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.     

Screening for the presence of the insertion sequence IS1 in the genus Bacteroides

Abstract A specific DNA probe, containing a conserved region of the insertion sequence IS1, was hybridised to dot blots of total genomic DNA from 2 oral and 5 intestinal Bacteroides spp. Using Escherichia coli K12 as a positive control and Pseudomonas aeruginosa as a negative control, DNA homologous to the probe could not be detected in Bacteroides corporis, Bacteroides intermedius, Bacteroides ovatus, Bacteroides vulgatus, Bacteroides thetaiotaomicron or 2 strains of Bacteroides fragilis . The total DNA included plasmid DNA of 30.2, 42.7 and 42.7 MDa from B. fragilis, B. intermedius and B. corporis , respectively.
IS1 is commonly found in members of the Enterobacteriaceae, and it was concluded that the 2 groups of bacteria are not closely related.     

Study of the antigenic relationships between strains of Bacteroides, intermedius, B. melaninogenicus, B. corporis, and B. denticola revealed by immunoblotting with rabbit antisera

Antigen profiles of saccharolytic oral black-pigmented Bacteroides have been developed by Western blotting. Visual comparisons indicated extensive cross-reactions between B. intermedius, B. melaninogenicus, B. denticola, and B. corporis. Porphyromonas gingivalis, P. asaccharolyticus, and B. buccae showed less cross-reaction. Quantitation of antigenic similarity was made from densitometric scans. Calculation of the Jackard coefficient gave results of 33-72% similarity among the saccharolytic pigmented species, with the two homology groups of B. intermedius separated at 53%. Species were separated below 70%. Subtraction of the profile of a cross-reacting strain from that of the homologous strain also allowed quantitation of similarities. These similarities were lower; the range between species was 4-62%, although the two homology groups of B. intermedius still separated at 50 and 58%. Species were separated below 63%. Sera absorbed with a cross-reacting strain gave reduced reactions with the homologous strain and cross-reacting strains, indicating several common antigens among the four species. The species-specific antigens demonstrated by sera absorbed with cells of cross-reacting species were relatively few (3-6) compared with cross-reacting antigens detected by non-absorbed sera (18-28). The method appears useful to quantitate antigenic similarities among Bacteroides species and strains and allows analysis and quantitation of individual humoral responses in animals to these bacteria.     

Rapid and correct identification of intestinal Bacteroides spp. with chromosomal DNA probes by whole-cell dot blot hybridization.

A dot blot hybridization procedure with 32P-labeled whole chromosomal DNA of the type strains as probes was developed as a rapid and simple method for identification of intestinal Bacteroides species. Bacterial cells were fixed onto membrane filters by slight suction, treated with 0.5 N NaOH, and hybridized with these probes. Of 65 Bacteroides strains isolated from 19 human fecal specimens, which were identified as B. fragilis, B. thetaiotaomicron, B. ovatus, B. caccae, B. uniformis, B. stercoris, B. vulgatus, B. distasonis, and B. merdae by conventional phenotypic characterization, 62 (95%) were correctly identified with this hybridization procedure.     

Rapid and correct identification of intestinal Bacteroides spp. with chromosomal DNA probes by whole-cell dot blot hybridization

A dot blot hybridization procedure with 32P-labeled whole chromosomal DNA of the type strains as probes was developed as a rapid and simple method for identification of intestinal Bacteroides species. Bacterial cells were fixed onto membrane filters by slight suction, treated with 0.5 N NaOH, and hybridized with these probes. Of 65 Bacteroides strains isolated from 19 human fecal specimens, which were identified as B. fragilis, B. thetaiotaomicron, B. ovatus, B. caccae, B. uniformis, B. stercoris, B. vulgatus, B. distasonis, and B. merdae by conventional phenotypic characterization, 62 (95%) were correctly identified with this hybridization procedure.     

Enumeration of Bacteroides species in human faeces by fluorescent in situ hybridisation combined with flow cytometry using 16S rRNA probes

Bacteroides is a predominant group of the faecal microbiota in healthy adults. To investigate the species composition of Bacteroides by fluorescent in situ hybridisation (FISH) combined with flow cytometry, we developed five species-specific probes targeting the 16S rRNA. Probes were designed to identify cells belonging to Bacteroides distasonis, B. fragilis, B. ovatus, B. vulgatus and B. putredinis. The species-specificity of the probes was assessed against a collection of reference strains from the Cytophaga-Flavobacterium-Bacteroides group. The results of the FISH experiments showed that the probes were specific as they only detected strains of the target species. Determining the fluorescence intensity of each probe relative to that of the EUB 338 probe (domain bacteria) further showed that each species probe easily accessed the targeted site. The probes were applied to quantify the Bacteroides species in faeces collected from 20 healthy adults. All five species were detected in the faecal samples. Cells hybridised with Bfra 998 were the most frequent as they were observed in 90% of individuals (18/20 samples, mean proportion of 3.9 +/- 2.2%). The cells hybridised with Bvulg 1017 were observed in 85% of individuals (17/20 samples) and represented with a mean proportion of 4.2 +/- 6.1%, the most abundant Bacteroides species in human faeces. Cells hybridising with probes for B. ovatus, B. distasonis and B. putredinis were less frequently detected. The large distribution of B. vulgatus and B. fragilis in human faeces is in accordance with previous reports based on culture or molecular studies. This work showed that fluorescent in situ hybridisation is a tool appropriate for a high-resolution analysis of the species composition of complex ecosystems and especially of the Bacteroides group within the faecal microbiota.     

A recent fixation of cfiA genes in a monophyletic cluster of Bacteroides fragilis is correlated with the presence of multiple insertion elements.

Small-subunit ribosomal DNA sequences of 16 strains of Bacteroides fragilis were determined and compared with previously published sequences. Three phylogenetic methods (the neighbor-joining, maximum-likelihood, and maximum-parsimony methods) as well as a bootstrap analysis were used to assess the robustness of each topology. All phylogenetic analyses demonstrated that the B. fragilis strains were clearly divided into two robust monophyletic units which corresponded to the cfiA-negative and cfiA-positive groups. Strains of two previously identified DNA homology groups separated similarly into the two monophyletic units. According to the intensity of the hybridization signal with a cfiA probe, the cfiA-positive cluster could be further divided into two groups. This difference might reflect the existence of two, probably closely related cfiA-type genes. In the strongly hybridizing cfiA-positive strains, the gene is capable of conferring high-level resistance to the carbapenems and to most beta-lactamase inhibitors as well, while in the weakly hybridizing cfiA-positive strains, only the latter type of resistance is known to occur. The presence of the cfiA-type genes within a monophyletic cluster of B. fragilis that apparently represents only a minority of the species B. fragilis is suggestive of a recent acquisition. The fact that this cluster is also the predominant pool of all known B. fragilis insertion elements, which have been found to play an important role in the expression of carbapenem resistance, raises the possibility that both genetic determinants, i.e., the resistance gene(s) and insertion elements, may have coevolved.     

Use of randomly cloned DNA fragments for identification of Bacteroides thetaiotaomicron.

Randomly cloned fragments of DNA from Bacteroides thetaiotaomicron were used as hybridization probes for differentiation of B. thetaiotaomicron from closely related Bacteroides species. HindIII digestion fragments of DNA from B. thetaiotaomicron (type strain) were inserted into plasmid pBR322 and labeled with [alpha-32P]dCTP by nick translation. These labeled plasmids were screened for hybridization to HindIII digests of chromosomal DNA from type strains of the following human colonic Bacteroides species: B. thetaiotaomicron, Bacteroides ovatus, reference strain 3452-A (formerly part of B. distasonis), Bacteroides uniformis, Bacteroides fragilis, Bacteroides vulgatus, Bacteroides distasonis, Bacteroides eggerthii, and reference strain B5-21 (formerly B. fragilis subsp. a). Two of the five cloned fragments hybridized only to DNA from B. thetaiotaomicron. Each of these two fragments hybridized to the same DNA restriction fragment in five strains of B. thetaiotaomicron other than the strain from which the DNA was cloned. One of the cloned fragments (pBT2) was further tested for specificity by determining its ability to hybridize to DNA from 65 additional strains of colonic Bacteroides.     

Numerical taxonomy of Bacteroides and other genera of Gram-negative anaerobic rods.

A numerical taxonomy was performed on 157 cultures (141 different strains) of species of Bacteroides, Polyphyromonas, Prevotella [not Prevotella (Labroue, 1976)] and Fusobacterium. Isolates were each tested for 111 phenotypic characters which included possession of constitutive enzymes, fermentation of specific carbohydrates, gas chromatographic analysis of metabolic end-products and of cellular carboxylic acid composition. Computation of similarity coefficients was followed by a single-linkage cluster analysis. At the 94% similarity level, the following groupings at genus level were apparent: (1) Bacteroides ureolyticus; (2) Fusobacterium mortiferum, F. necrogenes, F. necrophorum, F. nucleatum and F. varium; (3) B. caccae, B. distasonis, B. eggerthii, B. fragilis, B. merdae, B. ovatus, B stercoris, B. thetaiotaomicron, B. uniformus and B. vulgatus; (4) B. splanchnicus; (5) Porphyromonas asaccharolytica; (6) B. bivius (Prevotella bivia); (7) B. disiens (P. disiens); (8) B. intermedius (P. intermedia);and (9) B. melaninogenicus (P. melaninogenica). Single isolates of B. ruminicola (P. ruminicola), B. denticola (P. denticola) and B. capillosus did not cluster with other strains.     

Bacteroides gingivalis and Bacteroides intermedius recognize different sites on human fibrinogen.

Bacteroides (Porphyromonas) gingivalis and Bacteroides (Porphyromonas) intermedius have been implicated in the etiology of human periodontal diseases. These organisms are able to bind and degrade human fibrinogen, and these interactions may play a role in the pathogenesis of periodontal disease. In attempts to map the bacterial binding sites along the fibrinogen molecule, we have found that strains of B. gingivalis and B. intermedius, respectively, recognize spatially distant and distinct sites on the fibrinogen molecule. Isolated reduced and alkylated alpha-, beta-, and gamma-fibrinogen chains inhibited binding of 125I-fibrinogen to both Bacteroides species in a concentration-dependent manner. Plasmin fragments D and to some extent fragment E, however, produced a concentration-dependent inhibition of 125I-fibrinogen binding to B. intermedius strains but did not affect binding of 125I-fibrinogen to B. gingivalis strains. Radiolabeled fibrinogen chains and fragments were compared with 125I-fibrinogen with respect to specificity and reversibility of binding to bacteria. According to these criteria, gamma chain most closely resembled the native fibrinogen molecule in behavior toward B. gingivalis strains and fragments D most closely resembled fibrinogen in behavior toward B. intermedius strains. The ability of anti-human fibrinogen immunoglobulin G (IgG) to inhibit binding of 125I-fibrinogen to B. intermedius strains was greatly reduced by absorbing the IgG with fragments D. Absorbing the IgG with fragments D had no effect on the ability of the antibody to inhibit binding of 125I-fibrinogen to B. gingivalis strains. A purified staphylococcal fibrinogen-binding protein blocked binding of 125I-fibrinogen to B. intermedius strains but not to B. gingivalis strains.     

Enzyme-labeled oligonucleotide probes for detection of the genes for thermostable direct hemolysin (TDH) and TDH-related hemolysin (TRH) of Vibrio parahaemolyticus.

Alkaline phosphatase conjugated oligonucleotide probes were developed to detect the genes (tdh and trh) coding for the thermostable direct hemolysin (TDH) and TDH-related hemolysin (TRH) of Vibrio parahaemolyticus. Using dot blot hybridization, probes were tested with 94 clinical isolates of V. parahaemolyticus. Results agreed well with those obtained using radio-labeled recombinant DNA probes for the genes tdh and trh. Specificity and sensitivity of enzyme tdh probes for detection of the trh gene were 100 and 93%, respectively, and those of the trh probes for trh gene detection were 93 and 86%, respectively. The tdh probes also hybridized with tdh-like genes processed by all strains of V. hollisae, and some strains of V. mimicus and V. cholerae non-O1, but neither tdh nor trh probes reacted with other bacterial species isolated from diarrheal stools. However, some V. parahaemolyticus strains that were negative with the enzyme trh probe hybridized weakly with a radio-labeled trh DNA fragment probe at medium stringency, and a few strains that were negative in high stringency conditions with a radio-labeled trh DNA fragment probe hybridized with the enzyme trh probe. This suggests that some strains of V. parahaemolyticus may carry another gene resembling trh.     

Lactose metabolism and lactase gene sequence homologies amongst lactobacilli

A number of strains of Lactobacillus spp., including the thermophilic and mesophilic dairy species, were screened for the presence of β -galactosidase ( β -gal) and phospho- β -galactosidase (pbg) enzyme activities. The majority of lactose fermenting strains exhibited β -gal rather than pbg enzyme activity with the highest levels in the thermophilic dairy species.
Correlation between these enzymes and the presence of specific genetic determinants was sought using probes for β -gal and pbg genes from Lactobacillus casei ssp. casei strain 64H. Southern transfer and filter hybridization showed that the β-gal probe shared homology with one strain of Lact. casei ssp. casei only. Sequences homologous to the pbg gene were detected only in plasmid DNA from the same strain of Lact. casei ssp. casei and with plasmid DNA from an apparently unrelated strain of Lactobacillus which exhibited no pbg activity. Two other strains of Lact. casei ssp. casei appeared to show homology between their chromosomal DNA and the pbg gene probe. No other homologies were detected. Therefore, although lactase activity could be detected in many strains of Lactobacillus spp., the genetic determinants involved did not share extensive homology.     

Isolation of Rhizobium loti Strain-Specific DNA Sequences by Subtraction Hybridization

Mixed-phase (heterogeneous) and single-phase (homogeneous) DNA subtraction-hybridization methods were used to isolate specific DNA probes for closely related Rhizobium loti strains. In the heterogeneous method, DNA from the prospective probe strain was repeatedly hybridized to a mixture of DNA from cross-hybridizing strains (subtracter DNA) which was immobilized on an epoxy-activated cellulose matrix. Probe strain sequences which shared homology with the matrix-bound subtracter DNA hybridized to it, leaving unique probe strain sequences in the mobile phase. In the homogeneous method, probe strain sequences were hybridized in solution to biotinylated, mercurated subtracter DNA. Biotinylated, mercurated subtracer DNA and probe strain sequences hybridized to it were removed by two-step affinity chromatography on streptavidin-agarose and thiol-Sepharose. The specificity of the sequences remaining after subtraction hybridization by both methods was assessed and compared by colony hybridization with R. loti strains. Both methods allowed the rapid isolation of strain-specific DNA fragments which were suitable for use as probes.     

Cloning of chromosomal DNA encoding the F41 adhesin of enterotoxigenic Escherichia coli and genetic homology between adhesins F41 and K88.

The genetic determinant for production of the adhesive antigen F41 was isolated from a porcine enterotoxigenic Escherichia coli strain by cosmid cloning. The cloned DNA included sequences homologous to those of hybridization probes prepared from the K88 adhesive antigen operon. Transposon insertions which inactivated F41 production mapped to the same region of DNA showing homology with the K88 genes, demonstrating the genetic relatedness of F41 and K88. Hybridization of a K88 gene probe to plasmid and total DNA from the porcine E. coli isolate from which the F41 gene was cloned indicated that F41 is chromosomally encoded by this strain. This observation was extended to other F41-producing animal isolates. A large number of animal E. coli isolates were examined with K88, F41, and K99 gene probes and for mannose-resistant hemagglutination of human group O erythrocytes and K88 and F41 antigen production. All K88 and F41 antigen producers possessed genetic homology with the K88 and F41 gene probes. Most, but not all, F41-producing strains possessed homology to the K99 gene probe, reflecting the previously observed association of F41 and K99 antigen production. In the strains examined, homology with the K99 gene probe was plasmid associated, whereas homology with the F41 gene probe was chromosomal. The K88 antigen-producing strains showed no homology with the K99 probe. A number of strains possessed homology with the K88 and F41 gene probes and were mannose-resistant hemagglutination positive, but did not produce K88 or F41 antigens. This suggests that there are adhesins among animal isolates of E. coli which are genetically related to but antigenically distinct from K88 and F41.     

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.     

DNA-DNA hybridization studies and phenotypic characteristics of strains within the 'Streptococcus milleri group'

Twenty-five strains resembling 'Streptococcus milleri' were compared by DNA-DNA hybridization, by whole-cell-derived polypeptide patterns on SDS-PAGE, and by biochemical tests. Four homology groups were revealed by DNA-DNA hybridization. DNA homology groups 1, 2 and 3 were closely related and contained the type strains NCDO 2226 (Streptococcus constellatus), NCDO 2227 (Streptococcus intermedius) and NCTC 10713 (Streptococcus anginosus), respectively. DNA homology group 4 consisted of four strains received as variants of Streptococcus intermedius which were found not to be closely related to strains in groups 1-3. The data from SDS-PAGE polypeptide patterns and biochemical tests supported the recognition of three centres of variation within the 'Streptococcus milleri group' corresponding to DNA homology groups 1-3 and indicated that strains of DNA homology group 4 are members of an as yet undescribed species within the viridans streptococci.     

Extracellular Deoxyribonuclease Production by Anaerobic Bacteria

The production of extracellular deoxyribonuclease was examined with anaerobic organisms isolated from clinical specimens. Nuclease activity was extraordinarily common. All strains of Fusobacterium, including eight species, as well as Bacteroides fragilis and B. melaninogenicus, displayed enzyme activity. Whereas the gram-positive bacteria were generally less productive, all strains of Clostridium perfringens, Peptostreptococcus intermedius, and P. anaerobius specifically produced deoxyribonuclease. The test is taxonomically valuable, particularly in the characterization of gram-positive cocci, since a deoxyribonuclease-producing coccus indicates P. intermedius or P. anaerobius. Additionally, possession of the enzyme may prove to be a useful correlate of the potential pathogenicity of anaerobes.     

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.     

胡枝了属根瘤菌的多相分类研究

采用数值分类,全细胞可溶性蛋白电泳分析,ＤＮＡ,Ｇ＋Ｃｍｏｌ％和ＤＮＡ相关性的测定以及１６ＳｒＤＮＡＰＣＲ－ＲＦＬ分析等多相分类技术对来源于不同地区的１６种寄主的胡枝子根瘤菌进行了系统的分类研究,数值分类的结果表明,在６７％的相似性水平上,全部供试菌可以为快生型根瘤菌和慢性型根瘤菌两大群,在８０％的相似性水平上又可分为两个亚群。在此基础上,对各亚群的胡枝子根瘤菌进行了ＤＮＡ相关性的测定,以进一步证     

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.