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.     

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 Classification of Bacteroides melaninogenicus and Related Species

One hundred and seventy-five strains of Bacteroides melaninogenicus , 17 strains of B. oralis and six strains of B. ochraceus were studied in a series of biochemical, chemical tolerance and antibiotic disc resistance tests and by the gas-liquid chromatographic analysis of the acid end products of metabolism. Strains of B. melaninogenicus ss. asaccharolyticus formed a distinct group with clear differences from other B. melaninogenicus strains. B. melaninogenicus ss. intermedius strains formed a homogeneous group that could be readily identified. B. ochraceus was distinguished from other Bacteroides spp. by its ability to grow in air enriched with CO₂. Bacteriodes melaninogenicus ss. melaninogenicus and B. oralis gave very similar patterns of results with the tests used and invariably were indistinguishable; the capacity to produce black-pigmented colonies on blood-containing media may not be a valid criterion for dividing these similar strains into two species.     

Energy-dependent incorporation of sphingolipid precursors and fatty acids in Bacteriodes melaninogenicus.

Washed cells of Bacteroides melaninogenicus are unable to incorporate the sphingolipid precursor 3-ketodihydrosphingosine (3KDS) or dihydrosphingosine into the complete sphingolipids ceramide phosphorylethanolamine (CPE) and ceramide phosphorylglycerol (CPG), whereas growing cultures are able to do so. This result suggested that an energy source was required by washed cells to initiate the incorporation of 3KDS. Investigation of a number of energy sources for B. melaninogenicus showed that glutamine was active in driving the incorporation of 3KDS. This system shows saturation kinetics. Besides glutamine, only asparagine and reduced nicotinamide adenine dinucleotide (NADH) are effective; glutamate and other compounds are inactive. The glutamine-driven system is sensitive to 2,4-dinitrophenol, azide, N,N'- dicyclohexylcarbodiimide, and carbonyl cyanide m-chlorophenylhydrazone. Asparagine plus NADH shows a synergistic effect in stimulating the incorporation of 3KDS into CPE and CPG in washed cells. However, glutamine plus NADH and glutamine plus asparagine show no such synergy. The cytochrome-free mutant of B. melaninogenicus, strain S, incorporates 3KDS in a manner similar to the parent strain when glutamine is used to drive the reaction; NADH or asparagine, however, are ineffective when used with strain S. Vitamin K-depleted cells of B. melaninogenicus are similar to vitamin K-grown cells, when glutamine or NADH is used to drive the 3KDS incorporation. Glutamine and NADH are also effective in stimulating the incorporation of palmitate and acetate by washed cells of B, melaninogenicus. Increased incorporation of these fatty acids into CPE, CPG, 3KDS, and other phospholipids is significantly increased by the presence of glutamine or NADH. Thus, energization of the membrane of B. melaninogenicus by glutamine or the electron transport system by NADH or asparagine is required for sphingolipid and other phospholipid synthesis. The relationship of this energization to possible transport of sphingolipid precursors is discussed.     

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.     

Laboratory and clinical study of nitazole]

Nitazole, a drug from the nitrotiazole group, was shown to be active in vitro against bacteroides, peptococci, peptostreptococci, clostridia, staphylococci, colibacilli and streptococci. By its activity and antibacterial spectrum nitazole had some advantages over metronidazole, a drug from the nitroimidazole group. Experimental study of nitazole aerosole formulation in 4 models of purulent wounds of rabbits infected by Bacteroides fragilis, B. melaninogenicus, Clostridium perfringens 27 and Staphylococcus aureus 209P revealed its high therapeutic efficacy. In the treatment of 37 patients with purulent wounds of the soft tissues including 12 cases isolating anaerobic microbes, the clinical process of the acute suppuration in all the patients at the average reduced to the 5th-7th day. By the data of the bacteriological and cytological examinations the wound surface was ready for putting in stitches or free perforated cutaneous graft by the 10th-12th day. The drug tolerance was good. No adverse reaction were observed under the nitazole dressing in any case during the treatment of the wounds.     

Succinate as a Growth Factor for Bacteroides melaninogenicus

Rumen strains of the obligate anaerobe Bacteroides melaninogenicus normally require medium supplemented with both heme and vitamin K. Sodium succinate was found to be an additional growth factor in that this compound can replace the requirement for heme in the presence of vitamin K, allowing good growth of the organism, and succinate can also partially replace the requirement for vitamin K in the presence of heme. The addition of succinate to a medium supplemented with both vitamin K and heme increases the growth rate of the culture. This ability to stimulate growth was specific for succinate, and cells grown without heme but with vitamin K and succinate were insensitive to cyanide. These experiments demonstrate a central role for succinate in the metabolism of B. melaninogenicus.     

Growth of Bacteroidaceae in Stirred Fermentors

The conditions for increasing bacterial yields in cultures of Bacteroidaceae by the use of stirred fermentors and pH control were investigated by means of three representative species: Sphaerophorus necrophorus, Bacteroides fragilis, and B. melaninogenicus. A medium containing tryptone, yeast extract, and glucose or sucrose was used. Horse serum had to be added to obtain substantial growth of B. melaninogenicus. The optimal pH for growth rate and yield was 7.0 to 7.2. Lysis of the bacteria occurred when the glucose (or sucrose) was exhausted. The rate of lysis was very high in cultures of S. necrophorus, less so in B. fragilis and B. melaninogenicus. Pleomorphism, manifested as large sperical forms of the bacteria, was observed in the late logarithmic phase of S. necrophorus. Great differences in the length of the lag phase and of the mean generation time were found among the three bacterial species. The yield in static cultures of the three species without pH control was approximately 0.4 g of dry cells per liter, but was increased, in stirred fermentors with pH control, to 3.5 g (S. necrophorus), 2.7 g (B. fragilis), and 4.3 g (B. melaninogenicus) per liter. With an inoculum density of 5 to 10 mg (dry weight) per liter, these yields were obtained in approximately 10 (S. necrophorus), 25 (B. fragilis), and 35 hr (B. melaninogenicus), respectively.     

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.     

替硝唑体内抗厌氧菌感染作用的研究

本项研究观察了替硝唑对类杆菌,经腹腔感染小鼠的体内保护作用。结果显示替硝唑对脆弱类杆菌和产黑色素类杆菌感染的小鼠,均具有良好的保护作用,两株菌感染的半数有效剂量（ＥＤ５０）分别为１１．１５ｍｇ／ｋｇ和１３．０４ｍｇ／ｋｇ。与甲硝唑相比,两者无显著性差异。     

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.     

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.     

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.     

儿童乳牙根管感染的细菌学分析

对18例3～8岁儿童乳牙的根管感染以无菌技术进行定量取样,按种于12种选择性培养基和2种非选择性培养基上,进行需氧、微需氧和厌氧培养,并对细菌菌落计数。对牙髓拟杆菌和牙龈拟杆菌作半定量免疫荧光染色计数;并对其中9例病牙进行了菌相分析。检出的所有细菌中,厌氧菌占绝对优势;其中检出率较高的菌为:产黑色素拟杆菌属,厌氧革兰氏阳性球菌,微需氧革兰氏阳性球菌等.本试验证明,儿童乳牙根管感染是以厌氧菌为主的混合感染,其中以产黑色素拟杆菌属等最常见.     

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.     

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.     

Empirical antibiotic therapy of wounds complicated by anaerobic non-clostridial infection

Antibacterial activity of 14 drugs against clinical strains of asporogenic anaerobes causing wound infections in the soft tissues i. e. Bacteroides fragilis and Bacteroides melaninogenicus as well as anaerobic gram-positive++ cocci was assayed with the method of serial dilutions in agar. It was shown that among the investigated species B. fragilis had the most marked resistance since out of the 14 drugs only 8 were sufficiently active against it i.e. carbenicillin, levomycetin, lincomycin, dioxidine, metronidazole, thinidazole, nitrazole and erythromycin. The choice of drugs for treating infections caused by B. melaninogenicus and anaerobic grampositive cocci unlike those caused by B. fragilis offered no difficulty since practically++ all the investigated drugs were highly active against the causative agents. There was observed relationship between the frequency of asporogenic anaerobes and the wound genesis. The characteristic features of the species composition connected with localization of the suppurative foci were indicated. The detected specific antimicrobial profiles of the asporogenic anaerobes causing wound infections and the peculiarity of their participation in development of purulent infections of the soft tissues provided a differential approach to empirical antibacterial therapy prior to the pathogen bacteriological investigation and availability of the antibioticograms.     

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.     

In situ development and application of cDNA-AFLP to isolate genes of Candida oleophila (strain O) potentially involved in antagonistic properties against Botrytis cinerea

The yeast Candida oleophila (strain O) presents a high level of protective activity against Botrytis cinerea (gray mold) on postharvest apples. The cDNA-AFLP technique allows the comparison of mRNA populations extracted from cells grown in different conditions. In order to isolate yeast genes potentially involved in biological control properties, that technique was applied on strain O cells growing on apple wounds. The biological control properties of 8 C. oleophila strains and strain O were assessed in order to compare the gene expression of a non antagonistic strain against gene expression of strain O. In the absence of a non-antagonistic strain, an other comparison model was designed. It was based on the growth of strain O in different in situ conditions: strain O applied on apple wounds (O), strain O applied on apple wounds in presence of B. cinerea (B) and B. cinerea alone on apple wounds (F). A recovering technique, based on the washing of cells in the wound and a RNA extraction method followed by a DNase treatment were optimised before cDNA-AFLP application. Thirteen primer pairs were used. Their application resulted in an average of 54 and 55 bands for O and B respectively whereas no bands were observed for F. Among these bands, 8 were expressed more intensely in presence of the pathogen (1.1% of the fragments).