional catalase/peroxidase, and included all the genomovar III strains examined . These isolates represent most of the epidemic isolates that are often associated with the cepacia syndrome . The majority of the isolates from all the genomovars were resistant to extracellular O(-)(2), while resistance to intracellularly generated O(-)(2)was highly variable and could not be correlated with the detected levels of SOD activity . Altogether the results suggest that resistance to toxic oxygen metabolites from extracellular sources may be a factor involved in the persistence of B . cepacia in the airways of CF individuals. J Bacteriol, 2001 Mar, 183(5), 1511 - 6 Comparative specificities of two evolutionarily divergent hydrolases involved in microbial degradation of polychlorinated biphenyls; Seah SY et al.; 2-Hydroxy-6-oxo-6-phenylhexa-2,4-dienoate (HOPDA) hydrolase (BphD) is a key determinant in the aerobic transformation of polychlorinated biphenyls (PCBs) by Burkholderia sp . strain LB400 (S . Y . K . Seah, G . Labbe, S . Nerdinger, M . Johnson, V . Snieckus, and L . D . Eltis, J . Biol . Chem . 275:15701-15708, 2000) . To determine whether this is also true in divergent biphenyl degraders, the homologous hydrolase of Rhodococcus globerulus P6, BphD(P6), was hyperexpressed, purified to apparent homogeneity, and studied by steady-state kinetics . BphD(P6) hydrolyzed HOPDA with a k(cat)/K(m) of 1.62 (+/- 0.03) x 10(7) M(-1) s(-1) (100 mM phosphate {pH 7.5}, 25 degrees C), which is within 70% of that of BphD(LB400) . BphD(P6) was also similar to BphD(LB400) in that it catalyzed the hydrolysis of HOPDAs bearing chloro substituents on the phenyl moiety at least 25 times more specifically than those bearing chloro substituents on the dienoate moiety . However, the rhodococcal enzyme was significantly more specific for 9-Cl and 10-Cl HOPDAs, catalyzing the hydrolysis of 9-Cl, 10-Cl, and 9,10-diCl HOPDAs two- to threefold respectively, more specifically than HOPDA . Moreover, 4-Cl HOPDA competitively inhibited BphD(P6) more effectively than 3-Cl HOPDA, which is the inverse of what was observed in BphD(LB400) . These results demonstrate that BphD is a key determinant in the aerobic transformation of PCBs by divergent biphenyl degraders, but that there exists significant diversity in the specificity of these biphenyl hydrolases. Appl Environ Microbiol, 2001 Feb, 67(2), 982 - 5 Burkholderia cepacia genomovar III Is a common plant-associated bacterium; Balandreau J et al.; A polyphasic taxonomic study involving DNA-DNA hybridization, whole-cell protein electrophoresis, and 16S ribosomal DNA sequence analysis revealed that a group of Burkholderia cepacia-like organisms isolated from the rhizosphere or tissues of maize, wheat, and lupine belong to B . cepacia genomovar III, a genomic species associated with "cepacia syndrome" in cystic fibrosis patients . The present study also revealed considerable protein electrophoretic heterogeneity within this species and demonstrated that the B . cepacia complex consists of two independent phylogenetic lineages. Appl Environ Microbiol, 2001 Feb, 67(2), 725 - 32 Nitrogen fixation genes in an endosymbiotic Burkholderia strain; Minerdi D et al.; In this paper we report the identification and characterization of a DNA region containing putative nif genes and belonging to a Burkholderia endosymbiont of the arbuscular mycorrhizal fungus Gigaspora margarita . A genomic library of total DNA extracted from the fungal spores was also representative of the bacterial genome and was used to investigate the prokaryotic genome . Screening of the library with Azospirillum brasilense nifHDK genes as the prokaryotic probes led to the identification of a 6,413-bp region . Analysis revealed three open reading frames encoding putative proteins with a very high degree of sequence similarity with the two subunits (NifD and NifK) of the component I and with component II (NifH) of nitrogenase from different diazotrophs . The three genes were arranged in an operon similar to that shown by most archaeal and bacterial diazotrophs . PCR experiments with primers designed on the Burkholderia nifHDK genes and Southern blot analysis demonstrate that they actually belong to the genome of the G . margarita endosymbiont . They offer, therefore, the first sequence for the nif operon described for Burkholderia . Reverse transcriptase PCR experiments with primers designed on the Burkholderia nifH and nifD genes and performed on total RNA extracted from spores demonstrate that the gene expression was limited to the germination phase . A phylogenetic analysis performed on the available nifK sequences placed the endosymbiotic Burkholderia close to A . brasilense. Curr Microbiol, 2000 Jul, 41(1), 33 - 8 Induction of stress shock proteins DnaK and GroEL by phenoxyherbicide 2,4-D in Burkholderia sp . YK-2 isolated from rice field; Cho YS et al.; The purpose of this work was to investigate the induction of stress shock proteins in Burkholderia sp . YK-2 in response to the phenoxyherbicide 2,4-dichlorophenoxyacetic acid (2,4-D) . The stress shock proteins, which contribute to the resistance of the cytotoxic effect of 2,4-D, were induced at different 2,4-D concentrations in exponentially growing cultures of Burkholderia sp . YK-2 . This response involved the induction of a 43-kDa DnaK and 41-kDa GroEL proteins, characterized by SDS-PAGE and Western blot by use of the anti-DnaK and anti-GroEL monoclonal antibodies . The total stress shock proteins were analyzed by 2-D PAGE . Survival of Burkholderia sp . YK-2 with time in the presence of different concentrations of 2,4-D was monitored, and viable counts paralleled the induction of the stress shock proteins in this strain. PDA J Pharm Sci Technol, 1999 Jul-Aug, 53(4), 186 - 201 Application of membrane filtration for removal of diminutive bioburden organisms in pharmaceutical products and processes; Sundaram S et al.; In this report, we present results of a recent investigation in our laboratories demonstrated the effect of process conditions and/or drug product composition on the ability of 0.2 micron and 0.22 micron sterilizing grade filters to fully retain Ralstonia (formerly Burkholderia, formerly Pseudomonas) pickettii . R . pickettii is a opportunistic pathogen widely distributed in nature as well as clinical specimens and there have been several reports of nosocomial infections due to intrinsic manufacture-related R . pickettii contamination in filter-sterilized parenteral fluids . This study documents the penetration of 0.2 micron nylon 66 and 0.22 micron modified PVDF sterilizing grade filters by R . pickettii (grown and challenged) in a drug solution under conditions that simulated a pharmaceutical filling operation . Penetration was not observed for every filter disc tested, and this may be explained, in part, by the stochastic nature (i.e., governed by the rules of probability) of the retention mechanisms involved . Scanning electron microscopy revealed significant changes in the microorganism's size and morphology as a result of exposure to the drug solution; these changes are consistent with those reported for bacteria subjected to nutrient deprivation . The SEM analyses of R . pickettii challenge suspensions in the drug solution showed that the average cell length decreased from 1.25 +/- 0.27 microns to 0.84 +/- 0.17 micron between zero and 24 hours . In addition, significant changes were observed in the size (length) distributions, with approximately 35% of the cells at 24 hours being smaller than any cell observed at the start of the challenge . These data suggest that the significant reduction in bioburden size and morphology that occurred as a result of exposure to the drug solution may play a role in the reduced ability of the 0.2 micron and 0.22 micron filters tested in this study to retain these organisms . Under the same test conditions where penetration of 0.2/0.22 micron filters was observed, 0.1 micron rated membrane filters qualified with both B . diminuta and Acholeplasma laidlawii mycoplasma consistently provided sterile effluent . Bacterial penetration of 0.2 (or 0.22) micron sterilizing grade filters was not observed under identical test conditions with either R . pickettii in a standardized solution (saline lactose broth) routinely used in challenge testing filters, or with the standard test organism, B . diminuta, in the drug solution . This study thus supports the renewed emphasis on both product- and process specific validation as well as routine bioburden monitoring expressed by regulatory agencies, and the use of enhanced bacterial removal efficiency 0.1 micron rated filters to provide enhanced sterility assurance in pharmaceutical processes. Med Microbiol Immunol (Berl), 1999 Nov, 188(2), 91 - 7 Highly resistant Burkholderia pseudomallei small colony variants isolated in vitro and in experimental melioidosis; Haussler S et al.; Burkhloderia pseudomallei is the causative agent of melioidosis, a disease in which treatment failures and relapses are common . This study reports on slow growing B . pseudomallei 'small colony variants' (SCVs), isolated either in vitro after exposure to ceftazidime, ciprofloxacin or gentamicin or from the spleen and liver in a mouse model of melioidosis after treatment with ceftazidime . Interestingly, SCVs isolated by either method or antimicrobial agent showed a significant increase in the minimal inhibitory concentrations of various unrelated classes of antimicrobial agents . B . pseudomallei SCVs did not differ from their parental strains in standard biochemical profiles, nor by pulsed field gel electrophoresis or electron microscopy . Although the SCV phenotype was stable throughout numerous passages on antibiotic-free solid media, revertants with the parental colony morphology and, most importantly, with the parental susceptibility pattern occurred . These revertants led to rapid overgrowth of SCVs in liquid media without added antibiotics . Future studies will have to determine the clinical relevance of B . pseudomallei SCVs especially in treatment failure and relapse of infection. Int J Syst Evol Microbiol, 2000 Nov, 50 Pt 6, 2135 - 9 Pseudomonas antimicrobica Attafuah and Bradbury 1990 is a junior synonym of Burkholderia gladioli (Severini 1913) Yabuuchi et al . 1993; Coenye T et al.; Comparison of the 16S rDNA sequence of Pseudomonas antimicrobica LMG 18920T with published 16S rDNA sequences from other pseudomonads indicated that Pseudomonas antimicrobica belongs to the genus Burkholderia, with Burkholderia gladioli, Burkholderia glumae and Burkholderia plantarii as its closest neighbours . DNA-DNA hybridizations confirmed that Pseudomonas antimicrobica and Burkholderia gladioli represent the same species . Strain LMG 18920T and other Burkholderia gladioli strains were also indistinguishable by SDS-PAGE of whole-cell proteins and had similar biochemical characteristics . The whole-cell fatty acid composition, however, was different from that of other Burkholderia gladioli strains . It is concluded that Pseudomonas antimicrobica is a later synonym of Burkholderia gladioli . As Burkholderia gladioli is known to cause infections in patients with cystic fibrosis and chronic granulomatous disease, the eventual use of strain LMG 18920T as a biological control agent should be approached with caution. Appl Microbiol Biotechnol, 2000 Dec, 54(6), 778 - 85 Cloning and characterization of EstC from Burkholderia gladioli, a novel-type esterase related to plant enzymes; Reiter B et al.; By screening a genomic library of Burkholderia gladioli (formerly Pseudomonas marginata) for clones exhibiting esterolytic activity, the gene for a novel-type esterase (EstC) showing significant homology to plant enzymes could be isolated . High homology was found to two hydroxynitrile lyases originating from Hevea brasiliensis (tropical rubber tree) and Manihot esculenta (cassava), and to two proteins from Oryza sativa (rice) that are specifically induced upon infection by Pseudomonas syringae pv . syringae . The sequenced ORF encodes for a protein of 298 amino acids . The enzyme was efficiently overexpressed in Escherichia coli, purified and characterized with respect to enzymatic capabilities . The enzyme was able to hydrolyze a variety of esterase substrates of low to medium carbonic acid chain length, but no triglycerides were hydrolyzed . Despite the high sequence homology, no hydroxynitrile lyase activity could be recognized. Rev Argent Microbiol, 2000 Oct-Dec, 32(4), 196 - 8 {Bact-Alert system for hemocultures: evaluation of terminal subcultures}; Soloaga R et al.; Few laboratory microbiological procedures are as important as the isolation of microorganisms from blood . To evaluate the usefulness of the terminal subcultures, 5669 blood cultures giving negative results after 7 days of incubation in the Bact/Alert System (Organon Teknika) were studied . Bottles were distributed as follows: 1562 adult aerobic bottles, 119 adult anaerobic bottles, 3960 pediatric bottles and 28 FAN bottles . From 5669 blood cultures, 10 subcultures that yielded growth had not been detected by the system . These included 5 adult aerobic bottles and 5 pediatric bottles, 7 of these microorganisms were considered contaminants according to clinical data (2 Micrococcus spp, 1 staphylococci coagulase negative, 1 Burkholderia cepacia, 1 Peptoestreptococcus spp, 1 Corynebacterium spp, 1 Scedosporium spp) while the other 3 were considered true bacteremia (1 Pseudomonas aeruginosa, 1 Proteus mirabilis, 1 Streptococcus sanguis), although no one made any change in treatment on the basis of the previous isolation . Based on these results the routinary utilization of terminal subcultures is not advisable and should be used only for special cases or a second system of blood culture should be added according to clinical or epidemiological data. J Immunol, 2001 Jan 15, 166(2), 1097 - 105 Bystander activation of CD8+ T cells contributes to the rapid production of IFN-gamma in response to bacterial pathogens; Lertmemongkolchai G et al.; The bacterium Burkholderia pseudomallei causes a life-threatening disease called melioidosis . In vivo experiments in mice have identified that a rapid IFN-gamma response is essential for host survival . To identify the cellular sources of IFN-gamma, spleen cells from uninfected mice were stimulated with B . pseudomallei in vitro and assayed by ELISA and flow cytometry . Costaining for intracellular IFN-gamma vs cell surface markers demonstrated that NK cells and, more surprisingly, CD8(+) T cells were the dominant sources of IFN-gamma . IFN-gamma(+) NK cells were detectable after 5 h and IFN-gamma(+) CD8(+) T cells within 15 h after addition of bacteria . IFN-gamma production by both cell populations was inhibited by coincubation with neutralizing mAb to IL-12 or IL-18, while a mAb to TNF had much less effect . Three-color flow cytometry showed that IFN-gamma-producing CD8(+) T cells were of the CD44(high) phenotype . The preferential activation of NK cells and CD8(+) T cells, rather than CD4(+) T cells, was also observed in response to Listeria monocytogenes or a combination of IL-12 and IL-18 both in vitro and in vivo . This rapid mechanism of CD8(+) T cell activation may be an important component of innate immunity to intracellular pathogens. Curr Opin Hematol, 2001 Jan, 8(1), 17 - 22 Clinical aspects of chronic granulomatous disease; Johnston RB Jr; Data from a registry of 368 patients with chronic granulomatous disease (CGD) documenta shift in the most common infecting organisms away from staphylococci and enteric bacteria to Aspergillus species, although staphylococci remain a threat . A . nidulans appears to have a particular virulence in CGD . Burkholderia cepacia sepsis/pneumonia was the second most lethal infection in patients in the registry . Seventy-six percent of registry patients had the X-linked recessive (XLR) form of CGD . Chorioretinitis may be more common than previously appreciated, and boys with the XLR disease should probably have routine full eye exams . A new variant of CGD has been described that is caused by an inhibitory mutation in Rac2, which regulates activity of the neutrophil respiratory burst and actin assembly . Interferon-gamma, antibacterial prophylaxis, and, probably, antifungal prophylaxis with itraconazole reduce the rate of infection, and bone marrow transplantation can cure the disease if a histocompatible donor is available . Gene therapy can cure CGD in knockout mouse models . Having even a small percentage of phagocytes that are nicotinamide adenine dinucleotide phospate oxidase-positive can reduce the risk of serious infection, and procedures now under study appear close to achieving that goal, if not a cure. J Pediatr Hematol Oncol, 2000 Nov-Dec, 22(6), 581 - 7 Infections in E-beta thalassemia; Wanachiwanawin W; Infection is a major complication and the leading cause of death in thalassemia, especially E-beta thalassemia . The spectrum of infections in E-beta thalassemia include mild and severe infections, therapy-related infections such as Yersinia enterocolitica infection associated with desferrioxamine (DFO) therapy, and transfusion-transmitted disease, as well as unique infections such as with pythiosis . Prospective studies in Thailand indicate that patients with E-beta thalassemia had more frequent episodes of both mild and severe infections . The former included upper respiratory tract infection, acute gastroenteritis, cutaneous abscess, and gingivitis . Severe infections occurred more commonly in patients with splenectomy and included septicemia, pneumonia, biliary tract infection, salmonellosis, and urinary tract infection . Responsible organisms were Escherichia coli (26%), Klebsiella pneumoniae (23%), Salmonella (15%), and Streptococcus pneumoniae (13%) . Other organisms included Pseudomonas, Staphylococci, Burkholderia pseudomallei (melioidosis), and Aeromonas . Patients undergoing DFO therapy are at risk for Y . enterocolitica infection which may be localized to mesenteric nodes and tonsils or occur as a generalized form such as septicemia . Recently, we have seen a unique infection so-called vascular pythiosis . Patients usually presented with clinical features of vascular occlusion of lower limbs from ascending arteritis and thrombosis . The causative organism, Pythium insidiosum, is fungus-like, in the kingdom Stramenopila, and in the class Oomycetes . The mortality rate is high and the only effective treatment has been early amputation or possibly immunotherapy . The predisposing factors of infections in thalassemia include splenectomy, iron overload, anemia, and granulocyte dysfunctions . General management of infections in thalassemia consist of prevention, i.e., immunization with pneumococcal and hepatitis vaccines, oral penicillins especially in patients with splenectomy, removal of predisposing factors such as gallstones, iron overload, and appropriate antibiotics. J Med Microbiol, 2000 Dec, 49(12), 1075 - 8 Monoclonal antibody-based rapid identification of Burkholderia pseudomallei in blood culture fluid from patients with community-acquired septicaemia; Anuntagool N et al.; A monoclonal antibody-based latex agglutination (MAb-LA) test was employed for the rapid identification of Burkholderia pseudomallei in blood culture fluid from patients with community-acquired septicaemia . These patients were admitted to 12 hospitals in the northeastern part of Thailand which is a region known to be endemic for melioidosis . Blood samples were collected and immediately added to the blood culture bottles which were incubated in either automated (five hospitals) or manual (seven hospitals) culture systems . Of a total of 1369 culture-positive specimens, 204 specimens were culture-positive for B . pseudomallei . Of those, 194 (95%) were positive by MAb-LA and the type of blood culture system did not affect positivity rates . The performance of the MAb-LA test on these specimens was highly satisfactory compared with culture detection and confirmation by biochemical test, with 95.1% sensitivity, 99.7% specificity and 98.8% and 99.2% for positive and negative predictive values, respectively . The method described is highly reproducible, simple to perform even by inexperienced laboratory personnel and does not require expensive or elaborate equipment. Drugs, 2000 Nov, 60(5), 1053 - 64 The treatment of respiratory pseudomonas infection in cystic fibrosis: what drug and which way? Banerjee D, Stableforth D. Pseudomonas aeruginosa is a non-capsulate and non-sporing gram-negative bacillus that most commonly affects the lower respiratory system in humans . Burkholderia (previously Pseudomonas) cepacia has emerged as an important respiratory pathogen in patients with cystic fibrosis (CF) . The ability of P . aeruginosa to persist and multiply in moist environments and equipment, such as humidifiers in hospital wards, bathrooms, sinks and kitchens, maybe of importance in cross-infection . P . aeruginosa infections of the lower respiratory tract can range in severity from colonisation (without an immunological response) to a severe necrotising bronchopneumonia . Infection is seen in patients with CF and other chronic lung diseases such as non-CF bronchiectasis . In patients with CF, once P . aeruginosa is established in the airways it is almost impossible to eradicate, but prior to this, aggressive treatment can delay the development of chronic infection . 30 to 40% of the present paediatric population with CF will have chronic pseudomonal infection . B . cepacia has a particular predisposition to infect patients with CF and may be distinguished from P . aeruginosa by accelerated lung disease in about one- third of patients . Overwhelming septicaemia and necrotising pneumonia are well described (cepacia syndrome); events that are rare with P . aeruginosa . With the propensity for social cross-infection, segregation policies have been accepted as means of controlling outbreaks . A number of antipseudomonal agents are available . The most commonly used are the extended-spectrum penicillins, aminoglycosides, cephalosporins, fluoroquinolones, polymixins and the monobactams . An aminoglycoside with a beta-lactam penicillin is usually considered to be the first line treatment . No trial has shown any significant clinical advantage of any particular combination regimen over another . The emergence of resistance continues to be a concern . Pipericillin, piperacillin/tazobactam and meropenem have good but equivalent antibacterial activity against P . aeruginosa . However, B . cepacia is characterised by in vitro resistance to colistin (colomycin), aminoglycosides and ciprofloxacin but better susceptibility to ceftazidime . Nebulised delivery of antipseudomonal antibiotics is thought to prevent recurrent exacerbations, reduce antibiotic usage and maintain lung function, particularly in patients with CF . Colistin, tobramycin and gentamicin are currently the most commonly prescribed nebulised antibiotics . Much effort is directed at treating chronic P . aeruginosa infection but as chronic infection is seldom if ever eradicated when first established, prevention is preferable . Early intensive treatment for P . aeruginosa infection is advocated in order to maintain pulmonary function and postpone the onset of chronic P . aeruginosa infection. Indian J Pathol Microbiol, 1999 Oct, 42(4), 493 - 4 Burkholderia pseudomallei--abscess in an unusual site; Rao PS et al.; Meloidosis in a unusual site has been reported in a child . Complete identification of the organism has been presented. Clin Exp Immunol, 2000 Dec, 122(3), 324 - 9 Kinetic studies of the production of nitric oxide (NO) and tumour necrosis factor-alpha (TNF-alpha) in macrophages stimulated with Burkholderia pseudomallei endotoxin; Utaisincharoen P et al.; The mechanism by which Burkholderia pseudomallei survives in macrophages is not clearly understood . In this study, we demonstrated that the mouse macrophage cell line (RAW 264.7) treated with lipopolysaccharide (LPS) from B . pseudomallei (BP-LPS) produced significantly less NO and TNF-alpha compared with those stimulated with the LPS from Escherichia coli and Salmonella typhi . The time required for the BP-LPS to trigger substantial NO and TNF-alpha release was at least 30 min, compared with < 5 min for the E . coli-LPS . A time course study of inducible nitric oxide synthase (iNOS) protein expression also indicated that the time required for macrophages stimulated with the BP-LPS to up-regulate iNOS was longer . The longer time lag for the BP-LPS to activate macrophages was probably due to the delay in up-regulation of iNOS and TNF-alpha mRNA transcription . These results indirectly suggest that the delay of the mediators' production may be due to a reduced rate of signal transduction initiated by the interaction of BP-LPS with the macrophage cell surface . The use of MoAb to phosphorylated p38 in a Western blot analysis provided data compatible with the notion that the maximum level of phosphorylated p38 from the cells activated with BP-LPS was attained at a slower rate . These results suggest that the unique structure of BP-LPS exhibits a property which may interfere with macrophage cell activation. Infect Immun, 2001 Jan, 69(1), 34 - 44 Detection of bacterial virulence genes by subtractive hybridization: identification of capsular polysaccharide of Burkholderia pseudomallei as a major virulence determinant; Reckseidler SL et al.; Burkholderia pseudomallei, the etiologic agent of melioidosis, is responsible for a broad spectrum of illnesses in humans and animals particularly in Southeast Asia and northern Australia, where it is endemic . Burkholderia thailandensis is a nonpathogenic environmental organism closely related to B . pseudomallei . Subtractive hybridization was carried out between these two species to identify genes encoding virulence determinants in B . pseudomallei . Screening of the subtraction library revealed A-T-rich DNA sequences unique to B . pseudomallei, suggesting they may have been acquired by horizontal transfer . One of the subtraction clones, pDD1015, encoded a protein with homology to a glycosyltransferase from Pseudomonas aeruginosa . This gene was insertionally inactivated in wild-type B . pseudomallei to create SR1015 . It was determined by enzyme-linked immunosorbent assay and immunoelectron microscopy that the inactivated gene was involved in the production of a major surface polysaccharide . The 50% lethal dose (LD(50)) for wild-type B . pseudomallei is <10 CFU; the LD(50) for SR1015 was determined to be 3.5 x 10(5) CFU, similar to that of B . thailandensis (6.8 x 10(5) CFU) . DNA sequencing of the region flanking the glycosyltransferase gene revealed open reading frames similar to capsular polysaccharide genes in Haemophilus influenzae, Escherichia coli, and Neisseria meningitidis . In addition, DNA from Burkholderia mallei and Burkholderia stabilis hybridized to a glycosyltransferase fragment probe, and a capsular structure was identified on the surface of B . stabilis via immunoelectron microscopy . Thus, the combination of PCR-based subtractive hybridization, insertional inactivation, and animal virulence studies has facilitated the identification of an important virulence determinant in B . pseudomallei. J Bacteriol, 2001 Jan, 183(1), 358 - 66 Phylogeny of the major head and tail genes of the wide-ranging T4-type bacteriophages; Tetart F et al.; We examined a number of bacteriophages with T4-type morphology that propagate in different genera of enterobacteria, Aeromonas, Burkholderia, and Vibrio . Most of these phages had a prolate icosahedral head, a contractile tail, and a genome size that was similar to that of T4 . A few of them had more elongated heads and larger genomes . All these phages are phylogenetically related, since they each had sequences homologous to the capsid gene (gene 23), tail sheath gene (gene 18), and tail tube gene (gene 19) of T4 . On the basis of the sequence comparison of their virion genes, the T4-type phages can be classified into three subgroups with increasing divergence from T4: the T-evens, pseudoT-evens, and schizoT-evens . In general, the phages that infect closely related host species have virion genes that are phylogenetically closer to each other than those of phages that infect distantly related hosts . However, some of the phages appear to be chimeras, indicating that, at least occasionally, some genetic shuffling has occurred between the different T4-type subgroups . The compilation of a number of gene 23 sequences reveals a pattern of conserved motifs separated by sequences that differ in the T4-type subgroups . Such variable patches in the gene 23 sequences may determine the size of the virion head and consequently the viral genome length . This sequence analysis provides molecular evidence that phages related to T4 are widespread in the biosphere and diverged from a common ancestor in acquiring the ability to infect different host bacteria and to occupy new ecological niches. Clin Infect Dis, 2001 Jan, 32(1), E15 - 6 Epub 2000 Dec 11. Travel-associated Burkholderia pseudomallei infection (Melioidosis) in a patient with cystic fibrosis: a case report; Visca P et al.; In September 1997, a 25-year-old Italian woman with cystic fibrosis (CF) spent 3 weeks in Thailand . In August 1998, her pulmonary function rapidly declined, with productive cough and intermittent fever . Chest x-ray films revealed diffuse, small, patchy opacities in the upper lobes . Burkholderia pseudomallei (BP) was isolated from specimens of the patient's sputum and was identified by means of 16S rDNA sequencing . The diagnosis of melioidosis was serologically confirmed . Continuous therapy with ceftazidime and co-trimoxazole and maintenance with co-trimoxazole, doxycycline, and chloramphenicol resulted in eradication of BP . We present the issue of whether patients with CF represent a population particularly at risk for melioidosis. Diagn Microbiol Infect Dis, 2000 Nov, 38(3), 141 - 5 Characterization of Burkholderia pseudomallei isolated in Thailand and Malaysia; Radua S et al.; A total of 35 Burkholderia pseudomallei isolates from Thailand (16 clinical and eight soil isolates) and Malaysia (seven animal, two isolate each from clinical and soil) were investigated by their antimicrobial resistance, plasmid profiles and were typed by randomly amplified polymorphic DNA analysis . All isolates were found to be resistant to six or more of the 12 antimicrobial agents tested . Only two small plasmids of 1.8 and 2.4 megadalton were detected in two clinical isolates from Thailand . RAPD analysis with primer GEN2-60-09 resulted in the identification of 35 RAPD-types among the 35 isolates . The constructed dendrogram differentiated the 35 isolates into two main clusters and a single isolate . The wide genetic biodiversity among the 35 isolates indicate that RAPD-PCR can be a useful method to differentiate unrelated B . pseudomallei in epidemiological investigation. Vet Pathol, 2000 Nov, 37(6), 626 - 36 Mouse model of sublethal and lethal intraperitoneal glanders (Burkholderia mallei); Fritz DL et al.; Sixty male BALB/c mice were inoculated intraperitoneally with either a sublethal or a lethal dose of Burkholderia mallei China 7 strain, then killed at multiple time points postinoculation . Histopathologic changes were qualitatively similar in both groups and consisted of pyogranulomatous inflammation . In sublethal study mice, changes were first seen at 6 hours in mediastinal lymph nodes, then in spleen, liver, peripheral lymph nodes, and bone marrow at day 3 . These changes generally reached maximal incidence and severity by day 4 but decreased by comparison in all tissues except the liver . Changes were first seen in lethal study mice also at 6 hours in mediastinal lymph nodes and in spleens . At day 1, changes were present in liver, peripheral lymph nodes, and bone marrow . The incidence and severity of these changes were maximal at day 2 . In contrast to sublethal study mice, the incidence and severity of the changes did not decrease through the remainder of the study . The most significant difference between the two groups was the rapid involvement of the spleen in the lethal study mice . Changes indicative of impaired vascular perfusion were more frequently seen in the sublethal study mice . Our findings indicate that mice are susceptible to B . mallei infection and may serve as an appropriate model for glanders infection in a resistant host such as human beings . Additionally, by immunoelectron microscopy, we showed the presence of type I O-antigenic polysaccharide (capsular) antigen surrounding B . mallei. J Clin Microbiol, 2000 Dec, 38(12), 4305 - 9 Identification and detection of Stenotrophomonas maltophilia by rRNA-directed PCR; Whitby PW et al.; Stenotrophomonas maltophilia has recently emerged as an important nosocomial pathogen in immunocompromised patients, in transplant recipients, and in persons with cystic fibrosis (CF) . While this organism is nonpathogenic in healthy individuals, it is increasingly associated with morbidity and mortality in susceptible populations . Recent studies have indicated that for approximately 10% of CF patients with moderate lung disease, S . maltophilia can be cultured from respiratory tract secretions . Identification of S . maltophilia can be problematic, and analysis of isolates from the Burkholderia cepacia Research Laboratory and Repository showed that several isolates presumptively identified as B . cepacia by clinical microbiology laboratories were in fact S . maltophilia . To overcome the problems associated with definitive identification, we developed species-specific PCR (SS-PCR) primers, designated SM1 and SM4, directed to the 23S rRNA gene, and tested their utility to accurately identify S . maltophilia directly from sputum . The SS-PCR was developed and tested against a panel of 112 S . maltophilia isolates collected from diverse geographic locations . To test for specificity, 43 isolates from 17 different species were analyzed . PCR with the SM1-SM4 primer pair and isolated genomic DNA as a template resulted in amplification of a band from all S . maltophilia isolates and was uniformly negative for all other species tested, yielding a sensitivity and a specificity of 100% for the SS-PCR . The utility of the SS-PCR to directly identify S . maltophilia in sputum was examined . Thirteen expectorated sputum samples from CF patients were analyzed by SS-PCR . Three samples were PCR positive, in complete concordance with the conventional laboratory culture . Thus, we have developed an SS-PCR protocol that can rapidly and accurately identify S . maltophilia isolates and which can be used for the direct detection of this organism in CF patient sputum. Braz J Infect Dis, 1998 Apr, 2(2), 43 - 61 Microbial Pathogens Associated With Cystic Fibrosis: Special Focus on Pseudomonas aeruginosa; Barth AL et al.; Cystic fibrosis (CF) is the most common inherited lethal disease among Caucasians . The disease is characterized by a high sodium sweat concentration, malabsorption, malnutrition, and chronic bronchopulmonary infection . Although CF is a multisystem disorder it is the deterioration of lung function, due mainly to bacterial colonization, that is the major determinant contributing to the high morbidity and mortality of affected patients . A relatively large variety of microbial species can be recovered from the CF sputum but it is widely accepted that Staphylococcus aureus, Haemophilus influenzae, Pseudomonas aeruginosa, and Burkholderia cepacia are the most important organisms causing infection in CF patients . Among these, P.aruginosa is the most prevalent pathogen responsible for much of the severe chronic lung infection . Antibiotic therapy aimed at clearing or reducing, the bacterial load in the CF lung, even though temporary, increases the longevity of patients and is one of the mainstays in treatment the disease. Appl Environ Microbiol, 2000 Dec, 66(12), 5192 - 200 Zoospore homing and infection events: effects of the biocontrol bacterium Burkholderia cepacia AMMDR1 on two oomycete pathogens of pea (Pisum sativum L.); Heungens K et al.; Burkholderia cepacia AMMDR1 is a biocontrol agent that protects pea and sweet corn seeds from Pythium damping-off in field experiments . The goal of this work was to understand the effect of B . cepacia AMMDR1 on Pythium aphanidermatum and Aphanomyces euteiches zoospore homing events and on infection of pea seeds or roots . In vitro, B . cepacia AMMDR1 caused zoospore lysis, prevented cyst germination, and inhibited germ tube growth of both oomycetes . B . cepacia AMMDR1 also reduced the attractiveness of seed exudates to Pythium zoospores to nondetectable levels . However, when present at high levels on seeds, B . cepacia AMMDR1 had little net effect on zoospore attraction, probably because it also enhanced seed exudation . Seed-applied B . cepacia AMMDR1 dramatically reduced the incidence of infection by Pythium zoospores in situ compared with an antibiosis-deficient Tn5 mutant strain . This mutant strain also decreased Pythium infection incidence to some extent, but only when the pathogen inoculum potential was low . B . cepacia AMMDR1 did not affect attraction of Aphanomyces zoospores or Aphanomyces root rot incidence . These results suggest that B . cepacia AMMDR1 controls P . aphanidermatum largely through antibiosis, but competition for zoospore-attracting compounds can contribute to the effect . Differences in suppression of Aphanomyces and Pythium are discussed in relation to differences in the ecology of the two pathogens. FEMS Microbiol Lett, 2000 Dec 1, 193(1), 179 - 85 Studies on polyhydroxyalkanoate (PHA) accumulation in a PHA synthase I-negative mutant of Burkholderia cepacia generated by homogenotization; de Andrade Rodrigues MF et al.; In the genome of Burkholderia cepacia strain IPT64, which accumulates a blend of the two homopolyesters poly(3-hydroxybutyrate), poly(3HB), and poly(3-hydroxy-4-pentenoic acid), poly(3H4PE), from sucrose or gluconate as single carbon source, the polyhydroxyalkanoate (PHA) synthase structural gene was disrupted by the insertion of a chloramphenicol-resistant gene cassette (phaC1::Cm) . The suicide vector pSUP202 harboring phaC1::Cm was transferred to B . cepacia by conjugation . The inactivated gene was integrated into the chromosome of B . cepacia by homologous recombination . This mutant and also 15 N-methyl-N'-nitrosoguanidine (NMG)-induced mutants still accumulated low amounts of PHAs and expressed low PHA synthase activity . The analysis of the mutant phaC1::Cm showed that it accumulated about 1% of PHA consisting of 68.2 mol% 3HB and 31.8 mol% 3H4PE from gluconate . The wild-type, in contrast, accumulated 49.3% of PHA consisting of 96.5 mol% 3HB and 3 . 5 mol% 3H4PE . Our results indicated that the genome of B . cepacia possesses at least two PHA synthase genes, which probably have different substrate specificities. J Biotechnol, 2001 Nov 30, 84(2), 155 - 61 Expression of a mineral phosphate solubilizing gene from Erwinia herbicola in two rhizobacterial strains; Rodriguez H et al.; A genetic construction was carried out using the broad host range vector pKT230 and plasmid pMCG898, which encodes the Erwinia herbicola pyrroloquinoline quinone (PQQ) synthase, a gene involved in mineral phosphate solubilization (mps) . The final construction was transformed and expressed in Escherichia coli MC1061, and the recombinant plasmids were transferred to Burkholderia cepacia IS-16 and Pseudomonas sp . PSS recipient cells by conjugation . Clones containing recombinant plasmids produced higher clearing halos in plates with insoluble phosphate as the unique (P) source, in comparison with those of strains without plasmids, demonstrating the heterologous expression of the E . herbicola gene in the recipient strains . This genetic manipulation allowed the increase in mps ability of both strains, enhancing their potentialities as growth promoters of agricultural crops . These results represent the first report on the application of the recombinant DNA methodology for the obtaining of improved phosphate solubilizing ability from rhizobacterial strains for biofertilization purposes. Infect Control Hosp Epidemiol, 2000 Nov, 21(11), 739 - 41 Outbreak of nosocomial Burkholderia cepacia infection and colonization associated with intrinsically contaminated mouthwash; Matrician L et al.; From August 1996 through June 1998, 69 ventilated, intensive care unit patients at two Arizona hospitals had nosocomial respiratory tract cultures positive for Burkholderia cepacia . Intrinsically contaminated alcohol-free mouthwash was identified by pulsed-field gel electrophoresis as the source of the outbreak. Infect Immun, 2000 Dec, 68(12), 6554 - 60 Identification of a siderophore receptor required for ferric ornibactin uptake in Burkholderia cepacia; Sokol PA et al.; Ornibactins are linear hydroxamate siderophores produced by Burkholderia cepacia with peptide structures similar to that of pyoverdines produced by the fluorescent pseudomonads . The gene encoding the outer membrane receptor (orbA) was identified, sequenced, and demonstrated to have significant homology with hydroxamate receptors produced by other organisms . The orbA precursor was predicted to be a protein with a molecular mass of 81 kDa . An orbA mutant was constructed and demonstrated to be unable to take up (59)Fe-ornibactins or to grow in medium supplemented with ornibactins . Outer membrane protein profiles from the parent strain, K56-2, revealed an iron-regulated outer membrane protein of 78 kDa that was not detectable in the K56orbA::tp mutant . When this mutant harbored a plasmid containing the orbA gene, the 78-kDa protein was present in the outer membrane protein profiles and the mutant was able to utilize ornibactin to acquire iron . The orbA mutant was less virulent in a chronic respiratory infection model than the parent strain, indicating that ornibactin uptake and utilization are important in the pathogenesis of B . cepacia respiratory infections. Acta Trop, 2000 Nov 2, 77(2), 229 - 37 Genotype analysis of Burkholderia pseudomallei using randomly amplified polymorphic DNA (RAPD): indicative of genetic differences amongst environmental and clinical isolates; Leelayuwat C et al.; Burkholderia pseudomallei is the causative agent of melioidosis, an infectious disease common in the tropics . Melioidosis is most prevalent in the northeastern part of Thailand . The diseases has diverse clinical manifestations ranging from mild localized to fatal septicemic forms . The bacterial genetic factors contributing to the severity of melioidosis have not been completely identified . We have developed a genotyping method based upon randomly amplified polymorphic DNA (RAPD) analysis . Eighteen deca-oligo nucleotide primers with 70% GC content, eight previously published 60%GC RAPD primers, and four random deca oligomers were tested on nine strains of B . pseudomallei isolated from five patients with localized and four with septicemic melioidosis . The RAPD patterns were analyzed by polyacrylamide gel electrophoresis using a laser based automated fragment analyzer, GS2000 . Based upon the pattern complexity, seven pairs consisting of eight primers were chosen for further analysis . Six hundred and thirty-two samples, including duplicates/triplicates, of B . pseudomallei isolated from melioidosis patients and the environment were analyzed . Two controls were included in each run of the test samples . All the samples were tested and patterns analyzed by blinded technical staff . Apparently, the method is reproducible . This is indicated by the RAPD patterns of the two controls of between run assay . Interestingly, some RAPD patterns were more prevalent in the clinical isolates than the environmental specimens and vice versa . For example, Q162KKU4-0 and Q162KKU1-0 were found 3 . 5 and 3.3 times more often in the clinical specimens (P<0.025) . Likewise, Q162KKU1-1 and Q162KKU4-1 were found 18 and 37 times more often in the environment (P<0.0000001) . In addition, there was a bias in the distribution of arabinose positive strains and particular RAPD patterns; RAPD patterns of B . pseudomallei that were found frequently in septicemic patients were less likely to be arabinose positive . The data suggest the existence of bacterial genetic differences between the clinical and environmental isolates of B . pseudomallei . Further analysis of the RAPD patterns searching for common polymorphic DNA fragments and systemic comparative genomic analysis of B . pseudomallei in accordance with the clinical data should reveal genetic factors involved in severity and bacterial pathogenesis of B . pseudomallei in melioidosis. Acta Trop, 2000 Nov 2, 77(2), 157 - 65 Melioidosis research in China; Yang S; Research on melioidosis and its pathogen has been ongoing in China for more than two decades . It has been demonstrated that the natural foci are located predominantly in Hainan, Guangdong and Guangxi province, where there is a good correlation between soil isolation and the serum prevalence of antibodies to Burkholderia pseudomallei . The cases of melioidosis reported up to now are concentrated in the Hainan and Zhanjiang peninsula . Investigations on serotype, virulence, ecology, antibiotic susceptibility, whole cell analysis by gas chromatography, and genetics have led to a new understanding of the pathology of the disease . Immunological cross reactions between Burkholderia mallei and B . pseudomallei and the difference between melioidosis and glanders in horses is discussed. Microb Ecol, 2000 Aug, 40(3), 189 - 199 Aquifer Protist Response and the Potential for TCE Bioremediation with Burkholderia cepacia G4 PR1; Snyder RA et al.; Bacterivorous protists have been recovered from pristine and contaminated aquifer environments, but the ecological role of these organisms in bioremediation strategies has not been well defined . Burkholderia cepacia G4 PR1 constitutively expresses a toluene ortho-monooxygenase (tom) due to a secondary transposition of a Tn5 transposable element in a trichloroethylene (TCE) degradative plasmid (TOM) . Groundwater and sediment from a potential site for a TCE bioremediation field demonstration were used in laboratory microcosms to test the survival of this organism . In nonsterile aquifer sediment slurries, the bacterium was eliminated in a logrithmic decay concomitant with an increase in bacterivorous protists . A half-life for the organism calculated from extinction coefficients increased logarithmically with increasing inoculation density above 1 x 10(6) PR1 ml(-1) . For inoculation densities below this level, the half-life of PR1 increased exponentially with decreasing inoculation density . The lowest half-lives corresponded to densities of bacteria that stimulate response of bacterivores . In a column system designed to incorporate aquifer flow, repeated addition of PR1 resulted in a buildup of bacterivore populations and reduced half-life of the bacterium . Addition of TCE and growth substrate in the eluent resulted in prolonged survival of PR1 and apparent mineralization of TCE . The results indicate significant but predictable losses due to native bacterivores would occur within and beyond a treatment zone where PR1 would be added to the aquifer, and mineralization of TCE in contaminated groundwater might be possible with repeated inoculation and addition of nutrients. Microb Ecol, 2000 Aug, 40(3), 169 - 176 Bias Caused by Using Different Isolation Media for Assessing the Genetic Diversity of a Natural Microbial Population; Tabacchioni S et al.; The influence of isolation medium on the biodiversity of Burkholderia cepacia strains recovered from the rhizosphere of Zea mays was evaluated by comparing the genetic diversity of isolates obtained by plating serial dilutions of root macerates on the two selective media TB-T and PCAT . From each medium, 50 randomly chosen colonies were isolated . On the basis of the restriction patterns of DNA coding for 16S rRNA (16S rDNA) amplified by means of PCR (ARDRA), all strains isolated from TB-T medium were assigned to the B . cepacia species, whereas among PCAT isolates only 74% were assigned to the B . cepacia species . Genetic diversity among the PCAT and TB-T isolates was evaluated by the random amplified polymorphic DNA (RAPD) technique . The analysis of molecular variance (AMOVA) method was applied to determine the variance component for RAPD patterns . Most of the genetic diversity (90.59%) was found within the two groups of isolates, but an appreciable amount (9.41%) still separated the two groups (P < 0.001) . Mean genetic distances among PCAT isolates (10.39) and TB-T isolates (9.36) were significantly different (P < 0.0001) . The results indicate that the two different isolation media select for B . cepacia populations with a different degree of genetic diversity . Moreover, a higher degree of genetic diversity was observed among strains isolated from PCAT medium than among those isolated from TB-T medium. J Med Microbiol, 2000 Nov, 49(11), 993 - 1001 Cloning and analysis of genomic differences unique to Burkholderia pseudomallei by comparison with B . thailandensis; Brown NF et al.; Melioidosis is an infectious disease caused by Burkholderia pseudomallei . Genomic subtractive hybridisation was performed with the closely related avirulent species B . thailandensis to identify virulence genes of B . pseudomallei . The subtractive hybridisation products were highly specific for B . pseudomallei . Sequence analysis revealed a number of putative virulence factors, as well as apparently novel sequences of unknown function . The subtracted library contained DNA regions of relatively low G + C mol% content, which were distributed throughout the B . pseudomallei genome . The distribution of subtracted sequences amongst a collection of 22 B . pseudomallei isolates was found to be variable, with the exception of three strains which almost universally lacked the subtracted sequences . These three strains also differed in that they were highly haemolytic, indicating a possible separate virotype. Anal Biochem, 2000 Nov 15, 286(2), 187 - 92 The use of protocatechuate dioxygenase for maintaining anaerobic conditions in biochemical experiments; Patil PV et al.; The study of redox-active systems often requires the maintenance of anaerobic conditions . The glucose oxidase system has often been used to maintain anaerobic conditions, but it has some drawbacks, such as the production of H(2)O(2) and limitations on stability . Protocatechuate dioxygenase from Burkholderia cepacia and the substrate, protocatechuate, constitute an alternate effective oxygen-scrubbing system that can be used in a wide variety of biochemical experiments . We have shown its suitability for maintaining rigorous anaerobic environments in solutions of pH 6-9, at temperatures from 4 to 35 degrees C, and for periods of time up to 15 months . The enzyme system was shown to be stable under these conditions and effective for maintaining anaerobic conditions in titrations of FAD . It is also suitable for scrubbing various types of apparatus such as stopped-flow instruments for anaerobic experiments . Biotechnol Bioeng, 2000 Dec 20, 70(6), 693 - 8 Aerobic degradation of mixtures of chlorinated aliphatics by cloned toluene-o-xylene monooxygenase and toluene o-monooxygenase in resting cells; Shim H et al.; Recombinant strains of Escherichia coli constitutively expressing toluene-o-xylene monooxygenase (ToMO) of Pseudomonas stutzeri OX1 and toluene o-monooxygenase (TOM) of Burkholderia cepacia G4 were investigated for their ability to oxidize trichloroethylene (TCE), 1,1-dichloroethylene (1,1-DCE), cis-1,2-dichloroethylene (cis-DCE), trans-1,2-dichloroethylene (trans-DCE), vinyl chloride (VC), and chloroform (CF), individually as well as in various mixtures . ToMO oxidized all of these individual compounds well, whereas TOM did not degrade VC significantly (16-fold less) and degraded cis-DCE and trans-DCE less well (3.7- and 2.4-fold, respectively) . For mixtures of these chlorinated aliphatics, ToMO was again more robust than TOM . For example, in binary mixtures including TCE, ToMO degraded all three DCE isomers and CF, but the presence of TCE inhibited VC degradation; TOM degraded both TCE/1,1-DCE and TCE/trans-DCE, but not cis-DCE for TCE/cis-DCE, and the addition of CF or VC to TCE completely inhibited degradation of both compounds and TCE . The addition of CF or trans-DCE stimulated VC degradation in the presence of TCE for ToMO, and the addition of any of the three DCE isomers stimulated VC degradation for TOM . Significant degradation of all ternary mixtures of TCE and less chlorinated ethenes, as well as a mixture of TCE, three DCEs, and VC, was achieved with ToMO (but not TOM) . In mixtures of these chlorinated compounds, degradation was found to occur simultaneously rather than sequentially, and the mineralization of many of these compounds could be confirmed through detection of chloride ions. Appl Environ Microbiol, 2000 Nov, 66(11), 4673 - 8 Rhizosphere competitiveness of trichloroethylene-degrading, poplar-colonizing recombinant bacteria; Shim H et al.; Indigenous bacteria from poplar tree (Populus canadensis var . eugenei 'Imperial Carolina') and southern California shrub rhizospheres, as well as two tree-colonizing Rhizobium strains (ATCC 10320 and ATCC 35645), were engineered to express constitutively and stably toluene o-monooxygenase (TOM) from Burkholderia cepacia G4 by integrating the tom locus into the chromosome . The poplar and Rhizobium recombinant bacteria degraded trichloroethylene at a rate of 0.8 to 2.1 nmol/min/mg of protein and were competitive against the unengineered hosts in wheat and barley rhizospheres for 1 month (colonization occurred at a level of 1.0 x 10(5) to 23 x 10(5) CFU/cm of root) . In addition, six of these recombinants colonized poplar roots stably and competitively with populations as large as 79% +/- 12% of all rhizosphere bacteria after 28 days (0.2 x 10(5) to 31 x 10(5) CFU/cm of root) . Furthermore, five of the most competitive poplar recombinants (e.g., Pb3-1 and Pb5-1, which were identified as Pseudomonas sp . strain PsK recombinants) retained the ability to express TOM for 29 days as 100% +/- 0% of the recombinants detected in the poplar rhizosphere expressed TOM constitutively. FEMS Microbiol Lett, 2000 Nov 1, 192(1), 67 - 72 Sequencing and characterization of a novel serine metalloprotease from Burkholderia pseudomallei; Lee MA et al.; Burkholderia pseudomallei, a Gram-negative bacterium is found in the soil and water, mainly in Southeast Asia and Northern Australia . It is responsible for melioidosis in human and animals . The bacteria produce several potential virulent factors such as extracellular protease, hemolysin, lipase and lecithinase . The isolation of virulence genes and the study of their functions will contribute to our understanding of bacterial pathogenesis . Previous studies have implicated protease as a contributing virulence factor in the pathogenesis of some bacteria . Three out of 5000 clones screened from a genomic DNA library of B . pseudomallei were found to express protease activity . The clones were found to have the same sequence . The nucleotide sequence revealed an open reading frame (designated as metalloprotease A, mprA) encoding a 500-amino acid protein, MprA, with an estimated molecular mass of 50241 Da . The predicted amino acid sequence shares homology with the subtilisin family of serine proteases. Appl Microbiol Biotechnol, 2000 Sep, 54(3), 382 - 9 Phospholipid compositional changes of five pseudomonad archetypes grown with and without toluene; Fang J et al.; Bacterial physiological responses to toluene exposure were investigated in five reference pseudomonad strains that express different toluene degradation pathways: Pseudomonas putida mt-2, Pseudomonas putida F1, Burkholderia cepacia G4, Burkholderia pickettii PKO1, and Pseudomonas mendocina KR1 . The intact phospholipids of these archetypes, grown with and without toluene, were characterized using liquid chromatography/electrospray ionization/mass spectrometry . All strains showed significant changes in phospholipid content and composition as an adaptive response to toluene exposure, as well as considerable diversity in response mechanisms . For example, the phospholipid content of toluene-grown PKO1, F1, and KR1 were 10.9-34.7% of that found in succinate-grown strains, while the phospholipid content of mt-2 and G4 increased by 56% and 94%, respectively, when grown on toluene . In addition, PKO1, F1, and mt-2 responded to the presence of toluene by synthesizing more phosphatidylglycerol, whereas G4 and KR1 synthesized phospholipids with polyunsaturated fatty acids (C18:2) on one or both of the sn-2 positions . These changes in phospholipid composition and concentration probably reflect the sensitivity and degree of tolerance of these strains to toluene, and suggest that different mechanisms are utilized by dissimilar bacteria to maintain optimal lipid ordering in the presence of such environmental pollutants. Appl Microbiol Biotechnol, 2000 Sep, 54(3), 354 - 60 Molecular cloning and characterization of a chitosanase from the chitosanolytic bacterium Burkholderia gladioli strain CHB101; Shimosaka M et al.; A chitosanase was purified from the culture fluid of the chitino- and chitosanolytic bacterium Burkholderia gladioli strain CHB101 . The purified enzyme (chitosanase A) had a molecular mass of 28 kDa, and catalyzed the endo-type cleavage of chitosans having a low degree of acetylation (0-30%) . The enzyme hydrolyzed glucosamine oligomers larger than a pentamer, but did not exhibit any activity toward N-acetylglucosamine oligomers and colloidal chitin . The gene coding for chitosanase A (csnA) was isolated and its nucleotide sequence determined . B . gladioli csnA has an ORF encoding a polypeptide of 355 amino acid residues . Analysis of the N-terminal amino acid sequence of the purified chitosanase A and comparison with that deduced from the csnA ORF suggests post-translational processing of a putative signal peptide and a possible substrate-binding domain . The deduced amino acid sequence corresponding to the mature protein showed 80% similarity to the sequences reported from Bacillus circulans strain MH-K1 and Bacillus ehimensis strain EAG1, which belong to family 46 glycosyl hydrolases. J Med Microbiol, 2000 Oct, 49(10), 875 - 85 Preferential adherence of cable-piliated burkholderia cepacia to respiratory epithelia of CF knockout mice and human cystic fibrosis lung explants; Sajjan U et al.; The Burkholderia cepacia complex consists of at least five well-documented bacterial genomovars, each of which has been isolated from the sputum of different patients with cystic fibrosis (CF) . Although the world-wide prevalence of this opportunist pathogen in CF patients is low (1-3%), 'epidemic' clusters occur in geographically isolated regions . Prevalence in some of these clusters is as high as 30-40% . The majority of CF B . cepacia isolates belong to genomovar III, but the relationship between genomovar and virulence has not yet been defined . Because the initial stage of infection involves bacterial binding to host tissues, the present study investigated differences in the binding of representative isolates of all five genomovars to fixed nasal sections of UNC cftr (-/-) and (+/+) mice and to human lung explants, biopsy and autopsy tissue of CF and non-CF patients . Binding was highest for isolates of genomovar III, subgroup RAPD type 2, but only if the isolates expressed the cable pili phenotype . Antibodies to the 22-kDa adhesin of cable pili virtually abolished binding . Binding occurred only to cftr (-/-) nasal sections or to CF lung sections and was negligible in cftr (+/+) or human non-CF, histologically normal lung sections . Unlike normal epithelia, the hyperplastic epithelia of CF bronchioles were enriched in cytokeratin 13, a 55-kDa protein that has previously been shown to act as a receptor in vitro for cable-piliated B . cepacia . These findings may help to explain the high transmissibility ofCbl-positive, genomovar III strains of B . cepacia among CF patients. Br J Neurosurg, 1998 Apr, 12(2), 170 - 2 Brain abscess as the presenting feature of melioidosis; Lath R et al.; Central nervous system involvement in melioidosis is rare and there are only a few reports of the causative organism, Burkholderia pseudomallei, causing a brain abscess . We report a patient who presented to us with a brain abscess due to this organism and emphasize the need for a high degree of suspicion for this disease in tropical countries and treatment with the appropriate antibiotics, as the mortality associated with this disease is very high. Appl Environ Microbiol, 2000 Oct, 66(10), 4585 - 8 A rhamnolipid biosurfactant reduces cadmium toxicity during naphthalene biodegradation; Sandrin TR et al.; A model cocontaminated system was developed to determine whether a metal-complexing biosurfactant, rhamnolipid, could reduce metal toxicity to allow enhanced organic biodegradation by a Burkholderia sp . isolated from soil . Rhamnolipid eliminated cadmium toxicity when added at a 10-fold greater concentration than cadmium (890 microM), reduced toxicity when added at an equimolar concentration (89 microM), and had no effect at a 10-fold smaller concentration (8.9 microM) . The mechanism by which rhamnolipid reduces metal toxicity may involve a combination of rhamnolipid complexation of cadmium and rhamnolipid interaction with the cell surface to alter cadmium uptake. Appl Environ Microbiol, 2000 Oct, 66(10), 4503 - 9 Detection and identification of bacterial endosymbionts in arbuscular mycorrhizal fungi belonging to the family Gigasporaceae; Bianciotto V et al.; Intracellular bacteria have been found previously in one isolate of the arbuscular mycorrhizal (AM) fungus Gigaspora margarita BEG 34 . In this study, we extended our investigation to 11 fungal isolates obtained from different geographic areas and belonging to six different species of the family Gigasporaceae . With the exception of Gigaspora rosea, isolates of all of the AM species harbored bacteria, and their DNA could be PCR amplified with universal bacterial primers . Primers specific for the endosymbiotic bacteria of BEG 34 could also amplify spore DNA from four species . These specific primers were successfully used as probes for in situ hybridization of endobacteria in G . margarita spores . Neighbor-joining analysis of the 16S ribosomal DNA sequences obtained from isolates of Scutellospora persica, Scutellospora castanea, and G . margarita revealed a single, strongly supported branch nested in the genus Burkholderia. Biochem Biophys Res Commun, 2000 Sep 16, 276(1), 71 - 6 Enzymes leading to the nucleotide sugar precursors for exopolysaccharide synthesis in Burkholderia cepacia; Richau JA et al.; Based on the chemical composition of the exopolysaccharide produced by the cystic fibrosis bacterial isolate Burkholderia cepacia IST408, we postulated and confirmed, based on the specificity of enzymes detected in crude cell-free extracts, the pathway leading to the presumptive activated sugar precursors: UDP-D-glucose, UDP-D-galactose, UDP-D-glucuronic acid, GDP-D-mannose, and GDP-D-rhamnose . Results also suggest that regulation of the expression of the mucoid phenotype in B . cepacia does not occur at the level of synthesis of any of these enzymes . Biotechnol Bioeng, 2000 Nov 20, 70(4), 436 - 45 Use of 16S-rRNA to investigate microbial population dynamics during biodegradation of toluene and phenol by a binary culture; Rogers JB et al.; Interspecies interactions and changes in the rate and extent of biodegradation in mixed culture-mixed substrate studies were investigated . A binary mixed culture of Pseudomonas putida F1 and Burkholderia sp . JS150 degraded toluene, phenol, and their mixture . Both toluene and phenol can serve as sole sources of carbon and energy for both P . putida F1 and strain JS150 . To investigate the population dynamics of this system, a fluorescent in-situ hybridization method was chosen because of its ability to produce quantitative data, its low standard error, and the ease of use of this method . When the binary mixed culture was grown on toluene or phenol alone, significant interactions between the species were observed . These interactions could not be explained by a pure-and-simple competition model and were substrate dependent . Strain JS150 growth was slightly inhibited when grown with P . putida F1 on phenol, and P . putida F1 grew more rapidly than expected . Conversely, when the two species were grown together on toluene alone, P . putida F1 was inhibited while strain JS150 was unaffected . During growth of the mixed culture on a combination of toluene and phenol, the interactions were similar to that observed during growth on phenol alone; P . putida F1 growth was enhanced while strain JS150 was unaffected . Because of the observed interspecies interactions, monoculture kinetic parameters were not sufficient to describe the mixed culture kinetics in any experiment . This is one of the first reports of microbial population dynamics in which molecular microbial ecology and mathematical modeling have been combined . The use of the 16S-rRNA-based method allowed for observation and understanding of interspecies interactions that were not observable with standard culture-based methods . These results suggest the need for more investigations that account for both substrate and microbial interactions when predicting the fate of organic pollutants in real systems . Biotechnol Bioeng, 2000 Nov 20, 70(4), 428 - 35 Modeling substrate interactions during the biodegradation of mixtures of toluene and phenol by Burkholderia species JS150; Rogers JB et al.; The biodegradation kinetics of toluene, phenol, and a mixture of toluene and phenol by Burkholderia species JS150 was measured and modeled . Both of these compounds can serve as the sole source of carbon and energy for this microorganism . The single-substrate biodegradation kinetics was described well using the Monod model, with model constants of mu(max,T) = 0.39 h(-1) and K(S,T) = 0.011 mM for growth on toluene and mu(max,P) = 0.309 h(-1) and K(S,P) = 0.0054 mM for growth on phenol . Degradation of the mixture of toluene and phenol followed simultaneous utilization kinetics with toluene being the preferred substrate . Toluene was found to inhibit the rate of utilization of phenol while the presence of phenol had little effect on the rate of degradation of toluene . Of the kinetic models that were tested, one developed for microbial degradation of multiple substrates was able to describe substrate interactions and to model the mixture utilization by strain JS150 . Simple competitive, noncompetitive, or uncompetitive substrate kinetics were not sufficient to describe the observed inhibitory interactions. J Med Assoc Thai, 2000 Aug, 83(8), 856 - 60 Comparison between the antimicrobial susceptibility of Burkholderia pseudomallei to trimethoprim-sulfamethoxazole by standard disk diffusion method and by minimal inhibitory concentration determination; Lumbiganon P et al.; Melioidosis, an infection caused by Burkholderia pseudomallei, usually occurs in immunocompromised patients and requires prolonged antibiotic therapy . Previously, oral trimethoprim-sulfamethoxazole (TM/SM), an inexpensive and effective drug has been used as a maintenance therapy . The susceptibility of B . pseudomallei to TM/SM by the standard disk diffusion method is very low . However, some patients who were treated with TM/SM as a maintenance therapy despite the in vitro resistance showed good clinical responses . There were no data comparing the susceptibility of B . pseudomallei by the standard disk diffusion method with other quantitative susceptibility tests . The objective of this study was to determine the agreement between the antimicrobial susceptibility of B . pseudomallei to TM/SM by standard disk diffusion and minimal inhibitory concentration determination (MIC) . We performed the susceptibility test of 144 strains of B . pseudomallei to TM/SM by both the standard disk diffusion and microbroth dilution MIC . The sensitivity results were 53.5 per cent and 84.0 per cent respectively . The agreement between the 2 tests was very poor (Kappa = 0.14; 95% CI = -0.01 to 0.29) . The false resistant rate by the standard disk diffusion test was 67.9 per cent . Further in vitro susceptibility and clinical study are needed to define the interpretive criteria that correlate with clinical response. J Agric Food Chem, 2000 Sep, 48(9), 4314 - 9 Protection of tomato seed germination from the inhibitory effect of 2,4,5-trichlorophenoxyacetic acid by inoculation of soil with Burkholderia cepacia AC1100; Gangadhara KP et al.; The effect of 2,4,5-trichlorophenoxy acetic acid (2,4,5-T) on the germination and seedling vigor of different crop seeds was tested . Seeds of rice, maize, sorghum, finger millet, and horse gram were comparatively more tolerant to the chemical with no marked effect up to a concentration of 200 mg 2,4,5-T L(-)(1) as tested by the filter paper method . Tomato and brinjal (egg plant) were highly susceptible . Even at 5 and 10 mg 2,4,5-T L(-)(1), marked reduction in the germination and seedling vigor of tomato and egg plant, respectively, was observed . At 20 and 30 mg L(-)(1), the germination of tomato and egg plant seeds, respectively, were completely inhibited on filter paper, whereas the inhibitory concentrations in soil was 40 mg 2,4,5-T kg(-)(1) soil . Several abnormalities were observed in the chemically affected seedlings . Protease activity of the seeds germinating in the presence of the chemical was drastically reduced . Bioremediation of the chemically contaminated soil with Burkholderia cepacia AC1100, by inoculation of the soil 7 days before sowing the seeds, completely protected the seeds, resulting in normal germination and an improved seedling vigor. J AOAC Int, 2000 Jul-Aug, 83(4), 963 - 6 Molecular detection of Burkholderia cepacia in toiletry, cosmetic, and pharmaceutical raw materials and finished products; Jimenez L et al.; A polymerase chain reaction (PCR) assay was developed and compared with standard methods for rapid detection of Burkholderia cepacia, a major industrial contaminant, in cosmetic and pharmaceutical raw materials and finished products . Artificially contaminated samples were incubated for 24 h in trypticase soy broth containing 4% Tween 20 and 0.5% soy lecithin . DNA was extracted from each sample using a proteinase K-tris-EDTA-Tween 20 treatment at 35 degrees C . The extracted DNA was added to Ready-To-Go PCR beads and specific DNA primers for B . cepacia . The B . cepacia DNA primers coded for a 209-base pair (bp) fragment of the 16S rRNA ribosomal gene . No DNA amplification was observed in samples that were not spiked with B . cepacia . However, all contaminated samples showed the specific 209-bp fragment for B . cepacia . There was a 100% correlation between standard methods and the PCR assay . Standard microbiological methods required 5-6 days for isolation and identification of spiked microorganisms, whereas PCR detection and identification was completed in 27 h . PCR detection of B . cepacia allows for rapid quality evaluation of cosmetic and pharmaceutical raw materials and finished products. Infect Immun, 2000 Oct, 68(10), 5710 - 5 IS1414, an Escherichia coli insertion sequence with a heat-stable enterotoxin gene embedded in a transposase-like gene; McVeigh A et al.; Enteroaggregative Escherichia coli (EAEC) heat-stable enterotoxin 1 (EAST1) was originally discovered in EAEC but has also been associated with enterotoxigenic E . coli (ETEC) . Multiple genomic restriction fragments from each of three ETEC strains of human origin showed homology with an EAST1 gene probe . A single hybridizing fragment was detected on the plasmid of ETEC strain 27D that also encodes heat-stable enterotoxin Ib and colonization factor antigen I . We isolated and characterized this fragment, showing that it (i) carries an allele of astA nearly identical to that originally reported from EAEC 17-2 and (ii) expressed enterotoxic activity . Sequence analysis of the toxin coding region revealed that astA is completely embedded within a 1,209-bp open reading frame (ORF1), whose coding sequence is on the same strand but in the -1 reading frame in reference to the toxin gene . In vitro expression of the predicted M(r)- approximately 46,000 protein product of ORF1 was demonstrated . ORF1 is highly similar to transposase genes of IS285 from Yersinia pestis, IS1356 from Burkholderia cepacia, and ISRm3 from Rhizobium meliloti . It is bounded by 30-bp imperfect inverted repeat sequences and flanked by 8-bp direct repeats . Based on these structural features, pathognomonic of a regular insertion sequence, this element was designated IS1414 . Preliminary experiments to show IS1414 translocation were unsuccessful . Overlapping genes of the type suggested by the IS1414 core region have heretofore not been described in bacteria . It seems to offer a most efficient mechanism for intragenomic and horizontal dissemination of EAST1. Rev Latinoam Microbiol, 1999 Jan-Mar, 41(1), 17 - 23 {The interaction between corn and Burkholderia (Pseudomonas) cepacia}; Velazquez M et al.; Burkholderia (Pseudomonas) cepacia is a plant growth promoting rhizobacteria that could improve maize productivity . In this work we studied some properties of B . cepacia strain 0057 in relation to interaction with maize . Spermosphere effect, chemostatic response, indolic compounds and siderophores production were evaluated . We observed inhibitory effect on bacterial growth by maize seeds and their soluble extracts . Root exudates had positive chemiostatic effect . We founded indolic compounds production (20.1 micrograms/ml) and siderophore presence . We think that this microorganisms could improve maize production. Trans R Soc Trop Med Hyg, 2000 May-Jun, 94(3), 301 - 4 Melioidosis: acute and chronic disease, relapse and re-activation; Currie BJ et al.; In melioidosis-endemic regions the importance of re-activation of Burkholderia pseudomallei from latent foci remains unclear . This topic was assessed in a 10-year prospective study (1989-99) of melioidosis in the tropical north of the Northern Territory of Australia, together with other aspects of the nature of melioidosis . Incubation period from defined inoculating events was previously ascertained as 1-21 (mean 9) days . Of 252 total cases 244 (97%) were considered to be from recent acquisition of B . pseudomallei infection and 8 (3%) were considered to be re-activation from a latent focus . Acute illness occurred in 222 (88%) cases; 30 (12%) cases had chronic illness (symptomatic for > 2 months) . Of the 207 patients surviving the initial illness, 27 (13%) had a confirmed relapse (mean time from initial diagnosis of 8 months), with 5 relapsing twice . Of these 32 relapses, 15 (3 fatal) were associated with poor adherence to the eradication therapy antibiotics and 10 (none fatal) were failures of eradication with doxycycline monotherapy . Following initial intensive therapy with ceftazidime or meropenem for at least 14 days, eradication therapy with trimethoprim-sulphamethoxazole monotherapy for at least 3 months had been more successful. Gene, 2000 Aug 22, 254(1-2), 129 - 37 Characterization and mutagenesis of fur gene from Burkholderia pseudomallei; Loprasert S et al.; A homolog of the ferric uptake regulator gene (fur) was isolated from Burkholderia pseudomallei (Bp) by a reverse genetic technique . Sequencing of a 2.2kb DNA fragment revealed an open reading frame with extensive homology to bacterial Fur proteins . The cloned gene encodes a 16kDa protein that cross-reacts with a polyclonal anti-Escherichia coli Fur serum . The transcription start site was determined by the primer extension technique . Expression analysis of fur showed no increased fur mRNA levels in response to various stresses and iron conditions . A positive selection procedure involving the isolation of manganese-resistant mutants was used to isolate mutants that produce altered Fur proteins . Sequencing of a fur mutant revealed a nucleotide change (G to A) converting a conserved amino acid arginine-69 to histidine . The fur missense mutant produced an elevated level of siderophore that could be complemented by a multicopy plasmid carrying the Bp fur . Interestingly, Fur was found to play roles as a positive regulator of FeSOD and peroxidase . The mutant showed a decreased activity of FeSOD and peroxidase, which could be important in its pathogenicity and survival in macrophages. J Clin Microbiol, 2000 Sep, 38(9), 3165 - 73 DNA-Based diagnostic approaches for identification of Burkholderia cepacia complex, Burkholderia vietnamiensis, Burkholderia multivorans, Burkholderia stabilis, and Burkholderia cepacia genomovars I and III; Mahenthiralingam E et al.; Bacteria of the Burkholderia cepacia complex consist of five discrete genomic species, including genomovars I and III and three new species: Burkholderia multivorans (formerly genomovar II), Burkholderia stabilis (formerly genomovar IV), and Burkholderia vietnamiensis (formerly genomovar V) . Strains of all five genomovars are capable of causing opportunistic human infection, and microbiological identification of these closely related species is difficult . The 16S rRNA gene (16S rDNA) and recA gene of these bacteria were examined in order to develop rapid tests for genomovar identification . Restriction fragment length polymorphism (RFLP) analysis of PCR-amplified 16S rDNA revealed sequence polymorphisms capable of identifying B . multivorans and B . vietnamiensis but insufficient to discriminate strains of B . cepacia genomovars I and III and B . stabilis . RFLP analysis of PCR-amplified recA demonstrated sufficient nucleotide sequence variation to enable separation of strains of all five B . cepacia complex genomovars . Complete recA nucleotide sequences were obtained for 20 strains representative of the diversity of the B . cepacia complex . Construction of a recA phylogenetic tree identified six distinct clusters (recA groups): B . multivorans, B . vietnamiensis, B . stabilis, genomovar I, and the subdivision of genomovar III isolates into two recA groups, III-A and III-B . Alignment of recA sequences enabled the design of PCR primers for the specific detection of each of the six latter recA groups . The recA gene was found on the largest chromosome within the genome of B . cepacia complex strains and, in contrast to the findings of a previous study, only a single copy of the gene was present . In conclusion, analysis of the recA gene of the B . cepacia complex provides a rapid and robust nucleotide sequence-based approach to identify and classify this taxonomically complex group of opportunistic pathogens. Med J Malaysia, 1997 Jun, 52(2), 158 - 60 In-vitro susceptibility of Burkholderia pseudomallei to cefoperazone-sulbactam combination; Koay AS et al.; Melioidosis is endemic in Malaysia . Emerging resistance with new and current antimicrobial agents has underscored the need to look further for new antimicrobial agents for the treatment of melioidosis . Hence, we evaluated the in-vitro susceptibility of fifty locally isolated strains of Burkholderia pseudomallei, the causative agent of melioidosis to cefoperazone-sulbactam combination using the method of NCCLS . All the fifty strains tested were susceptible in-vitro to cefoperazone-sulbactam . The MIC90 of the organism for cefoperazone-sulbactam was 4 mg/L . The results of our findings suggested that cefoperazone-sulbactam may be useful in the treatment of melioidosis. Appl Environ Microbiol, 2000 Sep, 66(9), 4139 - 41 Identification and characteristics of a novel Burkholderia strain with broad-spectrum antimicrobial activity; Cain CC et al.; A Burkholderia strain isolated from soil is capable of inhibiting the growth of bacteria, plant-pathogenic fungi, pathogenic yeasts, and protozoa . Inhibition does not involve cell contact or the presence of living cells, suggesting that at least a substantial portion of the antimicrobial activity is due to the excretion of extracellular compounds. Appl Environ Microbiol, 2000 Sep, 66(9), 4098 - 104 Toluene-degrading bacteria are chemotactic towards the environmental pollutants benzene, toluene, and trichloroethylene; Parales RE et al.; The bioremediation of polluted groundwater and toxic waste sites requires that bacteria come into close physical contact with pollutants . This can be accomplished by chemotaxis . Five motile strains of bacteria that use five different pathways to degrade toluene were tested for their ability to detect and swim towards this pollutant . Three of the five strains (Pseudomonas putida F1, Ralstonia pickettii PKO1, and Burkholderia cepacia G4) were attracted to toluene . In each case, the response was dependent on induction by growth with toluene . Pseudomonas mendocina KR1 and P . putida PaW15 did not show a convincing response . The chemotactic responses of P . putida F1 to a variety of toxic aromatic hydrocarbons and chlorinated aliphatic compounds were examined . Compounds that are growth substrates for P . putida F1, including benzene and ethylbenzene, were chemoattractants . P . putida F1 was also attracted to trichloroethylene (TCE), which is not a growth substrate but is dechlorinated and detoxified by P . putida F1 . Mutant strains of P . putida F1 that do not oxidize toluene were attracted to toluene, indicating that toluene itself and not a metabolite was the compound detected . The two-component response regulator pair TodS and TodT, which control expression of the toluene degradation genes in P . putida F1, were required for the response . This demonstration that soil bacteria can sense and swim towards the toxic compounds toluene, benzene, TCE, and related chemicals suggests that the introduction of chemotactic bacteria into selected polluted sites may accelerate bioremediation processes. Pediatr Med Chir, 1999 Sep-Oct, 21(5 Suppl), 213 - 8 {Bacterial infections and resistance to antibiotics in cystic fibrosis}; Taccetti G et al.; Knowledge of the microbiology of pulmonary infections is critical for treatment of cystic fibrosis because sickness and mortality in this disease are mainly due to relapse occurring in the respiratory tract . The microbiology of pulmonary infections presents several singular aspects . Respiratory tract infections are caused by bacteria such as Staphylococcus aureus in the early years of life and Pseudomonas aeruginosa and Burkholderia cepacia thereafter . The patients, who are not immune compromised, are predisposed to chronic colonization and highly transmissible bacterial strains can cause cross-infections . Bacterial also develop resistance mechanisms which make them difficult to treat . Until recently the relationship between genetic defects and a predisposition to colonization was not noted, but recent studies have allowed us to form some interesting hypothesis . The present work analyzes the principal mechanisms of antibiotic resistance, with particular reference to classic cystic fibrosis pathogens, and looks at future prospects of respiratory tract infection treatment. Mol Gen Genet, 2000 Jul, 263(6), 1031 - 7 Members of the amylovora group of Erwinia are cellulolytic and possess genes homologous to the type II secretion pathway; Riekki R et al.; A cellulase-producing clone was isolated from a genomic library of the Erwinia rhapontici (Millard) Burkholder strain NCPPB2989 . The corresponding gene, named celA, encodes an endoglucanase (EC 3.2.1.4) with the extremely low pH optimum of 3.4 and a temperature optimum between 40 and 50 degrees C . A single ORF of 999 nt was found to be responsible for the Cel activity . The corresponding protein, named CelA, showed 67% identity to the endoglucanase Y of E . chrysanthemi and 51.5% identity to the endoglucanase of Cellulomonas uda, and thus belongs to the glycosyl hydrolase family 8 . The celA gene, or its homologue, was found to be present in all E . rhapontici isolates analysed, i