Cytochrome composition and oxygen-dependent respiration-driven proton translocation in Wolinella curva, Wolinella recta, Bacteroides ureolyticus, and Bacteroides gracilis.

The membrane fractions of the microaerobically grown type strains of Wolinella curva, Wolinella recta, Bacteroides ureolyticus, and Bacteroides gracilis contained membrane-bound cytochrome b, cytochrome c, and CO-binding cytochrome c. Soluble cytochrome c and CO-binding cytochrome c were also present. Although B. gracilis is oxidase negative, it possessed cytochrome c. With H2 or formate as the electron donor, proton efflux from anaerobic cells occurred upon addition of a pulse of oxygen. With formate as the electron donor, the H+/O ratios of W. curva, W. recta, B. ureolyticus, and B. gracilis were 0.75, 1.66, 2.06, and 2.04, respectively. With H2 as the electron donor, the H+/O ratios of W. curva, B. ureolyticus, and B. gracilis were 1.25, 1.97, and 2.36, respectively. Proton translocation was inhibited by the protonophore carbonylcyanide m-chlorophenylhydrazone. The results confirm that the organisms are not anaerobes but are microaerophiles capable of respiring with oxygen.     

Growth of Bacteroides fragilis group in agar and broth media

The rate of bacterial growth of four Bacteroides fragilis group organisms was determined in agar and broth media. Exponential bacterial growth occurred in agar media within 4 to 8 h, while such growth was delayed in broth media and occurred within 12-24 h after inoculation. This phenomenon may explain why antimicrobials which manifest an 'inoculum effect' may show increased resistance to antimicrobials when tested in agar media.     

Revision of Campylobacter, Helicobacter, and Wolinella taxonomy: emendation of generic descriptions and proposal of Arcobacter gen. nov.

Hybridization experiments were carried out between DNAs from more than 70 strains of Campylobacter spp. and related taxa and either 3H-labeled 23S rRNAs from reference strains belonging to Campylobacter fetus, Campylobacter concisus, Campylobacter sputorum, Campylobacter coli, and Campylobacter nitrofigilis, an unnamed Campylobacter sp. strain, and a Wolinella succinogenes strain or 3H- or 14C-labeled 23S rRNAs from 13 gram-negative reference strains. An immunotyping analysis of 130 antigens versus 34 antisera of campylobacters and related taxa was also performed. We found that all of the named campylobacters and related taxa belong to the same phylogenetic group, which we name rRNA superfamily VI and which is far removed from the gram-negative bacteria allocated to the five rRNA superfamilies sensu De Ley. There is a high degree of heterogeneity within this rRNA superfamily. Organisms belonging to rRNA superfamily VI should be reclassified in several genera. We propose that the emended genus Campylobacter should be limited to Campylobacter fetus, Campylobacter hyointestinalis, Campylobacter concisus, Campylobacter mucosalis, Campylobacter sputorum, Campylobacter jejuni, Campylobacter coli, Campylobacter lari, and "Campylobacter upsaliensis." Wolinella curva and Wolinella recta are transferred to the genus Campylobacter as Campylobacter curvus comb. nov. and Campylobacter rectus comb. nov., respectively. Bacteroides gracilis and Bacteroides ureolyticus are generically misnamed and are closely related to the genus Campylobacter. Campylobacter nitrofigilis, Campylobacter cryaerophila, and an unnamed Campylobacter sp. strain constitute a new genus, for which the name Arcobacter is proposed; this genus contains two species, Arcobacter nitrofigilis comb. nov. (type species) and Arcobacter cryaerophilus comb. nov. Wolinella succinogenes so far is the only species of the genus Wolinella. The genus Helicobacter is also emended; Campylobacter cinaedi and Campylobacter fennelliae are included in this genus as Helicobacter cinaedi comb. nov. and Helicobacter fennelliae comb. nov., respectively. The genus "Flexispira," with "Flexispira rappini" as the only species, is closely related to the genus Helicobacter. The free-living, sulfur-reducing campylobacters do not belong to any of these genera; they probably constitute a distinct genus within rRNA superfamily VI.     

A note on ultra-violet red fluorescence of anaerobic bacteria in vitro

Anaerobes other than the Bacteroides melaninogenicus group isolated from clinical material produce an ultra-violet red fluorescence when grown under certain conditions in vitro. These organisms include other members of the genus Bacteroides as well as strains of some species of Clostridium, Bifidobacterium and Actinomyces. The major fluorescent pigment was identified as protoporphyrin IX. Factors necessary for the production of fluorescence are the presence of blood or haem and a fermentable carbohydrate during growth on a solid medium. Fluorescence intensity was related to the concentration of blood and fermentable carbohydrate present but was independant of inoculum size. Certain commercially available blood agar bases designed specifically for the isolation of fastidious anaerobes from clinical material which contain added carbohydrate were shown to induce fluorescence in certain organisms. This may lead to the misidentification of some anaerobes as B. melaninogenicus.     

A note on ultra-violet red fluorescence of anaerobic bacteria in vitro

Anaerobes other than the Bacteroides melaninogenicus group isolated from clinical material produce an ultra-violet red fluorescence when grown under certain conditions in vitro. These organisms include other members of the genus Bacteroides as well as strains of some species of Clostridium, Bifidobacterium and Actinomyces. The major fluorescent pigment was identified as protoporphyrin IX. Factors necessary for the production of fluorescence are the presence of blood or haem and a fermentable carbohydrate during growth on a solid medium. Fluorescence intensity was related to the concentration of blood and fermentable carbohydrate present but was independent of inoculum size. Certain commercially available blood agar bases designed specifically for the isolation of fastidious anaerobes from clinical material which contain added carbohydrate were shown to induce fluorescence in certain organisms. This may lead to the misidentification of some anaerobes as B. melaninogenicus.     

Characterization of Bacteroides Species Isolated From Soft Tissue Infections in Cats

A total of 77 strains of Gram negative anaerobes belonging to the genus Bacteroides and isolated from 60 subcutaneous abscesses and 10 cases of pyothorax in cats, have been examined morphologically and biochemically. Colony pigmentation, gas chromatography and biochemical analysis placed them into two major categories-those which produced pigmented colonies (Group 1) and those which failed to produce pigmented colonies after 14 d on laked blood agar (Group 2). All 29 strains in Group 1 produced acetic, propionic, isobutyric, butyric, isovaleric and succinic acids but failed to ferment carbohydrates, and were classified as B. asaccharolyticus. All organisms in Group 2 produced acetic, propionic, isobutyric, isovaleric and succinic acids and were divided into four categories based on indole production and bile tolerance. Designation to species was then decided on the basis of phenylacetic acid production and sugar fermentation tests. This sequence of analysis of results enabled confident speciation of some groups of these organisms despite some biochemical variation of the cat strains when compared to human type strains.     

Growth of Bacteraides fragilis group in agar and broth media

B rook , I. 1990. Growth of Bacteroides fragilis group in agar and broth media. Journal of Applied Bacteriology 69 , 697–700.
The rate of bacterial growth of four . Bacteroides fragilis group organisms was determined in agar and broth media. Exponential bacterial growth occurred in agar media within 4 to 8 h, while such growth was delayed in broth media and occurred within 12–24 h after inoculation. This phenomenon may explain why antimicrobials which manifest an 'inoculum effect' may show increased resistance to antimicrobials when tested in agar media.     

Detection of conjugal transfer systems in oral, black-pigmented Bacteroides spp.

Oral, black-pigmented Bacteroides spp. are important pathogens in oral anaerobic infections and dental disease. We detected conjugation systems in isolates of Bacteroides denticola and Bacteroides intermedius that transferred tetracycline resistance (Tetr) and penicillin resistance to Bacteroides buccae and to Bacteroides fragilis, an intestinal Bacteroides species. A cloned Tetr gene from B. fragilis hybridized to the transferable Tetr locus in the oral strains, indicating that genetic exchange occurs between these two groups of anaerobes.     

Re-assessment of phenotypic identifications of Bacteroides putredinis to Alistipes species using molecular methods

Alistipes (previously Bacteroides) are strictly anaerobic gram-negative rods that resemble the Bacteroides fragilis group in that most species are bile-resistant and indole-positive; however, they are only weakly saccharolytic and most species produce light brown pigment only on laked rabbit blood agar. In this retrospective study, we re-identified 18 organisms previously identified phenotypically as "Bacteroides putredinis-like", but that did not produce pigment on routine media. The strains were identified with 16S rDNA sequencing and pigment production was evaluated on several different culture media. Only 12/18 strains had molecular identifications of Alistipes species, while the remaining strains phylogenetically resembled Butyricimonas and Odoribacter spp. Pigment production was not a reliable test for those Alistipes strains that are described as pigment producers.     

Oxygen Sensitivity of Various Anaerobic Bacteria

Anaerobes differ in their sensitivity to oxygen, as two patterns were recognizable in the organisms included in this study. Strict anaerobes were species incapable of agar surface growth at pO(2) levels greater than 0.5%. Species that were found to be strict anaerobes were Treponema macrodentium, Treponema denticola, Treponema oralis n. sp., Clostridium haemolyticum, Selenomonas ruminatium, Butyrivibrio fibrisolvens, Succinivibrio dextrinosolvens, and Lachnospira multiparus. Moderate anaerobes would include those species capable of growth in the presence of oxygen levels as high as 2 to 8%. The moderate anaerobes could be exposed to room atmosphere for 60 to 90 min without appreciable loss of viability. Species considered as moderate anaerobes were Bacteroides fragilis, B. melaninogenicus, B. oralis, Fusobacteria nucleatum, Clostridium novyi type A, and Peptostreptococcus elsdenii. The recognition of at least two general types of anaerobes would seem to have practical import in regard to the primary isolation of anaerobes from source material.     

Further studies on some physical and biochemical characteristics of asaccharolytic pigmented Bacteroides of feline origin

S. COLLINGS AND D.N. LOVE. 1992. The ultrastructure of the appendages of 24 strains of asaccharolytic pigmented Bacteroides spp. of cats was studied by transmission electron microscopy. All strains examined by thin section showed abundant fimbriae, outer membrane vesicles and capsules. Negative staining showed fimbriae which varied from long, fine and wavy in Bact. salivosus and cat Group 2 to shorter, less abundant and thicker fimbriae in cat strains of Bact. gingivalis as well as type strains of Porphyromonas gingivalis and P. asaccharolytica. Capsular material was very thick amorphous in human P. gingivalis , cat strains of Bact. gingivalis and in P. assaccharolytica but fine and fibrillary in preparations of Bact. salivosus and cat Group 2 organisms. Wet india ink preparations showed a large capsule although those of Bact. salivosus and Group 2 appeared largest. Five-day Group 2 broth cultures featured a thick ropy growth consistent with a large accumulation of extracellular slime. Enzymatic activities of the 24 strains measured by API ZYM system as well as the conventional biochemical tests showed it was possible to differentiate reliably Bact. salivosus from the other cat strains of asaccharolytic pigmented Bacteroides species and from human P. gingivalis and P. endodontalis by a combination of these tests. These tests suggest that Bact. salivosus is unlikely to belong to the genus Prevotella. Its place within the genus Porphyromonas is still to be determined.     

Further studies on some physical and biochemical characteristics of asaccharolytic pigmented Bacteroides of feline origin.

The ultrastructure of the appendages of 24 strains of asaccharolytic pigmented Bacteroides spp. of cats was studied by transmission electron microscopy. All strains examined by thin section showed abundant fimbriae, outer membrane vesicles and capsules. Negative staining showed fimbriae which varied from long, fine and wavy in Bact. salivosus and cat Group 2 to shorter, less abundant and thicker fimbriae in cat strains of Bact. gingivalis as well as type strains of Porphyromonas gingivalis and P. asaccharolytica. Capsular material was very thick amorphous in human P. gingivalis, cat strains of Bact. gingivalis and in P. assaccharolytica but fine and fibrillary in preparations of Bact. salivosus and cat Group 2 organisms. Wet india ink preparations showed a large capsule although those of Bact. salivosus and Group 2 appeared largest. Five-day Group 2 broth cultures featured a thick ropy growth consistent with a large accumulation of extracellular slime. Enzymatic activities of the 24 strains measured by API ZYM system as well as the conventional biochemical tests showed it was possible to differentiate reliably Bact. salivosus from the other cat strains of asaccharolytic pigmented Bacteroides species and from human P. gingivalis and P. endodontalis by a combination of these tests. These tests suggest that Bact. salivosus is unlikely to belong to the genus Prevotella. Its place within the genus Porphyromonas is still to be determined.     

Biphasic culture system for rapid Campylobacter cultivation

We developed a biphasic culture system consisting of 4 ml of brucella agar (BA) and 6 ml of brucella broth (BB) in 25-cm2 tissue culture flasks, which were incubated in air (BB/BAa) or in a gas mixture of 5% O2, 10% CO2, and 85% N2 (BB/BAg). These media were also used with a supplement consisting of ferrous sulfate, sodium metabisulfite, and sodium pyruvate and incubated as above (FB/FAa and FB/FAg, respectively). Highly satisfactory growth of Campylobacter jejuni 301 was obtained with all medium-gas phase combinations provided that the number of viable cells in the inoculum was large (greater than or equal to 10(6)/ml). The use of FB/FAa permitted the inoculum to be reduced to 100 cells per ml. With an adjusted gas phase (BB/BAg and FB/FAg), near-optimal growth was obtained from an inoculum of 1 to 10 cells per ml. Under most of these conditions the generation time was approximately 90 min. During the logarithmic growth phase, the cells retained their typical spiral morphology and high motility. These media also proved to be highly satisfactory for the cultivation of fresh isolates as well as other stock strains of Campylobacter. When the broth phase of the cultures, after addition of 15% glycerol, was quickly frozen and maintained at -70 degrees C, all strains thus far examined were readily recoverable and satisfactorily cultivated without additional passage.     

Biphasic culture system for rapid Campylobacter cultivation.

We developed a biphasic culture system consisting of 4 ml of brucella agar (BA) and 6 ml of brucella broth (BB) in 25-cm2 tissue culture flasks, which were incubated in air (BB/BAa) or in a gas mixture of 5% O2, 10% CO2, and 85% N2 (BB/BAg). These media were also used with a supplement consisting of ferrous sulfate, sodium metabisulfite, and sodium pyruvate and incubated as above (FB/FAa and FB/FAg, respectively). Highly satisfactory growth of Campylobacter jejuni 301 was obtained with all medium-gas phase combinations provided that the number of viable cells in the inoculum was large (greater than or equal to 10(6)/ml). The use of FB/FAa permitted the inoculum to be reduced to 100 cells per ml. With an adjusted gas phase (BB/BAg and FB/FAg), near-optimal growth was obtained from an inoculum of 1 to 10 cells per ml. Under most of these conditions the generation time was approximately 90 min. During the logarithmic growth phase, the cells retained their typical spiral morphology and high motility. These media also proved to be highly satisfactory for the cultivation of fresh isolates as well as other stock strains of Campylobacter. When the broth phase of the cultures, after addition of 15% glycerol, was quickly frozen and maintained at -70 degrees C, all strains thus far examined were readily recoverable and satisfactorily cultivated without additional passage.     

Tryptic Soy Bile-Kanamycin Test for the Identification of Bacteroides fragilis

A simple and practical test for the identification of Bacteroides fragilis is described. It utilizes two well-known properties of this species, i.e., stimulation of growth by bile and resistance to kanamycin. The test media are a tryptic-soy bile agar plate and a supplemented blood agar plate on which a kanamycin 1,000-mug/ml disk is placed. Incubation is for 24 h at 37 C in GasPak. The results of screening 190 strains, mostly clinical isolates, indicate that B. fragilis can be easily and reliably distinguished from other Bacteroides and from Fusobacterium species by its growth on tryptic-soy bile agar and resistance to kanamycin.     

Pathogenicity of Bacteroides fragilis group in rat intra-abdominal abscesses.

Attempts were made to study the pathogenicity of some strains of Bacteroides fragilis group in the rat intra-abdominal abscess model. Multiple intraabdominal abscesses were produced in 50 to 70% of animals when an inoculum containing 10(9) CFU/ml of any of the five species of Bacteroides fragilis group was injected. Rising homologous antibody titers determined by indirect fluorescent antibody test were observed till the 3rd week when tested last, indirectly confirming the multiplication of the organisms as also evident by viable count of bacteria in the abscesses. In some cases in addition to inoculated organisms some intestinal bacteria like Escherichia coli, Proteus mirabilis and Streptococcus spp. were also recovered from the abscess pus. Studies with the electron microscope showed presence of capsular polysaccharide only in Bacteroides fragilis and Bacteroides thetaiotaomicron. It was doubtful in Bacteroides distasonis and absent in Bacteroides ovatus and Bacteroides vulgatus, suggesting that virulence factor beside the capsular polysaccharide may be playing a role. Further studies are required to investigate the virulence factor responsible for the pathogenicity of noncapsulated species.     

Anaerobes isolated from clinical material over three years

Data on anaerobic bacteria isolated from clinical specimens at the bacteriology department within the 3-year period (1992-1994) were analysed. Anaerobic cultivation was carried out in all aspirates and swabs were transferred in transport media in syringes or blood cultures. Established growth occurred in all samples cultivated in thioglycollate broth after 4 days of incubation. Cultivation methods included enrichment media, GasPak jar, and API (BioMerieux) for final identification. A sulfite-reduction test using the Wilson-Blair medium and the Ellner-Smith sporulation medium was also used for the isolation of Clostridium perfringens. Anaerobes were diagnosed in 899 samples. Wound swabs (266 samples) and aspirates (106 samples) were the most common clinical material used. In total, 964 anaerobes were isolated: Peptostreptococcus species (299 strains), Eubacterium species (188 strains), Propionibacterium species (153 strains), Bacteroides fragilis(149 strains), Bacteroides species (95 strains) and Clostridium perfringens(80 strains).     

The Bacteroides glycocalyx as visualized by differential interference contrast microscopy

The glycocalyx of eight strains representing six species of Bacteroides was examined by differential interference contrast microscopy. Wet mounts in India ink were prepared from bacteria cultured in broth and on an agar medium; the wet mounts were observed by phase-contrast microscopy and differential interference contrast microscopy. With differential interference contrast microscopy, all bacteria demonstrated a glycocalyx, which included capsules surrounding single cells and microcolonies, strands of glycocalyx connecting cells and microcolonies, detached slime, and solid masses of glycocalyx in which innumerable bacteria were enmeshed. Bacteria showed comparable amounts of glycocalyx by visual observation with differential interference contrast microscopy whether grown on plates or in broth. Serial transfers of cultures did not diminish the amount of glycocalyx. Differential interference contrast microscopy proved to be a superior method to phase contrast for examining wet preparations of Bacteroides.     

Effect of growth conditions and storage on the specific activity of beta-lactamases of Bacteroides spp

The influences of growth conditions and cold storage on the specific activity of beta-lactamases of four strains of Bacteroides spp. was studied. Interbatch variation was observed in extracts prepared in an identical way on separate occasions but less variation was observed in extracts prepared from bacteria grown on Brain Heart Infusion agar supplemented with yeast extract, haemin and menadione, than in similar extracts of bacteria grown in broth or on other solid media. The loss of enzyme activity seen during the stationary phase of growth of some strains in broth was minimal during incubation for 48 h on agar. Storage of enzyme extracts at 4 degrees C was associated with loss of enzyme activity, but activity was retained during storage at -70 degrees C for up to 32 days. Freezing and thawing had little effect on enzyme activity.     

Susceptibility testing of anaerobic bacteria: A review of current methods and future prospects

The current NCCLS document, M11 A2, describes two methods for susceptibility testing of anaerobic bacteria. The reference method utilizes an agar dilution procedure, which is labor intensive and not convenient for testing individual patient isolates. The broth microdilution method does not support the growth of 15–40% clinical isolates and demonstrates poor correlation with the reference method for some members of the Bacteroides fragilis group with β-lactam agents and clindamycin. Etest is a new technique that incorporates an antibiotic gradient onto a plastic strip and utilizes agar media. This method is easily performed, permits growth of all anaerobes, and provides quantitative MICs for rapidly growing strains after overnight (20 hr) incubation. This method is convenient and reliable and enables the laboratory to provide the clinician with MIC data for individual patient isolates within a clinically relevant time period.