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.     

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.     

Chromosomal DNA probes for the identification of Bacteroides species

We compared 22 Bacteroides species by DNA-DNA homology studies using the S1 endonuclease method. None of the currently defined species shared more than 30% DNA homology with any other species examined with the exception of B. buccae and B. capillus (which along with B. pentosaceus are now considered a single species), which shared 86% of their DNA sequences. Two clusters showed weak genetic relationships, with DNA homology greater than 10%. The first cluster included B. coporis, B. disiens, B. bivius, B. intermedius and B. melaninogenicus. The second cluster included B. fragilis, B. eggerthii, B. ovatus, B. thetaiotaomicron and B. uniformis. Five of the oral species, B. asaccharolyticus, B. gingivalis, B. loescheii, B. intermedius and B. melanogenicus, were chosen for study as whole chromosomal probes in dot blot assays. These were tested against 243 clinical strains biochemically identified as Bacteroides species. The DNA probes correctly identified 94% of the clinical strains. DNA probe and biochemical identification was 100% for two of the five species. In contrast, only 86% of the strains biochemically identified as B. intermedius were identified by the DNA probe. The DNA probes gave a species identification to seven strains which could not be biochemically identified.     

Synthetic oligodeoxynucleotide probes for the rapid detection of bacteria associated with human periodontitis

Analysis of the subgingival microflora has recently implicated Actinobacillus (Haemophilus) actinomycetemcomitans and several black Bacteroides species in the aetiology of juvenile, adult and rapidly progressing periodontitis. Rapid bacteriological diagnosis has been hampered by the slow growth and fastidious nature of these bacteria. To construct diagnostic probes, dideoxy sequencing of the 16S rRNA molecules from A. (H.) actinomycetemcomitans, Haemophilus aphrophilus, Bacteroides gingivalis, Bacteroides intermedius subgroup II, Bacteroides asaccharolyticus and several closely related species was performed. Next, oligodeoxynucleotides, complementary to defined regions of the 16S rRNA exhibiting considerable evolutionary divergence, were synthesized for use as molecular probes. In a dot-blot hybridization assay, all strains from each of the species for which probes were constructed were correctly identified, with a detection limit of less than 5 x 10(3) organisms. No cross-hybridization to closely related species (except for H. aphrophilus and Haemophilus paraphrophilus) or contaminating bacteria was observed. Using a modified DNA/RNA hybridization technique, the detection could be performed in less than 12 h, as compared to 2-3 weeks using conventional bacteriological procedures.     

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.     

Molecular characterization of Bacillus anthracis using multiplex PCR, ERIC-PCR and RAPD

AIMS: To investigate the molecular characterization of Bacillus anthracis strains by multiplex PCR, enterobacterial repetitive intergenic consensus-PCR (ERIC-PCR) and random amplification of polymorphic DNA (RAPD). METHODS AND RESULTS: Three primers were used to amplify the cya, cap and cereolysinAB genes in the multiplex PCR. Two distinct ERIC-PCR and RAPD fragments, which separated B. anthracis into two groups, were used as probes in Southern hybridization experiments. The probes hybridized only to the cya+ B. anthracis strains identified by the multiplex PCR. Nucleotide sequence analysis of the two cloned fragments showed they were from the pXO1 plasmid of B. anthracis. CONCLUSION: Multiplex PCR simultaneously identified isolates of the Bacillus cereus group and the B. anthracis virulence factors. ERIC-PCR and RAPD, combined with the Southern hybridization analyses, differentiated B. anthracis strains and separated them from the closely related B. cereus group bacteria. SIGNIFICANCE AND IMPACT OF THE STUDY: ERIC-PCR and RAPD assay could be effective in differentiating virulent from avirulent B. anthracis. Our results also show that the amplification of the large plasmids was allowed in the ERIC-PCR and RAPD assay.     

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.     

Detection system for Escherichia coli-specific virulence genes: absence of virulence determinants in B and C strains.

We describe a rational approach to simultaneously test Escherichia coli strains for the presence of known virulence genes in a reverse dot blot procedure. Specific segments of virulence genes of E. coli designed to have similar hybridization parameters were subcloned on plasmids and subsequently amplified by PCR as unlabeled probes in amounts sufficient to be bound to nylon membranes. Various pathogenic isolates and laboratory strains of E. coli were probed for the presence of virulence genes by labeling the genomic DNA of these strains with digoxigenin and then hybridizing them to the prepared nylon membranes. These hybridization results demonstrated that besides the E. coli K-12 safety strain derivatives, E. coli B and C strains are also devoid of genes encoding any of the investigated virulence factors. In contrast, pathogenic E. coli control strains, used to evaluate the method, showed typical hybridization patterns. The described probes and their easy application on a single filter were shown to provide a useful tool for the safety assessment of E. coli strains to be used as hosts in biotechnological processes. This approach might also be used for the identification and characterization of clinically significant E. coli isolates from human and animal species.     

Design of species-specific oligonucleotide probes for the detection of Bacteroides and Parabacteroides by fluorescence in situ hybridization and their application to the analysis of mouse caecal Bacteroides-Parabacteroides microbiota

Aims: To develop species‐specific monitoring techniques for rapid detection of Bacteroides and Parabacteroides inhabiting the mouse intestine by fluorescence in situ hybridization. Methods and Results: The specificity of oligonucleotide probes was evaluated by fluorescence whole‐cell hybridization. Oligonucleotide probes specific for each species hybridized only with the target bacteria. Using these probes, caecal Bacteroides–Parabacteroides microbiota of conventional mice and specific pathogen‐free (SPF) mice from three different breeders were analysed. It was shown that Bacteroides acidifaciens Group‐1, Group‐2 and Group‐3 were dominant in conventional mice and SPF mice from two out of three breeders. Bacteroides vulgatus and Parabacteroides distasonis were detected in one of these two SPF breeding colonies in addition to Bact. acidifaciens. SPF mice of the remaining breeder harboured characteristic Bacteroides–Parabacteroides microbiota, consisting of Bacteroides sp. ASF519 and Bacteroides caccae. Conclusions: Bacteroides acidifaciens is the dominant and most typical species in the mouse Bacteroides–Parabacteroides microbiota. The Group‐3 was identified as a novel group and revealed to occupy a major niche together with Bact. acidifaciens Group‐1 and Group‐2. Significance and Impact of the Study: The species‐specific probe set developed in this study was the efficient tool for rapid detection of target bacterial groups inhabiting the mouse intestine. The results of this study provide important new information on the mouse Bacteroides–Parabacteroides community.     

In situ identification of symbiotic dinoflagellates,the genus Symbiodinium with fluorescence-labeled rRNA-targeted oligonucleotide probes

Fluorescence in situ hybridization has been used for the identification and analysis of populations of the dinoflagellate Symbiodinium that lives symbiotically in marine invertebrates. Conditions for in situ hybridization of Symbiodinium were optimized and used to identify the clade to which the isolate belongs using specific probes. The optimized in situ hybridization procedure used a combination of chlorophyll removal and permeabilization with hot ethanol. Incubation of the cells in 50% ethanol at 80 degrees C for 20 min rendered the cell wall permeable to Cy3-labeled probes. Symbiodinium clade-specific probes were designed based on 18S rRNA sequences. Symbiodinium A, B and C were distinguished by in situ hybridization with the specific probes SymA, SymB and SymC, respectively. The hybridization results using clade-specific probes corresponded with results obtained using restriction fragment length polymorphism (RFLP) analysis. Symbiodinium isolated from jellyfish Cassiopea sp. and sea anemone Aiptasia sp. were classified as belonging to clades A and B using the FISH procedure established in this study.     

Design and application of group-specific oligonucleotide probes for detecting and monitoring mouse clostridia

Clostridia dominate the rodent intestinal bacterial community and play an important role in physiological functions of the host. However, their ecology and diversity are still unclear. In our previous report, we showed that phylogenetically novel groups of clostridia inhabit the mouse intestine and contribute to the normalization of germfree mice. In this study, five new oligonucleotide probes were designed and applied to detect these clostridial groups that are essential for the normalization of germfree mice. Faecal microbiota of conventional mouse strains and specific pathogen-free mice from different breeding colonies were analysed by fluorescence in situ hybridization using these five probes. Our results showed that the composition of clostridia differed among mouse strains and also among mouse groups of the same inbred strain from different breeding colonies. These five new probes for mouse clostridia were able to detect the difference in clostridial diversity in each mouse group. In addition to Clostridium, we also analysed Bacteroides and Lactobacillus using previously described probes and the number or the frequency of occurrence of Bacteroides was shown to be different among mouse groups analysed. The oligonucleotide probe set including our newly developed and previously described probes used in this study can be applied to monitoring of significant groups of mouse intestinal microbiota.     

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.     

Characterization of Bacteroides gingivalis by direct fluorescent antibody staining and cellular fatty acid profiles

Bacteroides gingivalis is a newly proposed species which includes strains isolated from the mouth. Thirteen strains of B. gingivalis isolated from three geographic locations in the United States and France were examined with direct fluorescent antibody staining and analysis of total cellular fatty acids and compared with 16 strains of B. asaccharolyticus of nonoral origin by the same methods. Bacteroides gingivalis strains reacted with the B. gingivalis conjugate (fluorescein isothiocyanate labeled antibody reagent) only, while the B, asaccharolyticus strains reacted with the B. asaccharolyticus conjugate only. The B. gingivalis strains showed negative fluorescence with fluorescein isothiocyanate conjugates for other black-pigmented Bacteroides species. The specificity of the B. gingivalis conjugate was demonstrated by its failure to stain 88 strains of aerobic and anaerobic bacteria other than B. gingivalis. The fatty acid profiles of B. gingivalis and B. asaccharolyticus were readily distinguishable. The B. gingivalis profile was also distinguishable from those of other pigmenting Bacteroides species on the basis of concentration ratios among the characteristic components. These results support the species separation of B. gingivalis and B. asaccharolyticus. Further, they indicate the usefulness of cellular fatty acid profiles as an adjunct to the use of specific fluorescent antibody conjugates for identification of Bacteroides species.     

Discerning the Role of Bacteroides fragilis in Celiac Disease Pathogenesis

Celiac disease (CD) is associated with intestinal dysbiosis, which can theoretically lead to dysfunctions in host-microbe interactions and contribute to the disease. In the present study, possible differences in Bacteroides spp. and their pathogenic features between CD patients and controls were investigated. Bacteroides clones (n = 274) were isolated, identified, and screened for the presence of the virulence genes (bft and mpII) coding for metalloproteases. The proteolytic activity of selected Bacteroides fragilis strains was evaluated by zymography and, after gastrointestinal digestion of gliadin, by high-pressure liquid chromatography/electrospray ionization/tandem mass spectrometry. The effects of B. fragilis strains on Caco-2 cell culture permeability and inflammatory response to digested gliadin were determined. B. fragilis was more frequently identified in CD patients than in healthy controls, in contrast to Bacteroides ovatus. B. fragilis clones carrying virulence genes coding for metalloproteases were more abundant in CD patients than in controls. B. fragilis strains, representing the isolated clones and carrying metalloprotease genes, showed gelatinase activity and exerted the strongest adverse effects on the integrity of the Caco-2 cell monolayer. All B. fragilis strains also showed gliadin-hydrolyzing activity, and some of them generated immunogenic peptides that preserved or increased inflammatory cytokine production (tumor necrosis factor alpha) and showed increased ability to permeate through Caco-2 cell cultures. These findings suggest that increased abundance of B. fragilis strains with metalloprotease activities could play a role in CD pathogenesis, although further in vivo studies are required to support this hypothesis.     

Use of oligonucleotide probes to study the relatedness of delta-endotoxin genes among Bacillus thuringiensis subspecies and strains

Fifteen Bacillus thuringiensis strains representing 13 serotypes were screened with five oligodeoxyribonucleotide probes specific for certain regions of two published sequences and one unpublished sequence of B. thuringiensis delta-endotoxin genes. Of the 15 cultures, 14 hybridized with at least one probe; the B. thuringiensis subsp. thompsoni strain alone did not hybridize. Two B. thuringiensis subsp. kurstaki strains of commercial interest, HD-1 and NRD-12, were found to be so closely related as to be indistinguishable with this technique; the same situation was found with strains from B. thuringiensis subspp. dendrolimus and sotto. Five strains were identified as probably containing only one endotoxin gene. A probe specific for the gene from the B. thuringiensis subsp. kurstaki HD-73 strain hybridized to only 3 of the 15 cultures tested. The hybridization data suggest that the DNA sequences coding for the C-terminal region of the endotoxin protein are as well conserved as those coding for the N-terminal toxic portion.     

Species-specific oligonucleotide probes for five Bifidobacterium species detected in human intestinal microflora.

Portions of the 16S rRNA from closely related species of the genus Bifidobacterium that are found in the human intestinal microflora were sequenced in order to design species-specific oligonucleotide probes. Five oligonucleotide probes ranging from 16 to 19 bases in length and complementary to 16S rRNA sequences from Bifidobacterium adolescentis, B. bifidum, B. breve, B. infantis, and B. longum were synthesized. With crude high-molecular-weight RNA preparations as targets, these probes showed the desired species specificity, even down to a 1-nucleotide difference. For the practical evaluation of these probes, their specificity and sensitivity were tested against seven strains of the same species and 54 strains of heterologous bacteria with fixed whole cells as targets. The probes for B. adolescentis, B. breve, and B. longum showed efficient and specific hybridization. Although the probes for B. bifidum and B. infantis cross-reacted with a few bacterial strains not isolated from humans, these probes showed species specificity for human intestinal bacteria. These 16S rRNA probes should prove valuable for the identification and detection of human intestinal Bifidobacterium species.     

Species-specific oligonucleotide probes for five Bifidobacterium species detected in human intestinal microflora.

Portions of the 16S rRNA from closely related species of the genus Bifidobacterium that are found in the human intestinal microflora were sequenced in order to design species-specific oligonucleotide probes. Five oligonucleotide probes ranging from 16 to 19 bases in length and complementary to 16S rRNA sequences from Bifidobacterium adolescentis, B. bifidum, B. breve, B. infantis, and B. longum were synthesized. With crude high-molecular-weight RNA preparations as targets, these probes showed the desired species specificity, even down to a 1-nucleotide difference. For the practical evaluation of these probes, their specificity and sensitivity were tested against seven strains of the same species and 54 strains of heterologous bacteria with fixed whole cells as targets. The probes for B. adolescentis, B. breve, and B. longum showed efficient and specific hybridization. Although the probes for B. bifidum and B. infantis cross-reacted with a few bacterial strains not isolated from humans, these probes showed species specificity for human intestinal bacteria. These 16S rRNA probes should prove valuable for the identification and detection of human intestinal Bifidobacterium species.     

Use of oligonucleotide probes to study the relatedness of delta-endotoxin genes among Bacillus thuringiensis subspecies and strains.

Fifteen Bacillus thuringiensis strains representing 13 serotypes were screened with five oligodeoxyribonucleotide probes specific for certain regions of two published sequences and one unpublished sequence of B. thuringiensis delta-endotoxin genes. Of the 15 cultures, 14 hybridized with at least one probe; the B. thuringiensis subsp. thompsoni strain alone did not hybridize. Two B. thuringiensis subsp. kurstaki strains of commercial interest, HD-1 and NRD-12, were found to be so closely related as to be indistinguishable with this technique; the same situation was found with strains from B. thuringiensis subspp. dendrolimus and sotto. Five strains were identified as probably containing only one endotoxin gene. A probe specific for the gene from the B. thuringiensis subsp. kurstaki HD-73 strain hybridized to only 3 of the 15 cultures tested. The hybridization data suggest that the DNA sequences coding for the C-terminal region of the endotoxin protein are as well conserved as those coding for the N-terminal toxic portion.     

Colonic microbiota signatures across five northern European countries

The composition of the colonic microbiota of 91 northern Europeans was characterized by fluorescent in situ hybridization using 18 phylogenetic probes. On average 75% of the bacteria were identified, and large interindividual variations were observed. Clostridium coccoides and Clostridium leptum were the dominant groups (28.0% and 25.2%), followed by the Bacteroides (8.5%). According to principal component analysis, no significant grouping with respect to geographic origin, age, or gender was observed.