Relationships between serological groups and deoxyribonucleic acid homology groups in Bacteroides fragilis and related species.

The serological properties of antigens extracted from strains of Bacteroides fragilis and related species belonging to several different deoxyribonucleic acid homology groups were investigated. Antisera prepared against Formalin-treated whole cell suspensions of representative strains were tested against cell suspensions, cell wall preparations, and extracts of homologous and heterologous strains by using agglutination, immunodiffusion, and hemagglutination techniques. Serological results indicated that the species were antigenically distinct, although minor cross-reactions were observed. Homology groups, including the two B. fragilis subgroups, were relatively homogeneous, although the presence of serotypes within each homology group was suggested. Immunodiffusion tests demonstrated, however, that each possessed a mosaic antigen composition; at least 6 antigenic determinants could be demonstrated in B. fragilis 2553, and up to 10 were found in B. fragilis 2393. Hemagglutination tests using antigen extracts also indicate a mosaic of antigens in each strain.     

Strain differentiation in Bacteroides fragilis by ribotyping and computer-assisted gel analysis

It is important to distinguish between Bacteroides fragilis strains as their virulence may vary and as B. fragilis seems to be a heterogeneous species. The aim of our study was to evaluate ribotyping for differentiation of 46 strains of B. fragilis and for assessment of strain heterogeneity within and between the two DNA-DNA homology strain groups established in this species. Twenty-seven strains belonged to Johnson's DNA homology group I and eight to group II. Eleven strains had not been assigned to any group (NI group). DNA from all strains was cut with BglI, EcoRI and HindIII. Restriction fragment length polymorphisms were investigated using a non-radioactive digoxigenin-labelled cDNA probe transcribed from Escherichia coli 16S+23S rRNA. Ribotyping with BglI was most discriminatory, revealing a total of 26 different patterns by visual inspection of gels. EcoRI followed with 20 patterns and HindIII with 13 patterns. The gels from ribotyping were processed using the Dendron computer-assisted program. Strain clusters established using Dendron were not always in agreement with homology-based strain groups. Strains of the NI group fell into both homology groups. Ribotyping, as it is based on a relatively small portion of the genome, is useful for strain distinction in epidemiological studies with B. fragilis, whereas DNA-DNA homology, using the entire genome, is more reliable for taxonomy. The Dendron computer-assisted program, which enabled objective assessment of multiple banding patterns, increased the reliability of ribotyping.     

Fluorescent-antibody studies on selected strains of Bacteroides fragilis subspecies fragilis.

Antisera against seven strains of Bacteroides fragilis subspecies fragilis were produced from dense suspensions of whole cells. These sera exhibited high agglutination titers with homologous antigens. Reciprocal cross-reactions in agglutination tests with each immunizing strain yielded lower titers. Both the indirect and direct fluorescent-antibody techniques were used to evaluate these reagents in the serological identification of 24 defined strains of B. fragilis subspecies fragilis. Subspecies and even strain specificities were noted with particular antisera. A pooled antiserum and conjugate were prepared and studied. Study results showed that specific and high-titered antisera against strains within this subspecies can be produced by the methods described herein and that possibly more than one serotype exists within the seven strains studied. The development of more antibody pools will be necessary to encompass a wider antigenic coverage before the fluorescent-antibody technique can be relied upon altogether for serologically identifying isolates of B. fragilis subspecies fragilis. Test data showed that the indirect method of fluorescent-antibody staining with whole antiserum is an excellent means of identifying strains of this organism.     

Comparison of Bacteroides zoogleoformans strains isolated from soft tissue infections in cats with strains from periodontal disease in humans.

A total of 11 strains of Bacteroides zoogleoformans were isolated from 11 of 106 different cat subcutaneous "fight wound" abscesses and were among a total of 143 Bacteroides species isolated from these samples. They constituted 3.4% (11 of 325) of all anaerobic isolates. The cat strains and strains of B. zoogleoformans isolated from humans with periodontal disease were similar phenotypically as determined by biochemical reactions, polyacrylamide gel electrophoresis patterns of soluble proteins, and guanine plus cytosine ratios of DNA. Eight cat strains and five human strains tested had 45 to 54% DNA homology with the type strain of B. zoogleoformans. The eight cat strains and five human strains (excluding the type strain) were related by DNA homology at 70 to 77%. There was 85 to 90% intragroup DNA homology among the cat strains and 86 to 89% intragroup homology among the five human strains. The implications for epidemiology and human and animal ecology are discussed.     

Identification ofBacteroides fragilis by indirect immunofluorescence

Rabbit antiserum against live or heat-killed Bacteroides fragilis NCTC 9343 bacteria was titrated against hot phenol water-extracted polysaccharide antigens from five different species of the 'B. fragilis group' of bacteria using an enzyme immunoassay and shown to be specific for the B. fragilis NCTC 9343 polysaccharide. When the antiserum was used in indirect immunofluorescence, 97.1% of 244 B. fragilis strains were correctly identified. Only 8 of the other 312 Bacteroides strains were stained by the anti-B fragilis serum. The 'cross-reactive' Bacteriodes strains all belonged to the "B. fragilis group" of bacteria (i.e., B; distasonis, B. ovatus, B. thetaiotaomicron, B. uniformis, and B. vulgatus). None of the 425 aerobic enteric bacteria representing Salmonella, E. coli, Proteus, Yersinia, Shigella, Klebsiella, Enterobacter, Citrobacter, and Pseudomonas were positive using the anti-B. fragilis serum. Likewise, all the 59 gram-positive strains representing Streptococcus, Bacillus, Peptostreptococcus, Peptococcus, Propionibacterium, Lactobacillus, Eubacterium, and Clostridium did not stain. Our data shows in accordance with other findings [11], that B. fragilis strains possess a species-specific cell envelope antigen(s) which promises to be important for production of antisera, making a rapid identification of the species possible.     

Species identification of clinical isolates of Bacteroides by matrix-assisted laser-desorption/ionization time-of-flight mass spectrometry

Bacteroides fragilis and related species are important human pathogens involved in mixed infections of different origins. The B. fragilis group isolates are phenotypically very similar, grow more slowly than aerobic bacteria and, accordingly, are frequently misidentifed with classical or automated phenotypical identification methods. Recent taxonomic changes and new species accepted as members of the Bacteroides genus are not included in the different databases of commercially available identification kits. The use of matrix-assisted laser-desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) was therefore evaluated for the species identification of 277 clinical isolates of the Bacteroides genus. Species identification was carried out with MALDI Bruker Daltonik Biotyper software (Bruker Daltonik GmbH, Bremen, Germany) by comparing the mass spectrum of each strain with the mass spectra of the 3260 reference strains currently available. The results of conventional phenotypical identification of the isolates were used as a reference. 16S rRNA gene sequencing was performed for a selection of the strains that gave discrepant results and for all those inconclusively identified by MALDI-TOF MS; 270 isolates (97.5%) were unequivocally identified [log(score) ≥2.0] by comparison with the reference strains present in the MALDI Biotyper database. Of the 23 isolates for which the MALDI-TOF MS species identification differed from the conventional phenotypical identification, 11 were sequenced. The sequencing data confirmed the MALDI-TOF MS result in ten cases and, for the remaining isolate, the sequencing data did not lead to the determination of the species, but only to that of the genus ( Bacteroides sp.). The discriminating power and identification accuracy of MALDI-TOF MS proved to be superior to that of biochemical testing for Bacteroides thetaiotaomicron , Bacteroides ovatus and Bacteroides uniformis .     

Demonstration of bacteroides capsules by light microscopy and ultrastructural cytochemistry

Forty-six anaerobic gram-negative bacilli, including 26 members of the Bacteroides fragilis group (BFG), were examined for capsules by the India ink technic. Thirty-five were encapsulated, including all the BFG strains. As a follow-up, seven of these isolates and two previously studied reference strains (B. fragilis ATCC 23745 and Bacteroides vulgatus ATCC 8482) were examined for capsules by ultrastructural cytochemistry. Using the periodic acid thiocarbohydrazide silver proteinate (PATCSP) method of Thiéry, all the BFG examined were encapsulated. In addition to the reference strains, this included one strain of B. fragilis and four of Bacteroides thetaiotaomicron. One non-BFG strain showed no capsular material. Differences between these results and those reported previously with the ruthenium red technic may reflect species differences in the chemical composition of Bacteroides capsules.     

Detection of phages infecting Bacteroides fragilis HSP40 using a specific DNA probe

Nine bacteriophage isolates of Bacteroides fragilis, obtained from urban sewage and pig faeces samples using four different host strains (HSP40, RYC4023, RYC2056 and RYC3318), were compared on the basis of morphology, host range, DNA restriction patterns, DNA hybridisation and protein composition. All the phages are siphovirus and, as judged from cleavage by restriction endonucleases, their genome is composed of double-stranded DNA of similar size ( approximately 51-kb). Host range analysis differentiated two types of phages: (1) phages that clearly infect B. fragilis strains HSP40 (B40-2, B23-1, B23-2, B23-3 and B23-4, of which B40-8 is the phage type); and (2) the group of bacteriophages that were not infectious for HSP40 (B56-1, B56-2 and B18-1). Similarity in DNA restriction patterns and protein characteristics was found in the HSP40 infectious phages. Anti-B40-8 serum recognised only the proteins of the phages of this type. Although all phages showed similar major protein sizes, minor specific bands were detected. Bacteriophages B56-1, B56-2 and B18-1 showed heterogeneity in their DNA restriction profiles although some degree of DNA-DNA homology between all genomes was observed. Southern blot analysis with phage B40-8 DNA based probes identified a 1.5-kb DNA region homologous for all HSP40 phage isolates, but absent in the genome of the other phage isolates that did not infect this bacterial strain. The homologous region was used as a specific probe to specifically detect B. fragilis HSP40 phages.     

DNA probes for identification of clinically important Bacteroides species.

Conventional procedures for identifying gram-negative anaerobes such as Bacteroides spp. are cumbersome and time-consuming. A simpler approach would be to use DNA probes to identify these organisms. Since many different species of gram-negative anaerobes are isolated from clinical specimens, the most useful DNA probes would be probes that identify a few clinically significant groups of anaerobes. To obtain such probes, we cloned HindIII-digested chromosomal DNA from various Bacteroides species and then screened these probes, labeled with 32P by nick translation, for hybridization to DNA from various species of Bacteroides, Fusobacterium, and other gram-negative bacteria. We identified three DNA probes (pBFII-4, pBFII-5, and pBFII-6) that hybridized specifically to DNA from Bacteroides fragilis, the species that accounts for about half of all isolates from clinical specimens containing anaerobes. We identified one DNA probe (pBE-3) that hybridized to all members of the B. fragilis group, a group of species that resemble B. fragilis with respect to fermentation pattern and antibiotic resistances. We also identified one probe (pBO-21) that hybridized to DNA from all Bacteroides sp. strains as well as to DNA from strains of Fusobacterium necrophorum and Fusobacterium nucleatum. pBO-21, but not pBE-3, cross-hybridized with a cloned Bacteroides sp. 16S rRNA gene. The limit of detection for these probes was 10(6) bacteria. The probes could detect B. fragilis in blood culture medium and in mixed cultures with other gram-negative bacteria. Attempts to use biotin-labeled DNA probes instead of 32P-labeled probes were not successful because the Bacteroides sp. extracts contained material that bound the streptoavidin-peroxidase detection reagent.     

New medium for selection and presumptive identification of the Bacteroides fragilis group

A medium, Bacteroides fragilis bile-esculin (BBE) agar, was designed for the selection and, presumptive identification of the B. fragilis group. BBE agar contains bile, esculin, ferric ammonium citrate, hemin, and gentamicin in a Trypticase soy agar base. Growth in the presence of 20% bile and esculin hydrolysis, detected by blackening of the medium, provide presumptive evidence for the identification of the B. fragilis group. In addition to stimulating the growth of many strains of the B. fragilis group, hemin provides the option of testing isolates for catalase production. Gentamicin and bile prevent the growth of most organisms other than the esculin-positive bacteroides that can tolerate bile. Of 160 clinical isolates of the B. fragilis group tested on BBE agar, 159 grew well on the medium and 157 blackened it. Other anaerobes, Enterobacteriaceae, and enterococci either failed to grow on BBE agar or did not produce the characteristic morphology and blackening associated with isolates of the B. fragilis group. In a clinical laboratory trial, 687 specimens from patients were inoculated onto BBE agar plates. The B. fragilis group was recovered from 81 (11.8%) of these specimens in 24 to 48 h. Use of BBE agar in the clinical laboratory enables earlier recovery and identification of this important pathogen.     

Protein banding patterns of the outer membrane-enriched fraction of Bacteroides bivius.

Bacteroides bivius is a common anaerobe in female genital infections. Although protein banding patterns of outer membrane (OM) preparations of Bacteroides fragilis are well described and are homologous within the species, similar work has not been done with B. bivius. Our aims were to (i) characterize the OM banding patterns of B. bivius and compare them with those of other Bacteroides species and (ii) test clinical isolates of B. bivius against anti-B. bivius and anti-B. fragilis sera to identify different serogroups that might also exhibit different OM banding patterns. OM-enriched fractions of 27 clinical strains of B. bivius, 6 Bacteroides disiens strains, 10 B. fragilis strains, and 12 other Bacteroides strains were prepared, and sodium dodecyl sulfate-polyacrylamide gel electrophoresis was performed. Antisera to B. bivius ATCC 29303 and B. fragilis ATCC 25285 were raised in rabbits and tested against Bacteroides strains in an indirect enzyme-linked immunosorbent assay. All 27 B. bivius strains contained protein bands at 32, 27, 25, and 23 kilodaltons. This pattern was present in only 2 of 28 other strains; both of these were B. disiens. All B. bivius strains were reactive with the anti-B. bivius serum, while only 6 of 39 other strains (2 of 6 B. disiens) were reactive. Non-B. fragilis Bacteroides strains did not react with the anti-B. fragilis serum. Although there was marked homogeneity in the OM banding patterns of B. bivius, some B. disiens strains exhibited similar OM banding patterns. There appears to be some antigenic cross-reactivity between strains of B. bivius and B. disiens and very little with other Bacteroides species. These results may ultimately allow the development of rapid diagnostic tests for the B. bivius-B. disiens group.t     

A study of the antigenic composition of the fragilis group of Bacteroides

Representative strains of 22 serotypes of the fragilis group of Bacteroides and four non-fragilis control strains of B. melaninogenicus, B. disiens, B. bivius and Fusobacterium nucleatum were tested by SDS-PAGE and immunoblotting with hyperimmune rabbit sera. SDS-PAGE showed 25 polypeptide bands but, after immunoblotting, 24 antigenic bands were observed in various combinations in all the strains. Three of these were detected only in the control strains, whereas six others were present in different combinations in all strains of the fragilis group but were not present in the controls. Cluster analysis of the antigenic bands showed that the controls were antigenically different from the fragilis group strains. Strains of the fragilis group from the same geographic localities grouped in single clusters; most faecal isolates and NCTC strains appeared separate. There was no correlation between the species of Bacteroides and their antigenic structure. SDS-PAGE with immunoblotting is a superior technique for typing the fragilis group of Bacteroides. Specific antigens have been identified which may be used in the serodiagnosis of infection with these organisms.     

Cloning and characterization of the Bacteroides fragilis metalloprotease toxin gene.

Strains of Bacteroides fragilis that produce a ca. 20-kDa heat-labile protein toxin (termed B. fragilis toxin [BFT]) have been associated with diarrheal disease of animals and humans. BFT alters the morphology of intestinal epithelial cells both in vitro and in vivo and stimulates secretion in ligated intestinal segments of rats, rabbits, and lambs. Previous genetic and biochemical data indicated that BFT was a metalloprotease which hydrolyzed G (monomeric) actin, gelatin, and azocoll in vitro. In this paper, the cloning and sequencing of the entire B. fragilis toxin gene (bft) from enterotoxigenic B. fragilis (ETBF) 86-5443-2-2 is reported. The bft gene from this ETBF strain consists of one open reading frame of 1,191 nucleotides encoding a predicted 397-residue holotoxin with a calculated molecular weight of 44,493. Comparison of the predicted BFT protein sequence with the N-terminal amino acid sequence of purified BFT indicates that BFT is most probably synthesized by ETBF strains as a preproprotein. These data predict that BFT is processed to yield a biologically active toxin of 186 residues with a molecular mass of 20.7 kDa which is secreted into the culture supernatant. Analysis of the holotoxin sequence predicts a 20-residue amphipathic region at the carboxy terminus of BFT. Thus, in addition to the metalloprotease activity of BFT, the prediction of an amphipathic domain suggests that oligomerization of BFT may permit membrane insertion of the toxin with creation of a transmembrane pore. Comparison of the sequences available for the bft genes from ETBF 86-5443-2-2 and VPI 13784 revealed two regions of reduced homology. Hybridization of oligonucleotide probes specific for each bft to toxigenic B.fragilis strains revealed that 51 and 49% of toxigenic strains contained the 86-5433-2-2 and VPI 13784 bft genes, respectively. No toxigenic strain hybridized with both probes. We propose that these two subtypes of bft be termed bft-1 (VPI 13784) and bft-2 (86-5433-2-2).     

Accuracy and reproducibility of the 4-hour ATB 32A method for anaerobe identification.

The ATB 32A system (API System SA, La Balme les Grottes, Montalieu-Vercieu, France) was evaluated for use in the identification of 214 anaerobes. Organisms included 73 isolates of the Bacteroides fragilis group, 24 Bacteroides spp., 10 fusobacteria, 43 clostridia, 28 cocci, and 36 gram-positive, nonsporeforming rods. With the concomitant use of Gram stain, pigmentation, catalase testing, and aerobic growth, the ATB 32A system correctly identified 97% of the B. fragilis group isolates, 88% of Bacteroides spp., 50% of fusobacteria, 74% of clostridia, 100% of cocci, and 86% of the gram-positive, nonsporeforming rods. Overall, 188 strains (88%) were correctly identified, with 18 (8%) requiring extra tests, other than the four mentioned above, for correct identification. Eight strains were misidentified, including one Bacteroides sp., three fusobacteria, one Clostridium sp., and three gram-positive, nonsporeforming rods. Reproducibility was very good, with 12 of 14 strains (86%) tested in triplicate yielding identical correct results on each of three occasions and 2 strains (14%) yielding identical correct results on two occasions. There was a low-probability identification (including the correct species) on the third testing. The ATB 32A system represents a worthwhile advance in systems used for the identification of clinically significant anaerobic bacteria.     

Factors affecting production of catalase by Bacteroides.

Several variables affected the production of catalase by members of the "Bacteroides fragilis group" of anaerobic bacteria. Both media yielded higher catalase levels than the respective agar media. Addition of hemin to media after autoclave sterilization, rather than before, significantly increased production of catalase. Both of these variables could be related to the available hemin concentration present in the medium being tested. Significantly higher amounts of hemin were required for catalase production than were required for growth. For catalase production by B. fragilis ATCC 25285, 1 microgram of hemin per ml was required. Of the various media tested, the use of chopped meat broth resulted in the highest levels of catalase production (up to 50 to 60 U of catalase per mg of protein). Of the various species and DNA homology groups tested, strains of B. fragilis and Bacteroides distasonis were catalase positive. Strains of Bacteroides thetaiotaomicron, Bacteroides ovatus, and Bacteroides eggerthi possessed variable catalase activity. Bacteroides vulgatus, Bacteroides uniformis, and DNA homology groups "3452A" and "subsp. a" were catalase negative. A catalase well test, in which equal volumes of 3% H2O2 and chopped meat culture are mixed, is described and recommended for routine catalase tests.     

Reaction of clinical isolates and typed strains of the family Bacteroidaceae with rabbit anti-Bacteroides sera examined by radial immunodiffusion.

Antisera were produced which were reactive with Bacteroides fragilis, B. gingivalis, or B. melaninogenicus subsp. melaninogenicus. Anti-B. gingivalis serum exhibited strong reactions with strains of homologous species and reacted with only one other species, B. thetaiotaomicron. Antigens shared by organisms of the B. fragilis group and B. melaninogenicus subspecies of the Bacteroidaceae must be considered when serological methods are used for their identification.     

The isolation and identification of Bacteroides spp. from the normal human gingival flora

Gram-negative anaerobic bacilli were isolated on an enriched selective medium from specimens of subgingival plaque from 20 normal, healthy adults without periodontal disease. a heavy growth of Bacteroides and Fusobacterium spp. was obtained from all specimens and 10 representative colonies from each subject were selected for identification by conventional bacteriological methods. Results were obtained in these tests with 188 isolates; some strains of the B. melaninogenicus/oralis group would not grow in pure culture on solid media and showed satellitism in mixed culture, and some strains of fusobacteria did not survive repeated subculture. Most isolates (68%) belonged to the B. melaninogenicus/oralis group. The commonest species identified in this group were B. oralis (41%), B. melaninogenicus ss. melaninogenicus (26%) and ss. intermedius (17%). B. oralis was isolated from all subjects and at least one strain of B. melaninogenicus was isolated from 18 out of 20 subjects. Only eight strains of the B. fragilis group were detected and they probably represented transient carriage. Fusobacterium spp. and L. buccalis were isolated from 15 out of 20 subjects and accounted for 21% of the isolates studied, but most could not be identified specifically by the set of tests used. Asaccharolytic species of Bacteroides were isolated in small numbers from seven subjects.     

Pathogenicity of the Bacteroides fragilis group

The Bacteroides fragilis group is one of the most important pathogens in polymicrobial infections. The distribution of the different members of the B. fragilis group in clinical infections varies. Bacteroides fragilis accounts for 63 percent of all the group isolates, Bacteroides thetaiotaomicron for 14 percent, Bacteroides vulgatus and Bacteroides ovatus for seven percent each, Bacteroides distasonis for six percent and Bacteroides uniformis for two percent. All members of the group induced bacteremia that was associated with an average mortality of 27 percent. The B. fragilis group resist beta lactam antibiotics by producing the enzyme beta-lactamase. This enzyme can be detected in abscess fluid, and can interfere with the eradication of other bacteria that are susceptible to penicillins and cephalosporins. All members of the B. fragilis group can become encapsulated during an inflammatory process as was demonstrated in a subcutaneous abscess model in mice. Non-encapsulated strains can become encapsulated with the assistance of their aerobic counterparts. These encapsulated strains are more virulent to the host than non-encapsulated strains. This increased virulence can be demonstrated by a higher rate of induction of bacteremia, and a greater enhancement of growth of other bacteria in mixed infection. Antimicrobial therapy directed only at the eradication of the aerobic bacteria did not prevent encapsulation, or reduce the number of Bacteroides species. The virulence of all members of the B. fragilis group highlights the need to direct antimicrobial therapy against all members of this group.     

Antimicrobial susceptibility of Bacteroides fragilis group isolates in Europe

Objective  To evaluate the activity of old and newer antianaerobic drugs against clinical isolates of Bacteroides fragilis group strains from different parts of Europe.
Methods  Bacteroides fragilis group isolates from 37 laboratories in 19 countries were biochemically characterized. The MICs of seven antimicrobial agents were determined by the agar dilution method as recommended by the NCCLS. Production of beta-lactamase was detected by nitrocefin.
Results  There were 1284 B. fragilis group isolates included in the study. Abdominal infections and wounds were the most common sources of isolation and B. fragilis was the dominating species. Ninety-nine percent of the strains were resistant to ampicillin (breakpoint 2 mg/L), 6% to cefoxitin (64 mg/L), 15% to clindamycin (8 mg/L) and 9% to moxifloxacin (8 mg/L). Less than 1% were resistant to imipenem (16 mg/L), piperacillin-tazobactam (128 mg/L) and metronidazole (32 mg/L). Ninety-six percent of the isolates were beta-lactamase producers.
Conclusions  Antimicrobial resistance among the B. fragilis group is increasing.     

Preliminary study using species-specific oligonucleotide probe for rRNA of Bilophila wadsworthia.

Portions of the 16S RNA from a urease-positive Bilophila wadsworthia strain were sequenced, and a probe was constructed. The probe was end labeled with [32P]ATP and polynucleotide kinase and hybridized on a nylon filter (by dot blot hybridization) to the immobilized rRNA of 12 B. wadsworthia strains and eight other anaerobic isolates. The probe efficiently hybridized only to the Bilophila strains. Cross-reactivity at high RNA levels (2,000 ng) was observed with one strain of Bacteroides thetaiotamicron and one strain of Bacteroides fragilis (with 10x SET buffer [20x SET buffer is 0.5 M NaCl, 0.03 M Tris, and 2 mM EDTA]) but was not seen at lower RNA levels or with 5x SET buffer. When tested against mixed cultures of aerobic and anaerobic isolates representative of appendiceal abscess flora, the probe did not react with mixed cultures containing no Bilophila cells and could detect > or = 10(5) Bilophila CFU/ml when the mixture was seeded with Bilophila cells. This probe is of potential use in the rapid identification of pure isolates and in the direct identification of B. wadsworthia in clinical specimens.