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Alasdair M Cook - One of the best experts on this subject based on the ideXlab platform.
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Taurine Reduction in Anaerobic Respiration of
2020Co-Authors: Heike Laue, Karin Denger, Alasdair M CookAbstract:Organosulfonates are important natural and man-made compounds, but until recently (T. J. Lie, T. Pitta, E. R. Leadbetter, W. Godchaux III, and J. R. Leadbetter. Arch. Microbiol. 166:204‐210, 1996), they were not believed to be dissimilated under anoxic conditions. We also chose to test whether alkane- and arenesulfonates could serve as electron sinks in respiratory metabolism. We generated 60 anoxic enrichment cultures in mineral salts medium which included several potential electron donors and a single organic sulfonate as an electron sink, and we used material from anaerobic digestors in communal sewage works as inocula. None of the four aromatic sulfonates, the three unsubstituted alkanesulfonates, or the N-sulfonate tested gave positive enrichment cultures requiring both the electron donor and electron sink for growth. Nine cultures utilizing the natural products taurine, cysteate, or isethionate were considered positive for growth, and all formed sulfide. Two clearly different pure cultures were examined. Putative Desulfovibrio sp. strain RZACYSA, with lactate as the electron donor, utilized sulfate, aminomethanesulfonate, taurine, isethionate, and cysteate, converting the latter to ammonia, acetate, and sulfide. Strain RZATAU was identified by 16S rDNA analysis as Bilophila wadsworthia. In the presence of, e.g., formate as the electron donor, it utilized, e.g., cysteate and isethionate and converted taurine quantitatively to cell material and products identified as ammonia, acetate, and sulfide. Sulfite and thiosulfate, but not sulfate, were utilized as electron sinks, as was nitrate, when lactate was provided as the electron donor and carbon source. A growth requirement for 1,4-naphthoquinone indicates a menaquinone electron carrier, and the presence of cytochrome c supports the presence of an electron transport chain. Pyruvate-dependent disappearance of taurine from cell extracts, as well as formation of alanine and release of ammonia and acetate, was detected. We suspected that sulfite is an intermediate, and we detected desulfoviridin (sulfite reductase). We thus believe that sulfonate reduction is one aspect of a respiratory system transferring electrons from, e.g., formate to sulfite reductase via an electron transport system which presumably generates a proton gradient across the cell membrane.
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identification of Bilophila wadsworthia by specific pcr which targets the taurine pyruvate aminotransferase gene
Fems Microbiology Letters, 2006Co-Authors: Heike Laue, M C Claros, Ulrike Schumacher, Theo H M Smits, Ralf Hartemink, Alasdair M CookAbstract:The bile-resistant, strictly anaerobic bacterium Bilophila wadsworthia is found in human faecal flora, in human infections and in environmental samples. A specific PCR primer set for the gene encoding the first metabolic enzyme in the degradative pathway for taurine in B. wadsworthia, taurine:pyruvate aminotransferase (tpa), was developed and tested. In addition, enrichment cultures were started from faecal samples of primates and felines and shown to contain B. wadsworthia. These were subcultured on agar media and then identified by PCR fingerprinting. PCR for tpa was successful in all positive enrichment cultures and showed no amplification signal in a variety of other bacterial species. Therefore, this PCR method could be a promising tool for rapid detection of B. wadsworthia in biological samples.
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dissimilatory sulfite reductase desulfoviridin of the taurine degrading non sulfate reducing bacterium Bilophila wadsworthia rzatau contains a fused dsrb dsrd subunit
Journal of Bacteriology, 2001Co-Authors: Heike Laue, Michael W Friedrich, Jurgen Ruff, Alasdair M CookAbstract:A dissimilatory sulfite reductase (DSR) was purified from the anaerobic, taurine-degrading bacterium Bilophila wadsworthia RZATAU to apparent homogeneity. The enzyme is involved in energy conservation by reducing sulfite, which is formed during the degradation of taurine as an electron acceptor, to sulfide. According to its UV-visible absorption spectrum with maxima at 392, 410, 583, and 630 nm, the enzyme belongs to the desulfoviridin type of DSRs. The sulfite reductase was isolated as an a2b2gn (n > 2) multimer with a native size of 285 kDa as determined by gel filtration. We have sequenced the genes encoding the a and b subunits (dsrA and dsrB, respectively), which probably constitute one operon. dsrA and dsrB encode polypeptides of 49 (a) and 54 kDa (b) which show significant similarities to the homologous subunits of other DSRs. The dsrB gene product of B. wadsworthia is apparently a fusion protein of dsrB and dsrD. This indicates a possible functional role of DsrD in DSR function because of its presence as a fusion protein as an integral part of the DSR holoenzyme in B. wadsworthia. A phylogenetic analysis using the available Dsr sequences revealed that B. wadsworthia grouped with its closest 16S rDNA relative Desulfovibrio desulfuricans Essex 6. Bilophila wadsworthia is a strictly anaerobic, gram-negative bacterium (2) which belongs to the family Desulfovibrionaceae in the delta subdivision of the Proteobacteria, but does not
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biochemical and molecular characterization of taurine pyruvate aminotransferase from the anaerobe Bilophila wadsworthia
FEBS Journal, 2000Co-Authors: Heike Laue, Alasdair M CookAbstract:Bilophila wadsworthia RZATAU is a Gram-negative bacterium which converts the sulfonate taurine (2-aminoethanesulfonate) to ammonia, acetate and sulfide in an anaerobic respiration. Taurine:pyruvate aminotransferase (Tpa) catalyses the initial metabolic reaction yielding alanine and sulfoacetaldehyde. We purified Tpa 72-fold to apparent homogeneity with an overall yield of 89%. The purified enzyme did not require addition of pyridoxal 5 0 -phosphate, but highly active enzyme was only obtained by addition of pyridoxal 5 0 -phosphate to all buffers during purification. SDS/PAGE revealed a single protein band with a molecular mass of 51 kDa. The apparent molecular mass of the native enzyme was 197 kDa as determined by gel filtration, which indicates a homotetrameric structure. The kinetic constants for taurine were: Kma 7.1 mm, Vmaxa 1.20 nmol·s 21 , and for pyruvate: Kma 0.82 mm, Vmaxa 0.17 nmol·s 21 . The purified enzyme was able to transaminate hypotaurine (2-aminosulfinate), taurine, b-alanine and with low activity cysteine and 3-aminopropanesulfonate. In addition to pyruvate, 2-ketobutyrate and oxaloacetate were utilized as amino group acceptors. We have sequenced the encoding gene (tpa). It encoded a 50-kDa peptide, which revealed 33% identity to diaminopelargonate aminotransferase from Bacillus subtilis.
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purification properties and primary structure of alanine dehydrogenase involved in taurine metabolism in the anaerobe Bilophila wadsworthia
Archives of Microbiology, 2000Co-Authors: Heike Laue, Alasdair M CookAbstract:Alanine dehydrogenase [l-alanine:NAD+ oxidoreductase (deaminating), EC 1.4.1.4.] catalyses the reversible oxidative deamination of l-alanine to pyruvate and, in the anaerobic bacterium Bilophila wadsworthia RZATAU, it is involved in the degradation of taurine (2-aminoethanesulfonate). The enzyme regenerates the amino-group acceptor pyruvate, which is consumed during the transamination of taurine and liberates ammonia, which is one of the degradation end products. Alanine dehydrogenase seems to be induced during growth with taurine. The enzyme was purified about 24-fold to apparent homogeneity in a three-step purification. SDS-PAGE revealed a single protein band with a molecular mass of 42 kDa. The apparent molecular mass of the native enzyme was 273 kDa, as determined by gel filtration chromatography, suggesting a homo-hexameric structure. The N-terminal amino acid sequence was determined. The pH optimum was pH 9.0 for reductive amination of pyruvate and pH 9.0–11.5 for oxidative deamination of alanine. The apparent K m values for alanine, NAD+, pyruvate, ammonia and NADH were 1.6, 0.15, 1.1, 31 and 0.04 mM, respectively. The alanine dehydrogenase gene was sequenced. The deduced amino acid sequence corresponded to a size of 39.9 kDa and was very similar to that of the alanine dehydrogenase from Bacillus subtilis.
Sydney M Finegold - One of the best experts on this subject based on the ideXlab platform.
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In vitro activities of faropenem against 579 strains of anaerobic bacteria.
Antimicrobial Agents and Chemotherapy, 2002Co-Authors: Hannah M Wexler, Denise Molitoris, Shahera St. John, Ann Vu, Erik K. Read, Sydney M FinegoldAbstract:The activity of faropenem, a new oral penem, was tested against 579 strains of anaerobic bacteria by using the NCCLS-approved reference method. Drugs tested included amoxicillin-clavulanate, cefoxitin, clindamycin, faropenem, imipenem, and metronidazole. Of the 176 strains of Bacteroides fragilis group isolates tested, two isolates had faropenem MICs of 64 μg/ml and imipenem MICs of >32 μg/ml. Faropenem had an MIC of 16 μg/ml for an additional isolate of B. fragilis; this strain was sensitive to imipenem (MIC of 1 μg/ml). Both faropenem and imipenem had MICs of ≤4 μg/ml for all isolates of Bacteroides capillosus (10 isolates), Bacteroides splanchnicus (13 isolates), Bacteroides ureolyticus (11 isolates), Bilophila wadsworthia (11 isolates), Porphyromonas species (42 isolates), Prevotella species (78 isolates), Campylobacter species (25 isolates), Sutterella wadsworthensis (11 isolates), Fusobacterium nucleatum (19 isolates), Fusobacterium mortiferum/varium (20 isolates), and other Fusobacterium species (9 isolates). Faropenem and imipenem had MICs of 16 to 32 μg/ml for two strains of Clostridium difficile; the MICs for all other strains of Clostridium tested (69 isolates) were ≤4 μg/ml. Faropenem had MICs of 8 and 16 μg/ml, respectively, for two strains of Peptostreptococcus anaerobius (MICs of imipenem were 2 μg/ml). MICs were ≤4 μg/ml for all other strains of gram-positive anaerobic cocci (53 isolates) and non-spore-forming gram-positive rods (28 isolates). Other results were as expected and reported in previous studies. No metronidazole resistance was seen in gram-negative anaerobes other than S. wadsworthensis (18% resistant); 63% of gram-positive non-spore-forming rods were resistant. Some degree of clindamycin resistance was seen in most of the groups tested.
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In vitro activity of telithromycin (HMR 3647) against 502 strains of anaerobic bacteria
Journal of Antimicrobial Chemotherapy, 2001Co-Authors: Hannah M Wexler, Eric Molitoris, Denise Molitoris, Sydney M FinegoldAbstract:: In a previous study, we compared HMR 3004 with azithromycin, clarithromycin, erythromycin and roxithromycin against 502 anaerobic bacteria using NCCLS-approved procedures. This report extends this study by reporting the activity of telithromycin (HMR 3647) against these strains. Telithromycin inhibited 10% of Bacteroides fragilis, 50% of other B. fragilis group organisms and 93% of other Bacteroides spp. Telithromycin inhibited all Porphyromonas spp. and 98% of Prevotella spp. Activity against Bilophila wadsworthia (85-96%) was excellent. Telithromycin was not active against the Fusobacterium mortiferum/varium group. Telithromycin inhibited 100% of Clostridium perfringens, 46-56% of Clostridium difficile and Clostridium ramosum and approximately 90% of non-spore-forming Gram-positive bacilli.
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characterization of Bilophila wadsworthia isolates using pcr fingerprinting
Anaerobe, 1999Co-Authors: M C Claros, Sydney M Finegold, Hunt S Gerardo, Ulrike Schumacher, M Jacob, N Kleinkauf, Ellie J C Goldstein, Arne C RodloffAbstract:Abstract Bilophila wadsworthia , an under-appreciated anaerobic organism, was originally described in 1989. Ninety-nine Bilophila wadsworthia isolates, recovered form environmental and clinical specimens in Germany and in Southern California, were examined in this study. Many isolates were recovered in mixed culture with facultative aerobic and other anaerobic bacteria. All isolates were identified by standard laboratory procedures, including gas–liquid chromatography (GLC). A PCR fingerprint assay was established to compare the profiles of clinical and environmental isolates to the type strain (ATCC 49260) and to an environmental (sewage) reference strain (DSM 11045, RZATAU) for intra-species differences. Two primers, one universal primer, M13 core, and one tDNA primer, T3B, were used individually to analyse the strains. Homogeneous PCR fingerprint profiles were found for the majority of strains using the M13 core primer; two PCR groups were determined with T3B, one matching the type strain and one matching the environmental reference strain (DSM 11045, RZATAU). Two urease negative strains, WAL 11470 (blood isolate from California) and TUB 754 (intra-abdominal isolate from Germany) formed unique PCR fingerprint profiles with each of these primers. These results were confirmed by PCR fingerprinting using the T3A primer. These latter results suggest a possible genetic diversity in B. wadsworthia .
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In vitro activity of HMR 3004 (RU 64004) against 502 strains of anaerobic bacteria
Anaerobe, 1999Co-Authors: Hannah M Wexler, Eric Molitoris, Denise Molitoris, Sydney M FinegoldAbstract:Abstract The in vitro activity of HMR 3004 (RU 64004) was compared to azithromycin (AZ), clarithromycin (CL), erythromycin (ER) and roxithromycin (RO). Anaerobes (n =512) were tested using the NCCLS-approved Wadsworth brucella laked blood agar dilution method. Breakpoints for the inclusion of strains into the susceptible/intermediate categories were 4 μg/mL for all agents. Organisms tested included Bacteroides fragilis group species (206), Campylobacter gracilis (13), Bacteriodes species (13), Fusobacterium species (540 K), Bilophila wadsworthia (27),Peptostreptococcus (35), Porphyromonas sp. (12), Prevotella sp. (41), Sutterella wadsworthensis (16), Clostridium sp. (56) and Gram-positive non-sporeforming rods (29). HMR 3004 inhibited 78% of B. fragilis and 69% of B. fragilis groups species, respectively. Ninety-three percent of otherBacteroides were inhibited by HMR 3004, compared to 80% for AZ, 87% for ER, and 93% for CL and RO. HMR 3004 and AZ inhibited all Porphyromonas species, compared to 93–94% for the other agents. HMR 3004 inhibited 98% of Prevotella compared to 91%, 93%, 93% and 95% for ER, AZ, RO and CL, respectively. Activity against Biophila wadsworthia (85–96%) was excellent compared to poor or no activity for the other macrolides. All agents inhibited 100% of Clostridium perfringens and 50% of C. difficile and C. ramosum . None of the agents had activity against the Fusobacterium mortiferum/varium group. Approximately 90% of non-sporeforming Gram-positive rods were inhibited by all of the agents. Overall, HMR 3004 had the best activity against anaerobes, inhibiting 77% compared to AZ (46%), CL (73%), ER (47%) and RO (46%).
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Recently Described Clinically Important Anaerobic Bacteria: Medical Aspects
Clinical Infectious Diseases, 1997Co-Authors: Sydney M Finegold, Hannele Jousimies-somerAbstract:There is still inadequate information on the role of certain newly described or reclassified anaerobes in disease processes, on their normal sites of carriage, and on their antimicrobial susceptibilities. Herein, we summarize this information (most of the literature reviewed is from the past 5 years, but a few of the articles are C10 years old). Porphyromonas species had seemed to be relatively nonpathogenic, but recent work indicates that this belief is incorrect. P. gingivalis, P. levii ‐ like organisms, and P. endodontalis ‐ like organisms have been recovered from a variety of oral and extraoral infections. P. macacae has been recovered from infected cat bite wounds. Sutterella wadsworthensis, recently differentiated from Campylobacter gracilis, has been found in a variety of infections. Bilophila wadsworthia has also been recovered from a wide variety of infections. Newly described anaerobic cocci, gram-positive nonsporeforming rods, and clostridia have also been isolated from various infections. In recent years, a large number of new or reclassified taxa odontogenic abscesses, periimplantitis, a pulmonary infection, and a groin abscess (authors’ unpublished data), as summarized of anaerobes have been described [1 ‐ 4]. A number of these anaerobes cause infections in humans and/or animals. Data on in table 1. The nonpigmented Prevotella species are predominantly found in the normal oral and vaginal flora and are isothe normal sites of carriage of these ‘‘new’’ organisms, on their role in infection, and on their susceptibilities to antimicro- lated as part of a mixed flora from infections related to those sites; however, these organisms are also found in infections bial agents are not readily available. The clinically important anaerobes have recently been reviewed [5]. Herein, we present related to other sites. P. dentalis is a common isolate recovered
Heike Laue - One of the best experts on this subject based on the ideXlab platform.
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Taurine Reduction in Anaerobic Respiration of
2020Co-Authors: Heike Laue, Karin Denger, Alasdair M CookAbstract:Organosulfonates are important natural and man-made compounds, but until recently (T. J. Lie, T. Pitta, E. R. Leadbetter, W. Godchaux III, and J. R. Leadbetter. Arch. Microbiol. 166:204‐210, 1996), they were not believed to be dissimilated under anoxic conditions. We also chose to test whether alkane- and arenesulfonates could serve as electron sinks in respiratory metabolism. We generated 60 anoxic enrichment cultures in mineral salts medium which included several potential electron donors and a single organic sulfonate as an electron sink, and we used material from anaerobic digestors in communal sewage works as inocula. None of the four aromatic sulfonates, the three unsubstituted alkanesulfonates, or the N-sulfonate tested gave positive enrichment cultures requiring both the electron donor and electron sink for growth. Nine cultures utilizing the natural products taurine, cysteate, or isethionate were considered positive for growth, and all formed sulfide. Two clearly different pure cultures were examined. Putative Desulfovibrio sp. strain RZACYSA, with lactate as the electron donor, utilized sulfate, aminomethanesulfonate, taurine, isethionate, and cysteate, converting the latter to ammonia, acetate, and sulfide. Strain RZATAU was identified by 16S rDNA analysis as Bilophila wadsworthia. In the presence of, e.g., formate as the electron donor, it utilized, e.g., cysteate and isethionate and converted taurine quantitatively to cell material and products identified as ammonia, acetate, and sulfide. Sulfite and thiosulfate, but not sulfate, were utilized as electron sinks, as was nitrate, when lactate was provided as the electron donor and carbon source. A growth requirement for 1,4-naphthoquinone indicates a menaquinone electron carrier, and the presence of cytochrome c supports the presence of an electron transport chain. Pyruvate-dependent disappearance of taurine from cell extracts, as well as formation of alanine and release of ammonia and acetate, was detected. We suspected that sulfite is an intermediate, and we detected desulfoviridin (sulfite reductase). We thus believe that sulfonate reduction is one aspect of a respiratory system transferring electrons from, e.g., formate to sulfite reductase via an electron transport system which presumably generates a proton gradient across the cell membrane.
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identification of Bilophila wadsworthia by specific pcr which targets the taurine pyruvate aminotransferase gene
Fems Microbiology Letters, 2006Co-Authors: Heike Laue, M C Claros, Ulrike Schumacher, Theo H M Smits, Ralf Hartemink, Alasdair M CookAbstract:The bile-resistant, strictly anaerobic bacterium Bilophila wadsworthia is found in human faecal flora, in human infections and in environmental samples. A specific PCR primer set for the gene encoding the first metabolic enzyme in the degradative pathway for taurine in B. wadsworthia, taurine:pyruvate aminotransferase (tpa), was developed and tested. In addition, enrichment cultures were started from faecal samples of primates and felines and shown to contain B. wadsworthia. These were subcultured on agar media and then identified by PCR fingerprinting. PCR for tpa was successful in all positive enrichment cultures and showed no amplification signal in a variety of other bacterial species. Therefore, this PCR method could be a promising tool for rapid detection of B. wadsworthia in biological samples.
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dissimilatory sulfite reductase desulfoviridin of the taurine degrading non sulfate reducing bacterium Bilophila wadsworthia rzatau contains a fused dsrb dsrd subunit
Journal of Bacteriology, 2001Co-Authors: Heike Laue, Michael W Friedrich, Jurgen Ruff, Alasdair M CookAbstract:A dissimilatory sulfite reductase (DSR) was purified from the anaerobic, taurine-degrading bacterium Bilophila wadsworthia RZATAU to apparent homogeneity. The enzyme is involved in energy conservation by reducing sulfite, which is formed during the degradation of taurine as an electron acceptor, to sulfide. According to its UV-visible absorption spectrum with maxima at 392, 410, 583, and 630 nm, the enzyme belongs to the desulfoviridin type of DSRs. The sulfite reductase was isolated as an a2b2gn (n > 2) multimer with a native size of 285 kDa as determined by gel filtration. We have sequenced the genes encoding the a and b subunits (dsrA and dsrB, respectively), which probably constitute one operon. dsrA and dsrB encode polypeptides of 49 (a) and 54 kDa (b) which show significant similarities to the homologous subunits of other DSRs. The dsrB gene product of B. wadsworthia is apparently a fusion protein of dsrB and dsrD. This indicates a possible functional role of DsrD in DSR function because of its presence as a fusion protein as an integral part of the DSR holoenzyme in B. wadsworthia. A phylogenetic analysis using the available Dsr sequences revealed that B. wadsworthia grouped with its closest 16S rDNA relative Desulfovibrio desulfuricans Essex 6. Bilophila wadsworthia is a strictly anaerobic, gram-negative bacterium (2) which belongs to the family Desulfovibrionaceae in the delta subdivision of the Proteobacteria, but does not
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biochemical and molecular characterization of taurine pyruvate aminotransferase from the anaerobe Bilophila wadsworthia
FEBS Journal, 2000Co-Authors: Heike Laue, Alasdair M CookAbstract:Bilophila wadsworthia RZATAU is a Gram-negative bacterium which converts the sulfonate taurine (2-aminoethanesulfonate) to ammonia, acetate and sulfide in an anaerobic respiration. Taurine:pyruvate aminotransferase (Tpa) catalyses the initial metabolic reaction yielding alanine and sulfoacetaldehyde. We purified Tpa 72-fold to apparent homogeneity with an overall yield of 89%. The purified enzyme did not require addition of pyridoxal 5 0 -phosphate, but highly active enzyme was only obtained by addition of pyridoxal 5 0 -phosphate to all buffers during purification. SDS/PAGE revealed a single protein band with a molecular mass of 51 kDa. The apparent molecular mass of the native enzyme was 197 kDa as determined by gel filtration, which indicates a homotetrameric structure. The kinetic constants for taurine were: Kma 7.1 mm, Vmaxa 1.20 nmol·s 21 , and for pyruvate: Kma 0.82 mm, Vmaxa 0.17 nmol·s 21 . The purified enzyme was able to transaminate hypotaurine (2-aminosulfinate), taurine, b-alanine and with low activity cysteine and 3-aminopropanesulfonate. In addition to pyruvate, 2-ketobutyrate and oxaloacetate were utilized as amino group acceptors. We have sequenced the encoding gene (tpa). It encoded a 50-kDa peptide, which revealed 33% identity to diaminopelargonate aminotransferase from Bacillus subtilis.
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purification properties and primary structure of alanine dehydrogenase involved in taurine metabolism in the anaerobe Bilophila wadsworthia
Archives of Microbiology, 2000Co-Authors: Heike Laue, Alasdair M CookAbstract:Alanine dehydrogenase [l-alanine:NAD+ oxidoreductase (deaminating), EC 1.4.1.4.] catalyses the reversible oxidative deamination of l-alanine to pyruvate and, in the anaerobic bacterium Bilophila wadsworthia RZATAU, it is involved in the degradation of taurine (2-aminoethanesulfonate). The enzyme regenerates the amino-group acceptor pyruvate, which is consumed during the transamination of taurine and liberates ammonia, which is one of the degradation end products. Alanine dehydrogenase seems to be induced during growth with taurine. The enzyme was purified about 24-fold to apparent homogeneity in a three-step purification. SDS-PAGE revealed a single protein band with a molecular mass of 42 kDa. The apparent molecular mass of the native enzyme was 273 kDa, as determined by gel filtration chromatography, suggesting a homo-hexameric structure. The N-terminal amino acid sequence was determined. The pH optimum was pH 9.0 for reductive amination of pyruvate and pH 9.0–11.5 for oxidative deamination of alanine. The apparent K m values for alanine, NAD+, pyruvate, ammonia and NADH were 1.6, 0.15, 1.1, 31 and 0.04 mM, respectively. The alanine dehydrogenase gene was sequenced. The deduced amino acid sequence corresponded to a size of 39.9 kDa and was very similar to that of the alanine dehydrogenase from Bacillus subtilis.
Ellen Jo Baron - One of the best experts on this subject based on the ideXlab platform.
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Bilophila wadsworthia a unique gram negative anaerobic rod
Anaerobe, 1997Co-Authors: Ellen Jo BaronAbstract:Abstract Although comprising less than 0·01% of the normal human gastrointestinal microbiota,Bilophila wadsworthiais the third most common anaerobe recovered from clinical material obtained from patients with perforated and gangrenous appendicitis. Since its discovery in 1988,B. wadsworthiahas been recovered from clinical specimens associated with a variety of infections, including sepsis, liver abscesses, cholecystitis, Fournier's gangrene, soft tissue abscesses, empyema, osteomyelitis, Bartholinitis, and hidradenitis suppurativa. In addition, it has been found in the saliva and vaginal fluids of asymptomatic adults and even in the periodontal pockets of dogs. The organism is asaccharolytic, fastidious, and is easily recognized by its strong catalase reaction with 15% H2O2, production of hydrogen sulfide, and growth stimulation by bile (oxgall) and pyruvate. Approximately 75% of strains are urease positive. When grown on pyruvate-containing media, > 85% of strains demonstrate β-lactamase production. Ribosomal RNA-based phylogenetic studies showBilophilato be a homogeneous species, most closely related toDesulfovibriospecies. Both adherence to human cells and endotoxin have been observed, and preliminary work suggests that environmental iron has a role in expression of outer membrane proteins. Penicillin-binding proteins appear to mediate the organism's susceptibility to at least some β-lactam agents, which induce spheroplast formation that results in a haze of growth on agar dilution susceptibility test plates which is difficult to interpret.Bilophilastrains are inhibitedin vitroby most antibiotics.
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Effect of iron depletion on protein profiles of Bilophila wadsworthia
Clinical Infectious Diseases, 1995Co-Authors: Catherine Daniel, Ellen Jo Baron, René CourcolAbstract:Pathogenicity of microorganisms is a multifactorial phenomenon. Host environment plays a role in the development of the infectious process. The presence of key nutrients, such as iron, may be important for the expression of virulence factors. Iron is an essential component of a wide variety of biochemical processes in microorganisms, and its availability is of major importance in bacterial pathogenesis. In response to a low level of available iron in vivo, the creation of high-affinity iron-uptake systems and of a number of iron-regulated membrane proteins is induced. Although relationships between iron and virulence factors of aerobes have been better studied, a few such studies have been carried out with regard to anaerobes (mainly Bacteroides fragilis) [ 1, 2]. As discussed by Duerden [3], there is as yet no clear understanding of many of the virulence factors of anaerobes. Little is known about the mechanisms of iron uptake used by anaerobic bacteria, and nothing about those of Bilophila wadsworthia. The purpose of this work was to study the properties of this recently recognized anaerobe in relation to iron metabolism. B. wadsworthia is an anaerobic, gram-negative, asaccharolytic, bileresistant, and strongly catalase-positive bacillus [4]. This bacterium was first isolated from patients with gangrenous and perforative appendicitis. This study sought to determine whether B. wadsworthia expressed iron-regulated outer-membrane proteins (OMPs) in an iron-depleted environment and whether these proteins were immunogenic. Iron-free reagents and materials were used to prepare the ironfree medium. In this work, all glassware was deferrated. Chemicals used to prepare the medium were of highest purity. The medium was one previously described, to which oxgall (2%) and pyruvate (10 mM) were added [5]. The medium components were deferrated with Chelex-100 resin (Bio-Rad Laboratories, Hercules, CA), except for MgSO4, amino acids, and vitamins. Medium without iron contained
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effect of iron depletion on protein profiles of Bilophila wadsworthia
Clinical Infectious Diseases, 1995Co-Authors: Catherine Daniel, Ellen Jo Baron, René CourcolAbstract:Pathogenicity of microorganisms is a multifactorial phenomenon. Host environment plays a role in the development of the infectious process. The presence of key nutrients, such as iron, may be important for the expression of virulence factors. Iron is an essential component of a wide variety of biochemical processes in microorganisms, and its availability is of major importance in bacterial pathogenesis. In response to a low level of available iron in vivo, the creation of high-affinity iron-uptake systems and of a number of iron-regulated membrane proteins is induced. Although relationships between iron and virulence factors of aerobes have been better studied, a few such studies have been carried out with regard to anaerobes (mainly Bacteroides fragilis) [ 1, 2]. As discussed by Duerden [3], there is as yet no clear understanding of many of the virulence factors of anaerobes. Little is known about the mechanisms of iron uptake used by anaerobic bacteria, and nothing about those of Bilophila wadsworthia. The purpose of this work was to study the properties of this recently recognized anaerobe in relation to iron metabolism. B. wadsworthia is an anaerobic, gram-negative, asaccharolytic, bileresistant, and strongly catalase-positive bacillus [4]. This bacterium was first isolated from patients with gangrenous and perforative appendicitis. This study sought to determine whether B. wadsworthia expressed iron-regulated outer-membrane proteins (OMPs) in an iron-depleted environment and whether these proteins were immunogenic. Iron-free reagents and materials were used to prepare the ironfree medium. In this work, all glassware was deferrated. Chemicals used to prepare the medium were of highest purity. The medium was one previously described, to which oxgall (2%) and pyruvate (10 mM) were added [5]. The medium components were deferrated with Chelex-100 resin (Bio-Rad Laboratories, Hercules, CA), except for MgSO4, amino acids, and vitamins. Medium without iron contained <0.04 tM Fe+++. Iron was added to a concentration of 100 iM in preparation of the ironcontaining medium. The final pH was adjusted to 7.0-7.1. Four strains of B. wadsworthia originally isolated from appendicitis case specimens were studied, including the reference strain ATCC 49260. They were all incubated for 14 days in 200-mL flask cultures (in media with and without iron) at 37?C in an anaerobic atmosphere. Detection of siderophores in culture supernatants was attempted by means of the method described by Schwyn and Neilands [6]. Preparation of the outer membrane was accomplished according to the method described by Sprott et al. [7]. Cells harvested by centrifugation were disrupted ultrasonically. Unbroken cells were discarded by centrifugation. The supernatant was treated with 2% Triton
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bactericidal activity of selected antimicrobial agents against Bilophila wadsworthia and bacteroides gracilis
Clinical Infectious Diseases, 1993Co-Authors: Ellen Jo Baron, G Ropers, P Summanen, René CourcolAbstract:Antimicrobial agents tested were ampicillin/sulbactam (final concentrations, 16/8 tug/mL), ticarcillin/clavulanate (128/2 gg/mL), imipenem (8 gg/mL), cefoxitin (32 ltg/mL), chloramphenicol (16 ,g/mL), clindamycin (4 ,g/mL), and metronidazole (16 ,g/mL). Although all antimicrobial agents tested inhibited growth of all Bilophila strains during the first 24 hours, bactericidal activity was variable; only metronidazole was uniformly bactericidal. Most strains of Bilophila showed 1-2 log increases in growth at 6 hours with clindamycin and chloramphenicol. With chloramphenicol, some Bilophila strains tested showed regrowth starting at 30 hours. B. gracilis strains were generally more susceptible to all agents tested. Metronidazole, ticarcillin/ clavulanate, chloramphenicol, and imipenem were most active. Several strains of B. gracilis were not killed by ampicillin/sulbactam, clindamycin, or cefoxitin. Activity was variable among strains and antimicrobial agents. Bacteroides gracilis and Bilophila wadsworthia are asaccharolytic, nonmotile gram-negative bacilli that form part of the normal flora of the human oral cavity and gastrointestinal tract. Both species are involved in polymicrobic infections [1-3]. B. gracilis has recently been characterized as a microaerobic organism [4], although it originally was thought to be anaerobic and is usually grown under anaerobic conditions. B. wadsworthia requires strictly anaerobic conditions. These species are fastidious; they require supplementary factors for optimal growth in vitro and exhibit characteristic slow colony formation. For these reasons, results of conventional susceptibility tests, which rely on adequate growth in a standardized medium, are difficult to interpret when these species are assayed [5-8]. Results obtained with
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clinical importance of Bilophila wadsworthia
European Journal of Clinical Microbiology & Infectious Diseases, 1992Co-Authors: Sydney M Finegold, Paula H Summanen, Hunt S Gerardo, Ellen Jo BaronAbstract:Bilophila wadsworthia is an anaerobic, gram-negative, asaccharolytic, urease-positive, bile-resistant, catalase-positive bacillus, originally recovered from infections in patients with gangrenous and perforated appendicitis. Additional isolations from clinical specimens, including pleural fluid, joint fluid, blood and pus from a scrotal abscess, mandibular osteomyelitis and axillary hidradenitis suppurativa are described here.Bilophila is found as normal flora in feces and, occasionally, in saliva and in the vagina. Isolates from humans are usually β-lactamase positive and therefore resistant to certain β-lactam antibiotics. Two percent of strains are also resistant to clindamycin.
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identification of Bilophila wadsworthia by specific pcr which targets the taurine pyruvate aminotransferase gene
Fems Microbiology Letters, 2006Co-Authors: Heike Laue, M C Claros, Ulrike Schumacher, Theo H M Smits, Ralf Hartemink, Alasdair M CookAbstract:The bile-resistant, strictly anaerobic bacterium Bilophila wadsworthia is found in human faecal flora, in human infections and in environmental samples. A specific PCR primer set for the gene encoding the first metabolic enzyme in the degradative pathway for taurine in B. wadsworthia, taurine:pyruvate aminotransferase (tpa), was developed and tested. In addition, enrichment cultures were started from faecal samples of primates and felines and shown to contain B. wadsworthia. These were subcultured on agar media and then identified by PCR fingerprinting. PCR for tpa was successful in all positive enrichment cultures and showed no amplification signal in a variety of other bacterial species. Therefore, this PCR method could be a promising tool for rapid detection of B. wadsworthia in biological samples.
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characterization of Bilophila wadsworthia isolates using pcr fingerprinting
Anaerobe, 1999Co-Authors: M C Claros, Sydney M Finegold, Hunt S Gerardo, Ulrike Schumacher, M Jacob, N Kleinkauf, Ellie J C Goldstein, Arne C RodloffAbstract:Abstract Bilophila wadsworthia , an under-appreciated anaerobic organism, was originally described in 1989. Ninety-nine Bilophila wadsworthia isolates, recovered form environmental and clinical specimens in Germany and in Southern California, were examined in this study. Many isolates were recovered in mixed culture with facultative aerobic and other anaerobic bacteria. All isolates were identified by standard laboratory procedures, including gas–liquid chromatography (GLC). A PCR fingerprint assay was established to compare the profiles of clinical and environmental isolates to the type strain (ATCC 49260) and to an environmental (sewage) reference strain (DSM 11045, RZATAU) for intra-species differences. Two primers, one universal primer, M13 core, and one tDNA primer, T3B, were used individually to analyse the strains. Homogeneous PCR fingerprint profiles were found for the majority of strains using the M13 core primer; two PCR groups were determined with T3B, one matching the type strain and one matching the environmental reference strain (DSM 11045, RZATAU). Two urease negative strains, WAL 11470 (blood isolate from California) and TUB 754 (intra-abdominal isolate from Germany) formed unique PCR fingerprint profiles with each of these primers. These results were confirmed by PCR fingerprinting using the T3A primer. These latter results suggest a possible genetic diversity in B. wadsworthia .
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taurine reduction powers rapid growth of Bilophila wadsworthia a liquid minimal salts medium for clinical research
Anaerobe, 1999Co-Authors: Heike Laue, Ulrike Schumacher, Alasdair M CookAbstract:Abstract Clinical isolates of Bilophila wadsworthia grew rapidly (1–3 days) in the liquid taurine-minimal-salts medium developed for B. wadsworthia RZATAU, whereas growth on Bacteroides Bile Esculin Agar takes up to 1 week. Though rapid growth of B. wadsworthia was achieved, and no other pure cultures grew, the medium was not selective for the organism in human faeces or in intra-abdominal specimens. We hope, however, that our understanding of the physiological and biochemical characteristics of the organism supplies a tool for further research.
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susceptibility of the anaerobic gram negative non sporulating rod Bilophila wadsworthia to beta lactams beta lactamase inhibitors meropenem metronidaz
Zentralblatt Fur Bakteriologie-international Journal of Medical Microbiology Virology Parasitology and Infectious Diseases, 1998Co-Authors: Ulrike Schumacher, Bernd SingleAbstract:Summary The susceptibility of eighty-seven strains of Bilophila wadsworthia to five beta-lactams, two beta-lactamase inhibitors, meropenem, metronidazole, clindamycin and two quinolones was determined. Tests were performed by the modified reference agar dilution technique using triphenyltetrazolium chloride for endpoint reading. The test strains showed a reduced susceptibility to the beta-lactams, penicillin G (MIC90 4 μg/ml), ampicillin (MIC90 32 μg/ml), piperacillin (MIC90 64 μg/ml), cephalothin (MIC90 2 μg/ml and cefotaxim (MIC90 4 μg/ml). The activity of ampicillin was increased by addition of the beta-lactamase inhibitor, sulbactam (MIC90 2 μg/ml), as was the activity of piperacillin by the addition of tazobactam (MIC90 4 μg/ml) 90.8% of the strains were found to produce beta-lactamase by the nitrocefin tube method. All strains were shown to be highly susceptible to meropenem, metronidazole and clindamycin (MICs ≤ 1 μg/ml). Sparfloxacin (MIC90 1 μg/ml) and ciprofloxacin (MIC90 0.5 μg/ml) were found to be active against most of the strains tested.
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comparison of the etest and a microbroth dilution system sceptor to a reference agar dilution method for susceptibility testing of Bilophila wadsworthia
Clinical Microbiology and Infection, 1997Co-Authors: Ulrike SchumacherAbstract:Objective To compare the Etest and a microbroth dilution system (Sceptor) to a reference agar dilution method for susceptibility testing of Bilophila wadsworthia. Methods The susceptibility of 15 clinical isolates of Bilophila wadsworthia was determined by the National Committee for Clinical Laboratory Standards (NCCLS) agar dilution method using triphenyltetrazolium chloride for endpoint determination. The results were compared with the results obtained by the E test and a commercial microbroth dilution system (Sceptor). Results Comparison of the MICs obtained by the reference method and the Etest revealed few discrepancies, with piperacillin and metronidazole being the only exceptions. The overall agreement was 70% within one dilution step. The discrepancies did not result in major interpretative errors. The overall essential agreement using susceptibility categories was 98% for the E test and 99% for the microdilution system. Conclusions Both methods may be considered as acceptable alternatives for testing individual isolates of B. wadsworthia.
