The Hydrolysis of Dextran by Gram Negative Non-sporing Anaerobic Bacilli

The ability of Gram negative anaerobic bacilli to hydrolyse dextran was determined in liquid and solid media containing Blue Dextran 2000. Released blue chromophore in the liquid medium was detected spectrophotometrically. Results obtained with 334 strains of Bacteroidaceae grown on the solid medium indicated that most strains did not hydrolyse the substrate. Hydrolysis of Blue Dextran 2000 occurred with certain strains of Bacteroides thetaiotaomicron , B. melaninogenicus ss. melaninogenicus, B. oralis, B. ovatus and B. ochraceus.     

Numerical taxonomy of some non-saccharolytic and saccharolytic Bacteroides species

Various Bacteroides spp. were examined by physiological tests, presence of specific enzymes, antibiotic sensitivity, menaquinone composition and a few miscellaneous tests. The data matrix containing 58 strains and 55 unit characters was examined using Gower's similarity coefficients (S _G ) and included matching negative character states and multistate characters. The highly saccharolytic strains were separated from the less saccharolytic and non-fermentative strains at the 55% similarity level; while at the slightly higher level of 63% strains of Capnocytophaga (formerly Bact. ochraceus ) were recovered as a compact phenon distinct from other saccharolytic species. The phenogram was divided into 6 clusters at 72% similarity level. Most of the ' Bact. fragilis group' of species clustered in one phenon while Bact. melaninogenicus ssp. melaninogenicus, Bact. bivius and a new species, Bact. denticola , formed another group. Another phenon comprised the saccharolytic non-pigmented species closely related to Bact. oralis such as Bact. buccalis and Bact. pentosaceus. The less saccharolytic strains of Bact. melaninogenicus ssp. intermedius and Bact. disiens were recovered in a distinct phenon. The low affinity (less than 55% similarity) between the two subspecies of Bact. melaninogenicus emphasised the need for reclassifying these taxa into separate species. The non-fermentative and very weakly saccharolytic strains formed good taxospecies. The separation of this cluster into three subclusters is in excellent agreement with chemotaxonomic data now available.     

Numerical taxonomy of some non-saccharolytic and saccharolytic Bacteroides species

Various Bacteroides spp. were examined by physiological tests, presence of specific enzymes, antibiotic sensitivity, menaquinone composition and a few miscellaneous tests. The data matrix containing 58 strains and 55 unit characters was examined using Gower's similarity coefficients (SG) and included matching negative character states and multistate characters. The highly saccharolytic strains were separated from the less saccharolytic and non-fermentative strains at the 55% similarity level; while at the slightly higher level of 63% strains of Capnocytophaga (formerly Bact. ochraceus) were recovered as a compact phenon distinct from other saccharolytic species. The phenogram was divided into 6 clusters at 72% similarity level. Most of the 'Bact. fragilis group' of species clustered in one phenon while Bact. melaninogenicus ssp. melaninogenicus, Bact. bivius and a new species, Bact. denticola, formed another group. Another phenon comprised the saccharolytic non-pigmented species closely related to Bact. oralis such as Bact. buccalis and Bact. pentosaceus. The less saccharolytic strains of Bact. melaninogenicus ssp. intemedius and Bact. disiens were recovered in a distinct phenon. The low affinity (less than 55% similarity) between the two subspecies of Bact. melaninogenicus emphasised the need for reclassifying these taxa into separate species. The non-fermentative and very weakly saccharolytic strains formed good taxospecies. The separation of this cluster into three subclusters is in excellent agreement with chemotaxonomic data now available.     

Effect of Bile and Desoxycholate on Gram-Negative Anaerobic Bacteria

The bile tests for characterizing gram-negative anaerobic bacilli were reevaluated in prereduced anaerobically sterilized peptone-yeast-glucose broth, in thioglycollate broth, and on blood agar plates. Blood agar plates were unsatisfactory. The combination of 20% bile with 0.1% desoxycholate inhibited Fusobacterium, Bacteroides melaninogenicus, and B. oralis and sometimes Sphaerophorus necrophorus, but not B. fragilis or other Sphaerophorus species studied. Ten per cent bile with 0.05% desoxycholate was less satisfactory. There was no significant difference between fresh and commercial powdered bile. Desoxycholate (0.1% in thioglycollate broth) inhibited B. fragilis, Fusobacterium, B. melaninogenicus, B. oralis, and S. necrophorus, but not S. varius or S. mortiferus/S. ridiculosus. The bile and desoxycholate tests are simple to perform and helpful for characterization and classification of gram-negative anaerobic bacilli.     

Comparison of the Biochemical Properties of Bacteroides melaninogenicus from Human Dental Plaque and Other Sites

A total of 45 strains of black-pigmented bacteroides, including Bacteroides melaninogenicus subsp. melaninogenicus, Bacteroides melaninogenicus subsp. intermedius and Bacteroides melaninogenicus subsp. asaccharolyticus , have been examined for morphological and physiological characteristics. They were also tested for the range of acidic metabolites, the typical basic amino acid of the mucopeptide, the base composition of DNA, the electrophoretic mobility of malate dehydrogenase and the susceptibility to certain antibiotics. The subspecies most commonly isolated from supragingival human dental plaque are B. melaninogenicus subsp. intermedius and B. melaninogenicus subsp. melaninogenicus . A list of tests for the differentiation of the three subspecies is given, but the separation of B. melaninogenicus subsp. asaccharolyticus from the other two subspecies of B. melaninogenicus is nevertheless recommended.     

Deoxyribonucleic acid base composition of certain species of the genus Bacteroides

The moles percent guanine plus cytosine content of the DNA (% G + C) of 15 Bacteroides strains representing six species was determined. One group, including three strains of Bacteroides ruminicola, two strains of B. melaninogenicus, two strains of B. succinogenes, and one strain of B. oralis (JI), had a % G + C of 47.6--50.3 and a second group including two strains of B. amylophilus, four strains of B. fragilis, and one strain of B. succinogenes had a lower % G + C of 40.3--42.7. The taxonomic relationships among these bacteroides species were discussed.     

Deoxyribonucleic acid homologies among strains of Bacteroides melaninogenicus and related species

Saccharolytic, black-pigmented Bacteroides strains, which at present belong to the species Bacteroides melaninogenicus were classified on the basis of deoxyribonucleic acid (DNA) base ratios and DNA hybridization studies. These strains were divided into several DNA homology groups, which showed no or low mutual DNA homology. A DNA homology group with a percentage guanine plus cytosine (G + C) of 42–43% was formed by three strains of Bact. melaninogenicus subsp. melaninogenicus ; the type strain of this subspecies, strain ATCC 25845, had about 60% DNA homology with this group. Strain ATCC 15930, which has been assigned to this subspecies, had a percentage G + C of 47% and showed no DNA homology with the former group. All strains of Bact. melaninogenicus subsp. intermedius had a percentage G + C of 39–45%. A DNA homology group was formed by eight strains of this subspecies. The type strain of Bact. melaninogenicus subsp. intermedius , ATCC 25611, showed relatively low DNA homology with this main DNA homology group. A strain of Bact. melaninogenicus subsp. intermedius serotype C1 showed no DNA homology with the other strains tested. Furthermore two strains labelled 'Bact. melaninogenicus subsp. levii' were found to form a distinct DNA homology group. On the basis of the DNA homology results, the strains, which at present are classified in the species Bact. melaninogenicus , were clearly distinguished from strains of Bact. asaccharolyticus and Bact. gingivalis , and also from strains of related non-pigmented Bacteroides species.     

Antibiotic susceptibility patterns as aids in classification and characterization of gram-negative anaerobic bacilli

Patterns of susceptibility of gram-negative anaerobic bacilli to antibiotics have been found to be distinctive and of significant help in classification and identification. Five major groups of gram-negative anaerobic bacilli have been defined on the basis of morphological and biochemical criteria. Antibiotic sensitivity patterns conform to these groupings and provide additional taxonomic criteria. The Bacteroides fragilis group is resistant to penicillin G, whereas the other groups are generally sensitive. B. fragilis strains are relatively sensitive to erythromycin, whereas the Sphaerophorus necrophorus group is resistant. B. melaninogenicus strains, B. oralis, and Fusobacterium are all more sensitive to kanamycin and neomycin than the other two groups. Kanamycin is more active against Fusobacterium strains than neomycin, but less active against all other groups. Colistin or polymyxin B is useful for distinguishing between the resistant B. fragilis and the sensitive S. necrophorus. Antibiotic susceptibility determinations may be more readily performed in clinical laboratories than certain biochemical tests recommended for differentiation of the gram-negative anaerobic bacilli and may serve as helpful adjuncts to morphological and biochemical observations in classifying and characterizing these organisms. The use of standardized procedures for antibiotic susceptibility tests is essential if comparable results are to be obtained in different laboratories.     

Determination of Bacteroides melaninogenicus serogroups by fluorescent antibody staining

Fluorescein isothiocyanate-labeled antibody reagents (conjugates) were prepared to one strain of each of the three subspecies of Bacteroides melaninogenicus: B. melaninogenicus subsp. melaninogenicus, B. melaninogenicus subsp. asaccharolyticus, and B. melaninogenicus subsp. intermedius. These three conjugates were specific; thus, they provided a new serological classification of B. melaninogenicus. The three serogroups were designated A, B, and C. Most test strains (98%) isolated from human clinical specimens were assigned to a specific serogroup by immunofluorescence, and the serogroup of these test strains corroborated the biochemical characterization of the three subspecies of B. melaninogenicus. The conjugates failed to cross-react with other anaerobes or aerobes tested. This fluorescent antibody technique provided a more rapid classification of the three subspecies of B. melaninogenicus than did conventional biochemical methods.     

The Characterization of Clinically Important Gram Negative Anaerobic Bacilli by Conventional Bacteriological Tests

One hundred and sixty-five reference strains and laboratory isolates of Gram negative, non-sporing, anaerobic bacilli were subjected to a series of simple laboratory tests that were initially selected for their discriminatory value. Conventional biochemical tests, tests of resistance to antibiotics, and tolerance to dyes and bile salts were included. These tests allowed a clear separation of strains into three main groups: Bacteroides fragilis, B. melaninogenicus and Fusobacterium spp. Certain tests were found useful for identifying recognized subspecies of B. fragilis and B. melaninogenicus . A scheme for the identification of unknown laboratory isolates of Gram negative anaerobic bacilli is presented.     

Neuraminidase production by Bacteroides species

Abstract 128 strains of Bacteroides isolated from clinical specimens were surveyed for their ability to produce neuraminidase. All strains of Bacteroides fragilis and the B. fragilis group were neuraminidase-positive, as were strains of B. oralis and B. bivius . All strains of B. capillosus, B. ruminicola, B. disiens, B. multiacidus and B. uniformis did not produce a detectable neuraminidase. When human erythrocytes were exposed to cell extracts of neuraminidase-producing Bacteroides , and then tested with peanut ( Arachis hypogeae ) lectin, agglutination occurred. It was concluded that the production of neuraminidase by clinical isolates of Bacteroides may be associated with the pathophysiology of severe Bacteroides infections.     

Identification of clinical isolates of selected species of Bacteroides: production of phenylacetic acid

A total of 132 human clinical isolates were identified and tested for the production of phenylacetic acid. With the exception of B. vulgatus, B. melaninogenicus ss. melaninogenicus, and B. melaninogenicus ss. intermedius, all other species under study produced phenylacetic acid. This property can thus serve as a basis for faster characterization of the strains in clinical laboratories.     

牙髓紫卟啉杆菌血清学特性分析

牙髓类杆菌曾是重要的产黑色素类杆菌群菌株,最近重新命名为牙髓紫卟啉杆菌,与牙髓尖周感染和牙源性脓肿有特殊关系。本文用牙髓紫卟啉杆菌ATCC 35406国际标准菌株免疫Balb/c小鼠制得免疫血清,ELISA法检测该抗血清的特异性。发现与中间型类杆菌、产黑色素类杆菌、赖氏类杆菌、躯体类杆菌、牙类杆菌、牙龈紫卟啉杆菌、脆弱类杆菌、黄褐二氧化碳嗜纤维菌、生痰二氧化碳嗜纤维菌、衣氏放线菌反应均阴性,而同不解糖紫卟啉杆菌呈现弱阳性反应,说明矛髓紫卟啉杆菌与不解糖紫卟啉杆菌具有某种相同抗原结构,存在血清交叉反应,血清学关系较近。牙髓紫卟啉杆茵多克隆抗体直接用于临床该菌的检出与鉴定,尚存一定缺陷。     

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.     

Serological properties and immunobiological activities of lipopolysaccharides from black-pigmented and related oral Bacteroides species

Lipopolysaccharides (LPS) from five species of oral Bacteroides, B. gingivalis strains 381 and ATCC 33277, B. oralis ATCC 33269, B. loescheii ATCC 15930, B. intermedius ATCC 25611 and B. corporis ATCC 33547, were extracted from whole cells by the phenol/water procedure, and subsequently purified by treatment with nuclease and ultracentrifugation. The LPS were composed of hexoses, glucosamine, fatty acids and phosphorus. Heptose and 2-keto-3-deoxyoctonate were not detected. The LPS preparations from B. gingivalis strains 381 and ATCC 33277 presented very similar SDS-polyacrylamide gel electrophoresis patterns when stained with ammoniacal silver. They produced a fused precipitin band against an antiserum to B. gingivalis 381 LPS in immunodiffusion tests. Antisera raised against the LPS from B. loescheii and B. intermedius reacted with the LPS prepared from all the oral Bacteroides strains except those of B. gingivalis. All the LPS preparations were mitogenic for spleen cells of BALB/c (nu/nu) mice, but not for thymus cells from C3H/HeN mice. The LPS induced marked mitogenic responses and polyclonal B cell activation for spleen cells of not only C3H/HeN (LPS responder) mice, but also C3H/HeJ (LPS nonresponder) mice. The mitogenic responses were not suppressed significantly upon addition of polymyxin B to the reaction mixture. These LPS also enhanced interleukin-1 production by murine peritoneal macrophages and mouse cell line J744. 1 macrophages. Hydrolysis of B. gingivalis ATCC 33277 LPS in 1 m-HCl at 100 degrees C for 1 h yielded lipid and polysaccharide. The lipid portion was largely composed of fatty acids and glucosamine, and was mitogenic for spleen cells from C3H/HeJ as well as C3H/HeN mice, while the polysaccharide portion induced no significant mitogenic responses under similar experimental conditions.     

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.     

Use of fluorescence microscopy for monitoring periodontal disease state

Samples of subgingival plaque from patients with periodontal disease and control subjects were stained with the Fluoretec Fluorescent test kits (Pfizer Inc., New York) developed for the rapid detection of members of the Bacteroides fragilis and B. melaninogenicus groups of anaerobes. The same fluorescent fields were also examined by dark-field microscopy for the total count of bacteria. Bacteroides fragilis and B. melaninogenicus were found in plaque samples of healthy subjects and periodontally diseased patients with no significant difference in percent of total flora. Oral spirochetes also fluoresced with the antisera used. Samples from healthy sites showed virtually no spirochetes; spirochetes were present in diseased sites. Tests with other antisera also showed that fluorescein-labelled antibodies can be adsorbed nonspecifically to the surface of spirochetes. Such a phenomenon can be used to monitor an individual's periodontal disease state.     

Multivariate analyses of fatty acid data from whole-cell methanolysates of Prevotella, Bacteroides and Porphyromonas spp

The genus Bacteroides contains a number of biochemically and physiologically heterogeneous groups of organisms and needs taxonomic revision. In this study cellular fatty acids from a number of Bacteroides spp. were identified and quantified using gas chromatography and gas chromatography-mass spectrometry. The chemical data were then subjected to principal components analysis. In B. fragilis, which is the type species of the genus Bacteroides, C3-OH-iso17 was the predominant fatty acid (38.0%) and Cante15 was present in higher amounts (32.7%) than Ciso15 (14.6%). B. fragilis thus differed from all the other species examined: Prevotella (Bacteroides) buccae, P. (B.) oralis, P. (B.) oris, P. (B.) disiens, P. (B.) veroralis, P. (B.) heparinolytica and Porphyromonas (Bacteroides) endodontalis. Principal components analysis also enabled the closely related P. buccae, P. oralis and P. oris to be differentiated.