A selective medium for Fusobacterium spp

A new selective medium (JVN) for the isolation of Fusobacterium spp. from clinical material is described. The medium incorporates josamycin, vancomycin and norfloxacin (at 3, 4 and 1 microgram/ml, respectively) as the selective agents, plus 5% defibrinated horse blood in Fastidious Anaerobe Agar Base (Lab M). This formula allowed luxuriant growth of all 82 strains (eight recognized species) of fusobacteria tested, while significantly inhibiting 51/51 (100%) strains of facultative anaerobes and 45/51 (88%) strains of other obligate anaerobes. JVN medium allowed the successful isolation of strains of Fusobacterium naviforme, F. nucleatum and F. necrophorum from the gingivae of 9/16 healthy volunteers, and strains of F. varium and F. mortiferum from faecal suspensions seeded with these organisms.     

A selective medium for Fusobacterium spp.

J.S. BRAZIER, D.M. CITRON AND E.J.C. GOLDSTEIN, 1991. A new selective medium (JVN) for the isolation of Fusobacterium spp. from clinical material is described. The medium incorporates josamycin, vancomycin and norfloxacin (at 3, 4 and 1 μg/ml, respectively) as the selective agents, plus 5% defibrinated horse blood in Fastidious Anaerobe Agar Base (Lab M). This formula allowed luxuriant growth of all 82 strains (eight recognized species) of fusobacteria tested, while significantly inhibiting 51/51 (100%) strains of facultative anaerobes and 45/51 (88%) strains of other obligate anaerobes. JVN medium allowed the successful isolation of strains of Fusobacterium naviforme, F. nucleatum and F. necrophorum from the gingivae of 9/16 healthy volunteers, and strains of F. varium and F. mortiferum from faecal suspensions seeded with these organisms.     

Pleomorphism of fusobacteria isolated from the cockroach hindgut.

Fusobacteria are commonly isolated from the hindgut of the cockroach Eublaberus posticus . Eleven strains isolated from E. posticus by us were keyed to four species, Fusobacterium necrophorum, F. varium , F. gonidiaformans , and F. prausnitzii , using current taxonomic criteria. With the exception of F. gonidiaformis , all species showed rods with swollen centers and large bodies. The pleomorphism of F. varium was examined by phase microscopy and scanning and transmission electron microscopy. The pleomorphic process begins with a gradual swelling at the center of the rod until a large round body is formed. Some of these round bodies then fragment, giving rise to rod-shaped cells. When 10% yeast extract was added to growth media, pleomorphism was not induced. A dialyzable factor was found to account for this observation. Fermentation of [1-14C]glutamic acid gives rise to butyrate labeled in the carboxyl carbon, indicating that butyrate is formed by the hydroxyglutarate pathway which may be characteristic for the genus Fusobacterium.     

Proteomic investigation of glucose metabolism in the butyrate-producing gut anaerobe Fusobacterium varium

A proteome survey and MS analysis were conducted to investigate glucose metabolism in Fusobacterium varium, a butyrate-producing constituent of the indigenous human gut microflora. The bacterium was capable of catabolizing glucose as the main energy source via the Embden-Meyerhof-Parnas pathway. 2-DE analyses revealed that the apparent concentrations of the six identified glycolytic enzymes (pyruvate kinase, enolase, glucose-6-phosphate isomerase, phosphoglycerate kinase, triosephosphate isomerase, and glyceraldehyde-3-phosphate dehydrogenase) were specifically increased in response to the presence of glucose in the chemically defined minimal growth medium, and did not diminish when the medium was additionally supplemented with L-glutamate, an amino acid readily fermented by members of the Fusobacterium genus. A substrate pool depletion study revealed that the sugar, and not the amino acid, is the more efficient growth substrate. Both proteomics and substrate pool depletion studies revealed that F. varium can simultaneously utilize both glucose and L-glutamate as energy sources. Enzymes involved in L-glutamate metabolism were also identified, including an NAD-dependent glutamate dehydrogenase and two enzymes of the methylaspartate pathway of L-glutamate catabolism (glutamate mutase and methylaspartate ammonia-lyase). Their apparent intracellular concentrations were elevated when the bacterium was cultured in media supplemented with excess L-glutamate. Our observation that the apparent concentrations of specific proteins were elevated in response to a particular growth substrate supplied as an energy source provides the first evidence for the presence of a nutrient-responsive mechanism governing intracellular protein concentration in F. varium.     

Metabolic footprinting of the anaerobic bacterium Fusobacterium varium using 1H NMR spectroscopy

Metabolic footprinting of the anaerobic bacterium Fusobacterium varium demonstrated the accumulation of six carboxylic acids as metabolic end-products and revealed specific growth requirements and utilization capabilities towards amino acids. Guided by (1)H NMR determinations of residual amino acids in spent medium, a modified chemically defined minimal medium (CDMM*) was developed by minimizing the amino acid composition while satisfying nutritional requirements to support abundant growth of F. varium. Quantitative determinations of carboxylate salts and residual substrates were readily performed by (1)H NMR analysis of lyophilized residues from CDMM* cultures without interference from initial medium components. Only small concentrations of alanine, arginine, glycine, isoleucine, leucine, methionine, proline and valine were required to support growth of F. varium, whereas larger quantities of aspartate, asparagine, cysteine, glutamine, glutamate, histidine, lysine, serine and threonine were utilized, most likely as energy sources. Both bacterial growth and the distribution of carboxylate end-products depended on the composition of the chemically defined medium. In cultures provided with glucose as the primary energy source, the accumulation of butyrate and lactate correlated with growth, consistent with the regeneration of reduced coenzyme formed by the oxidative steps of glucose catabolism.     

Intergeneric protoplast fusion between Fusobacterium varium and Enterococcus faecium for enhancing dehydrodivanillin degradation.

Intergeneric protoplast fusion between Fusobacterium varium (Pcs Glu+) and Enterococcus faecium (Pcr Glu-) was performed under strictly anaerobic conditions to improve dehydrodivanillin (DDV) degradation. The fusion frequency obtained from the selective medium (Pc+ Glu-) was about 0.9 X 10(-5) to 1.3 X 10(-5). The seven fusants isolated were all gram-negative anaerobes with rod shapes like that of F. varium and with main phenotypical properties of cocci like those of E. faecium such as esculin and starch hydrolysis, milk clotting, and lactate production. Five fusants showed enhanced DDV degradation activities that were 2 to 4 times higher than those of parental strains. Genetic relatedness between a fusant (FE7) and the parents was estimated by DNA-DNA Southern blot hybridization with 32P-labeled chromosomal DNA fragments of F. varium and E. faecium as respective probes. The fusant FE7 presented a very high cross-hybridization with both probes, indicating a high DNA homology between the fusant and both parental strains. Almost all the fusants obtained here have stably kept the properties described above for about 2 years. These results suggest that intergeneric gene transfer takes place through protoplast fusion and that the fusants that were obtained are stable recombinants.     

Proteomic investigation of amino acid catabolism in the indigenous gut anaerobe Fusobacterium varium

The butyrate-producing anaerobe Fusobacterium varium is an integral constituent of human gut microflora. Unlike many gut microorganisms, F. varium is capable of fermenting both amino acids and glucose. Although F. varium has been implicated in beneficial as well as pathological bacterium-host interactions, its genome has not been sequenced. To obtain a better understanding of the metabolic processes associated with amino acid fermentation by F. varium, we used a gel-based proteomic approach to examine the changes in the soluble proteome accompanying the utilization of eight different growth substrates: glucose, L- and D-glutamate, L-histidine, L- and D-lysine, and L- and D-serine. Using LC-MS/MS to analyze approximately 25% of the detected protein spots, we were able to identify 47 distinct proteins. While the intracellular concentrations of enzymes characteristic of a catabolic pathway for a specific amino acid were selectively increased in response to the presence of an excess of that amino acid in the growth medium, the concentrations of the core acetate-butyrate pathway enzymes remained relatively constant. Our analysis revealed (i) high intracellular concentrations of glutamate mutase and beta-methylaspartate ammonia-lyase under all growth conditions, underscoring the importance of the methylaspartate pathway of glutamate catabolism in F. varium (ii) the presence of two enzymes of the hydroxyglutarate pathway of glutamate degradation in the proteome of F. varium ((R)-2-hydroxyglutaryl-CoA dehydratase and NAD-specific glutamate dehydrogenase) specifically when L-glutamate was the main energy source (iii) the presence of genes in the genome of F. varium encoding each of the enzymes of the hydroxyglutarate pathway (iv) the presence of both L- and D-serine ammonia-lyases (dehydratases) which permit F. varium to thrive on either L- or D-serine, respectively, and (v) the presence of aspartate-semialdehyde dehydrogenase and dihydrodipicolinate synthase, consistent with the ability of F. varium to synthesize meso-2,6-diaminopimelic acid as a component of its peptidoglycan. Proteins involved in other cellular processes, including oxidation-reduction reactions, protein synthesis and turnover, and transport were also identified.     

Intrageneric relationships of members of the genus Fusobacterium as determined by reverse transcriptase sequencing of small-subunit rRNA.

The phylogenetic interrelationships of 14 members of the genus Fusobacterium were investigated by performing a comparative analysis of the 16S rRNA sequences of these organisms. The sequence data revealed considerable intrageneric heterogeneity. The four species Fusobacterium nucleatum (including F. nucleatum subsp. nucleatum, F. nucleatum subsp. polymorphum, "F. nucleatum subsp. fusiforme," and "F. nucleatum subsp. animalis"), Fusobacterium alocis, Fusobacterium periodonticum, and Fusobacterium simiae, which colonize oral cavities, exhibited high levels of sequence homology with each other and formed a distinct group within the genus. Fusobacterium mortiferum, Fusobacterium varium, and Fusobacterium ulcerans also formed a phylogenetically coherent group, as did the two species Fusobacterium gonidiaformans and Fusobacterium necrophorum. Fusobacterium russii and Fusobacterium necrogenes displayed no specific relationship with any of the other fusobacteria. The sequence data are discussed in the context of previous physiological and chemical findings.     

Intergeneric protoplast fusion between Fusobacterium varium and Enterococcus faecium for enhancing dehydrodivanillin degradation

Intergeneric protoplast fusion between Fusobacterium varium (Pcs Glu+) and Enterococcus faecium (Pcr Glu-) was performed under strictly anaerobic conditions to improve dehydrodivanillin (DDV) degradation. The fusion frequency obtained from the selective medium (Pc+ Glu-) was about 0.9 X 10(-5) to 1.3 X 10(-5). The seven fusants isolated were all gram-negative anaerobes with rod shapes like that of F. varium and with main phenotypical properties of cocci like those of E. faecium such as esculin and starch hydrolysis, milk clotting, and lactate production. Five fusants showed enhanced DDV degradation activities that were 2 to 4 times higher than those of parental strains. Genetic relatedness between a fusant (FE7) and the parents was estimated by DNA-DNA Southern blot hybridization with 32P-labeled chromosomal DNA fragments of F. varium and E. faecium as respective probes. The fusant FE7 presented a very high cross-hybridization with both probes, indicating a high DNA homology between the fusant and both parental strains. Almost all the fusants obtained here have stably kept the properties described above for about 2 years. These results suggest that intergeneric gene transfer takes place through protoplast fusion and that the fusants that were obtained are stable recombinants.     

Protoplast formation and regeneration of dehydrodivanillin-degrading strains of Fusobacterium varium and Enterococcus faecium.

Two strains of rumen anaerobes isolated from dehydrodivanillin-degrading cultures were identified as Fusobacterium varium and Enterococcus faecium. These organisms degraded dehydrodivanillin synergistically to 5-carboxymethylvanillin and vanillic acid. Specific conditions for protoplast formation and cell wall regeneration for both bacteria were determined, under strictly anaerobic conditions, to be as follows. The cell wall of each bacterium in yeast extract medium was loosened by adding penicillin G during early log-phase growth. The cell wall of F. varium was lysed by lysozyme (1 mg/ml) in glycerol (0.2 M)-phosphate buffer (0.05 M; pH 7.0). The addition of NaCl (0.08 M) with lysozyme was necessary for lysis of E. faecium in this solution. Almost all cells were converted to protoplasts after 2 h of incubation at 37 degrees C. Regeneration of both protoplasts was 20 to 30% on an agar-containing yeast extract medium.     

Protoplast formation and regeneration of dehydrodivanillin-degrading strains of Fusobacterium varium and Enterococcus faecium

Two strains of rumen anaerobes isolated from dehydrodivanillin-degrading cultures were identified as Fusobacterium varium and Enterococcus faecium. These organisms degraded dehydrodivanillin synergistically to 5-carboxymethylvanillin and vanillic acid. Specific conditions for protoplast formation and cell wall regeneration for both bacteria were determined, under strictly anaerobic conditions, to be as follows. The cell wall of each bacterium in yeast extract medium was loosened by adding penicillin G during early log-phase growth. The cell wall of F. varium was lysed by lysozyme (1 mg/ml) in glycerol (0.2 M)-phosphate buffer (0.05 M; pH 7.0). The addition of NaCl (0.08 M) with lysozyme was necessary for lysis of E. faecium in this solution. Almost all cells were converted to protoplasts after 2 h of incubation at 37 degrees C. Regeneration of both protoplasts was 20 to 30% on an agar-containing yeast extract medium.     

Pathways of glutamate catabolism among Fusobacterium species.

Glutamate is a major source of energy for Fusobacterium species but its mode of catabolism has not hitherto been elucidated. Cell suspensions of F. nucleatum and F. varium, as representative species from the oral cavity and gastrointestinal tract, respectively, both decarboxylated position-labelled glutamate but by different pathways. 14CO2 was released only from C-5 by F. nucleatum whereas F. varium decarboxylated glutamate at either C-1 or C-5. In both species, 2 mols of glutamate fermented yielded 2 mols of acetate and 1 mol of butyrate, suggesting the possibility of three metabolic pathways: the 2-oxoglutarate, mesaconate and 4-aminobutyrate pathways. Enzymes representative of the three pathways were assayed for in cell-free extracts of fusobacteria. All species tested possessed high levels of both glutamate dehydrogenase and 2-oxoglutarate reductase, indicating the presence of the 2-oxoglutarate pathway. Enzymes representative of the mesaconate pathway were detected in F. sulci, F. ulcerans, F. mortiferum and F. varium, while the latter two species also possessed the 4-aminobutyrate pathway. The pathways of glutamate catabolism therefore bore no relationship to the site of isolation of the fusobacteria tested but instead correlated with their chemotaxonomic properties. Thus, F. varium, F. mortiferum, F. ulcerans and F. sulci, which possess a peptidoglycan structure based on diaminopimelic acid, have either two or three pathways for glutamate catabolism whereas F. nucleatum and other species that have a lanthionine-based murein metabolized glutamate solely by the 2-oxoglutarate pathway.     

Glutamate racemization and catabolism in Fusobacterium varium

The pathways of glutamate catabolism in the anaerobic bacterium Fusobacterium varium, grown on complex, undefined medium and chemically defined, minimal medium, were investigated using specifically labelled (13)C-glutamate. The metabolic end-products acetate and butyrate were isolated from culture fluids and derivatized for analysis by nuclear magnetic resonance and mass spectrometry. On complex medium, labels from L-[1-(13)C]glutamate and L-[4-(13)C]glutamate were incorporated into C1 of acetate and equally into C1/C3 of butyrate, while label derived from L-[5-(13)C]glutamate was not incorporated. The isotopic incorporation results and the detection of glutamate mutase and 3-methylaspartate ammonia lyase in cell extracts are most consistent with the methylaspartate pathway, the best known route of glutamate catabolism in Clostridium species. When F. varium was grown on defined medium, label from L-[4-(13)C]glutamate was incorporated mainly into C4 of butyrate, demonstrating a major role for the hydroxyglutarate pathway. Upon addition of coenzyme B(12) or cobalt ion to the defined medium in replicate experiments, isotope was located equally at C1/C3 of butyrate in accord with the methylaspartate pathway. Racemization of D-glutamate and subsequent degradation of L-glutamate via the methylaspartate pathway are supported by incorporation of label into C2 of acetate and equally into C2/C4 of butyrate from D-[3-(13)C]glutamate and the detection of a cofactor-independent glutamate racemase in cell extracts. Together the results demonstrate a major role for the methylaspartate pathway of glutamate catabolism in F. varium and substantial participation of the hydroxyglutarate pathway when coenzyme B(12) is not available.     

Seroprevalence of Fusobacterium varium in ulcerative colitis patients in Japan

The etiology of ulcerative colitis (UC) is unknown, while an exacerbating factor of this disease is associated with infectious agents. Recently, Fusobacterium varium has been found in the mucosa of a significant number of patients with UC. The aim of this study was to estimate the prevalence of F. varium infection based on serology, evaluate the relationship between F. varium seropositivity and UC, and determine the clinical characteristics of infected UC individuals. Seropositive patients were determined by immunoblotting with F. varium ATCC 8501 antigen. We also identified cross-reactive protein spots by peptide mass mapping analysis. These protein spots showed putative caseinolytic protease protein, putative translation elongation factor G, and putative enolase. Immunoblotting with F. varium antigen revealed signals with sera from 45 (40.2%) of the 112 UC patients and 20 (15.6%) of the 128 healthy controls, respectively ( P <0.01). In terms of disease activity, seropositive UC patients were more likely to have clinically severe disease than seronegative UC patients. Disease location in seropositive patients was more extensive than the seronegative patients. In conclusion, F. varium is a predominant infection in the UC population and is a potential pathogen of UC.     

The effect of tube closures and media on the enrichment of Campylobacter jejuni

Enrichment of C. jejuni in metal, Morton-style capped tubes gave no growth. Cotton wool or sponge rubber plugged tubes yielded enrichment of C. jejuni to 1.6 × 10³ cfu per ml from an inoculation of 0.18 cfu per ml in 10 ml medium.Enrichment of C. jejuni from egg melange in cotton plugged tubes and bottles showed that the ratio of egg melange to broth should not exceed 4:1 in bottles and 2:3 in tubes. When enriching from incubating fertile eggs infected by C. jejuni a decreasing quantitative and qualitative recovery was experienced with increasing time of egg incubation. Five enrichment broths and two selective plating media were compared in this experiment. The medium of Doyle and Roman (Appl. Environ. Microbiol. 43, 1343–1353 (1982)) and a routine enrichment broth plus rifampin (brucella broth containing per litre: 50 ml lysed horse blood, 10 mg rifampin, 5000 IU polymyxin B, 10 mg vancomycin, 5 mg trimethoprim lactate and the reductants of George et al. (J. Clin. Microbiol. 8, 36–41 (1978)) were superior to BNP broth. All media showed decreased enrichment of C. jejuni with increasing time of egg incubation, when growth was only of the order of 2 × 10² cfu per ml at day 15 of egg incubation. Rifampin was required to suppress contamination by Proteus species and Gram-positive cocci.     

Growth and Metabolism of Lactic Acid Bacteria during and after Malolactic Fermentation of Wines at Different pH

A new medium, called novobiocin-brilliant green-glucose (NBG) agar, was developed for the isolation of Salmonella spp. and evaluated against other conventionally used media including bismuth sulfite, xylose-lysine decarboxylase, brilliant green-sulfa, hektoen enteric, and salmonella-shigella agars. NBG had recovery rates comparable to the other enteric media tested with pure cultures as well as with naturally contaminated amphibian and reptile waters and fecal specimens. However, NBG, hektoen enteric, and salmonella-shigella agars failed to differentiate Salmonella typhi from a fecal specimen even after enrichment in selenite F. Although Citrobacter freundii could grow and resembled salmonellae on NBG, at no time was the recovery of Salmonella spp. colonies jeopardized by the presence of C. freundii in either seeded or naturally contaminated samples. Confirmation rates of typical colonies from NBG agar also compared favorably to the other media tested; however, bismuth sulfite, although selective, was found to have varied differential characteristics for Salmonella spp. As a result, many more colonies had to be picked, which caused bismuth sulfite agar to have the lowest confirmation rate of the media tested. The distinct advantage that NBG agar offers over the conventional method tested, including bismuth sulfite, is the consistent differential reaction of all Salmonella subgroups including biochemically atypical strains. The medium is inexpensive, easy to prepare, and can be stored for at least 2 weeks at 4 degrees C without loss of selective or differential properties.     

Growth and Metabolism of Lactic Acid Bacteria during and after Malolactic Fermentation of Wines at Different pH

A new medium, called novobiocin-brilliant green-glucose (NBG) agar, was developed for the isolation of Salmonella spp. and evaluated against other conventionally used media including bismuth sulfite, xylose-lysine decarboxylase, brilliant green-sulfa, hektoen enteric, and salmonella-shigella agars. NBG had recovery rates comparable to the other enteric media tested with pure cultures as well as with naturally contaminated amphibian and reptile waters and fecal specimens. However, NBG, hektoen enteric, and salmonella-shigella agars failed to differentiate Salmonella typhi from a fecal specimen even after enrichment in selenite F. Although Citrobacter freundii could grow and resembled salmonellae on NBG, at no time was the recovery of Salmonella spp. colonies jeopardized by the presence of C. freundii in either seeded or naturally contaminated samples. Confirmation rates of typical colonies from NBG agar also compared favorably to the other media tested; however, bismuth sulfite, although selective, was found to have varied differential characteristics for Salmonella spp. As a result, many more colonies had to be picked, which caused bismuth sulfite agar to have the lowest confirmation rate of the media tested. The distinct advantage that NBG agar offers over the conventional method tested, including bismuth sulfite, is the consistent differential reaction of all Salmonella subgroups including biochemically atypical strains. The medium is inexpensive, easy to prepare, and can be stored for at least 2 weeks at 4 degrees C without loss of selective or differential properties.     

Identification of fusobacteria in a routine diagnostic laboratory

A scheme for differentiating Fusobacterium spp. and Leptotrichia spp. from Bac-teroides spp, was devised after examining 114 strains of fusobacteria and asac-charolytic bacteroides (17 reference strains and 97 clinical isolates). Sensitivity to a 300 μg/ml plate of phosphomycin and an acid reaction on a lysine plate were found to be reliable for differentiating Fusobacterium spp. and L. buccalis from Bacteroides Using a short set of simple cultural and biochemical tests, isolates could be identified as F. necrophorum, F. necrogenes, F. nucleatum, F. varium or L. buccalis . These tests were: indole, lecithinase, phosphatase, DNase and gas production, aesculin and casein hydrolysis, greening of casein/methylene blue agar, nitrite reduction, bile tolerance and haemolysis on horse blood agar.     

Identification of fusobacteria in a routine diagnostic laboratory

A scheme for differentiating Fusobacterium spp. and Leptotrichia spp. from Bacteroides spp. was devised after examining 114 strains of fusobacteria and asaccharolytic bacteroides (17 reference strains and 97 clinical isolates). Sensitivity to a 300 micrograms/ml plate of phosphomycin and an acid reaction on a lysine plate were found to be reliable for differentiating Fusobacterium spp. and L. buccalis from Bacteroides. Using a short set of simple cultural and biochemical tests, isolates could be identified as F. necrophorum, F. necrogenes, F. nucleatum, F. varium or L. buccalis. These tests were: indole, lecithinase, phosphatase, DNase and gas production, aesculin and casein hydrolysis, greening of casein/methylene blue agar, nitrite reduction, bile tolerance and haemolysis on horse blood agar.     

Development of a selective medium for isolation of Helicobacter pylori from cattle and beef samples

Helicobacter pylori has been isolated from the human stomach with media containing only minimal selective agents. However, current research on the transmission and sources of infection requires more selective media due to the higher numbers of contaminants in environmental, oral, and fecal samples. The objective of this study was to develop and evaluate detection techniques that are sufficiently selective to isolate H. pylori from potential animal and food sources. Since H. pylori survives in the acidic environment of the stomach, low pH with added urea was studied as a potential selective combination. H. pylori grew fairly well on H. pylori Special Peptone plating medium supplemented with 10 mM urea at pH 4. 5, but this pH did not sufficiently inhibit the growth of contaminants. Various antibiotic combinations were then compared, and a combination consisting of 10 mg of vancomycin per liter, 5 mg of amphotericin B per liter, 10 mg of cefsulodin per liter, 62,000 IU of polymyxin B sulfate per liter, 40 mg of trimethoprim per liter, and 20 mg of sulfamethoxazole per liter proved to be highly selective but still allowed robust colonies of H. pylori to grow. This medium was highly selective for recovering H. pylori from cattle and beef samples, and it is possible that it could be used to enhance the recovery of this bacterium from human and environmental samples, which may be contaminated with large numbers of competing microorganisms.