Detection of cfxA and cfxA2, the beta-lactamase genes of Prevotella spp., in clinical samples from dentoalveolar infection by real-time PCR

While most bacteria involved in dentoalveolar infection are highly susceptible to penicillin, some Prevotella strains exhibit resistance to this agent through the production of beta-lactamase. The production of beta-lactamase by Prevotella spp. is in turn associated with the expression of the genes cfxA and cfxA2. The aim of the present study was to determine the prevalence of cfxA and cfxA2 in Prevotella strains by use of real-time PCR and to assess the performance of this molecular method for the direct detection of the genes in 87 clinical samples (pus and root canal exudates) from dentoalveolar infection. Production of beta-lactamase by each isolate was determined using a nitrocefin disk. beta-Lactamase production was seen in 31% of Prevotella isolates, while all isolates of other species were beta-lactamase negative. The penicillin resistance of isolates strongly correlated with the production of beta-lactamase. Real-time PCR was found to detect the cfxA and cfxA2 genes from at least five cells per reaction mixture (5 x 10(3) CFU/ml of pus). Using real-time PCR, the presence of cfxA and cfxA2 was evident for all 48 beta-lactamase-positive Prevotella strains. In contrast, neither beta-lactamase-negative Prevotella (n = 91) or non-Prevotella (n = 31) strains were positive for the genes. In this study, 31 of the 87 samples yielded beta-lactamase-positive Prevotella results, and cfxA and cfxA2 were detected in all 31 samples. Of the 56 culture-negative samples, 8 (14%) were positive for cfxA and cfxA2 by the real-time PCR. This sensitive and specific molecular method offers a rapid clinical test for aiding in the selection of an appropriate antibiotic for treatment of dentoalveolar infection. Although penicillin remains largely effective in the treatment of dentoalveolar infection, beta-lactamase-stable antibiotics should be considered in cases in which beta-lactamase-positive Prevotella strains are involved.     

Expression of the pilin gene from Bacteroides nodosus in Escherichia coli.

Bacterial plasmids that direct the expression in Escherichia coli of the pilin of Bacteroides nodosus were constructed. The quantity of pilin produced was greater than that of the pilin synthesized by B. nodosus, but no surface structural pili were present; pilin was found associated with the inner membrane of E. coli. Vaccination of sheep with E. coli containing pilin elicited increases in agglutinating and enzyme-linked immunosorbent assay antibody titers, which in turn were lower than the titers in sheep immunized with pilin from B. nodosus. The E. coli-produced pilin vaccine initially appeared to delay the progression of infection in immunized sheep after a challenge with virulent homologous B. nodosus, but at a later time the severity of foot rot was similar to that in sheep vaccinated with a placebo.     

Determination of bft gene subtypes in Bacteroides fragilis clinical isolates

A rapid multiplex PCR approach was developed to detect the bft gene subtypes in Bacteroides fragilis clinical isolates. This technique could be used to look at the epidemiology of enterotoxigenic strains of B. fragilis in clinical infections and whether there is a correlation between disease and the presence of B. fragilis enterotoxin.     

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).     

Presence of katG gene in resistant Mycobacterium tuberculosis.

It has been reported recently that isoniazid resistant strains of Mycobacterium tuberculosis have lost the katG gene which encodes the catalase-peroxidase enzyme. A 35 mer oligonucleotide probe specific for the katG gene of M tuberculosis, 3' end-labelled with digoxigenin, was constructed and hybridised with DNA extracted from 26 clinical isolates of M tuberculosis under high stringency conditions. Twenty two of these isolates were resistant to 0.2 microgram/ml isoniazid and 20 to 1.0 microgram/ml isoniazid. Semiquantitative detection of catalase did not show any discrimination between isoniazid sensitive and resistant strains. The katG gene was present in all clinical strains of M tuberculosis. Therefore, complete deletion of the katG gene does not seem to be the mechanism of isoniazid resistance in M tuberculosis strains isolated from patients in India.     

Immunochemistry of the cell surfaces of Bacteroides bivius and Bacteroides disiens

Outer membranes were extracted from seven strains of Bacteroides bivius and six strains of B. disiens by the Sarkosyl method. Lipopolysaccharides (LPS) were extracted from the same strains by the Proteinase K method, and from three strains of each species by an aqueous phenol method. Analysis of the outer-membrane proteins by SDS-PAGE demonstrated that, within a species, very similar patterns with many shared or common bands were produced, but there were sufficient differences between species to allow separation. Immunoblotting with antisera raised against whole cells of each of the type strains showed that many antigens were shared between species. Smooth LPS was present in both species. By immunoblotting, the O-antigen of B. disiens was shown to be common to all six strains, and there was no cross-reaction between the B. disiens antiserum and B. bivius LPS. The O-antigen of B. bivius was not detected by immunoblotting with homologous antiserum, but antiserum to B. bivius reacted with a series of common low molecular mass antigens that were present in LPS preparations from strains of both species.     

Isolation of metronidazole-resistant Bacteroides fragilis carrying the nimA nitroreductase gene from a patient in Washington State

Members of the Bacteroides fragilis group are among the most common anaerobic bacterial isolates in clinical specimens. Metronidazole, a 5-nitroimidazole, is often used as empirical therapy for anaerobic infections. Susceptibility testing is not routinely performed because of nearly universal susceptibility of Bacteroides spp. to this agent. We report a case of metronidazole-resistant Bacteroides fragilis in the United States and demonstrate the presence of the nimA gene, encoding a nitroreductase previously shown to mediate resistance to 5-nitroimidazole antimicrobial agents in B. fragilis strains from Europe and Africa. Because clinical failures in Bacteroides infections have been associated with the use of inactive antimicrobial agents, clinicians need to be aware of the possibility of metronidazole-resistant B. fragilis strains in the United States and the importance of susceptibility testing in selected situations.     

Cytotoxic activity of Bacteroides gingivalis and Bacteroides asaccharolyticus

Culture filtrates of all eight strains of Bacteroides gingivalis and all five strains of B. asaccharolyticus were toxic for Vero cells. Cytotoxicity was in general greater with material from cultures of B. gingivalis than from B. asaccharolyticus but none of the culture filtrates from eight strains of B. melaninogenicus showed activity in this test. The toxic material was released during prolonged incubation and more detailed study of preparations from one strain indicated that it had a molecular weight of less than 3500 and was heat stable.     

Superoxide dismutase in Bacteroides fragilis and related Bacteroides species.

Superoxide dismutase (SOD) activity was demonstrated in cell-free extracts of Bacteroides fragilis, Bacteroides vulgatus, Bacteroides distasonis, Bacteroides ovatus, and Bacteroides thetaiotaomicron. The strains were grown under anaerobic conditions in Trypticase soy broth, and the specific activity of SOD in the extracts was, in most strains, higher than in cell-free extracts of Escherichia coli B grown under anaerobic conditions. Isoelectric focusing of the extracts in polyacrylamide gel demonstrated distinct forms of SOD in the different species.     

Frequency of isolation and antibiotic sensitivity of Bacteroides related to Bacteroides bivius and Bacteroides oralis

274 strains of the Bacteroides oralis-bivius group are studied: 112 are identified to Bacteroides bivius or B. disiens, 73 to Bacteroides oralis and 49 to Bacteroides oris or Bacteroides buccae. These strains are isolated from clinical sample: gynecologic suppurations or respiratory tract infections. The susceptibility of 63 strains to 7 antibiotics were determined. Tested antibiotics were: cefalotin, cefoxitine, cefotaxime, mezlocillin, clindamycin, metronidazole and colistine. Cefalotine has a poor activity against these strains.     

Prevalence of fragilysin gene in Bacteroides fragilis isolates from blood and other extraintestinal samples

Of 166 Bacteroides fragilis isolates, 26.2% of 103 isolates from blood and 20.6% of 63 extraintestinal isolates harbored the fragilysin gene (difference not statistically significant). Clinical characteristics and evolution were comparable in patients with B. fragilis bacteremia with or without this enterotoxin. Fragilysin seems not to be an important virulence factor in B. fragilis disease.     

Bacteroides endodontalis and other black-pigmented Bacteroides species in odontogenic abscesses.

Twenty-eight odontogenic abscesses were examined for the presence of black-pigmented Bacteroides spp. Of the 28 samples, 26 were found to contain one or more species of black-pigmented Bacteroides. Abscesses were divided into three categories according to the tissue of origin: endodontal, periodontal, and pericoronal. Four abscesses which developed after extraction were also examined. It was found that Bacteroides endodontalis, a newly described species of asaccharolytic black-pigmented Bacteroides, was isolated almost exclusively from periapical abscesses of endodontal origin. B. intermedius proved to be the most frequently isolated species in all of the samples. B. gingivalis was present in all of the periodontal abscesses studied, as well as in two endodontal abscesses. B. melaninogenicus was recovered once from a pericoronal abscess. Precautions for the isolation of B. endodontalis are discussed.     

Polymerase chain reaction amplification of the constant and variable regions of the Bacteroides nodosus fimbrial gene.

Serogrouping of Bacteroides nodosus is based on antigenic differences in fimbriae of the different New Zealand prototype strains. Because of the time needed to isolate and grow pure cultures of B. nodosus and the difficulty in distinguishing between different serogroups because of cross-agglutination, a new DNA-based diagnostic approach based on the fimbrial gene sequence of B. nodosus was developed. Published nucleotide sequences of the fimbrial genes for serogroups A, G, D, and H showed conservation at the 5' end, coding for the N terminus, and variability at the 3' end, coding for the C terminus. The polymerase chain reaction was used to amplify both the constant and variable regions of the fimbrial genes. Constant-region oligonucleotide primers were used to amplify a 100-base-pair fragment from the constant regions of the fimbrial genes of 10 New Zealand serogroups. Serogroup-specific oligonucleotide primers for serogroups A and H allowed amplification of a 282-base-pair fragment from serogroup A and a 363-base-pair fragment from serogroup H. Thus, amplification of the constant and variable regions of the fimbrial gene allows rapid detection and grouping of B. nodosus.     

Bacteroides ureolyticus (NTU) medium for the selective recovery of Bacteroides gracilis

Bacteroides gracilis is a gram-negative anaerobic bacillus which requires formate and fumarate for growth; it has been implicated in periodontal disease and serious infections of the head and neck. In this study, Bacteroides ureolyticus (NTU) medium was tested for its ability to allow the growth of B. gracilis and other formate-fumarate requiring gram-negative anaerobes and to enable the recovery of these organisms from clinical specimens. All reference strains grew on NTU medium with the exception of Wolinella recta and formate-fumarate requiring organisms were isolated from 18 of 20 samples of subgingival dental plaque from patients with chronic periodontitis. B. gracilis was the commonest species isolated (14 of the 29 isolates); B. ureolyticus was not found.     

Lipopolysaccharides of Bacteroides intermedius (Prevotella intermedia) and Bacteroides (Porphyromonas) gingivalis induce interleukin-8 gene expression in human gingival fibroblast cultures.

Lipopolysaccharides (LPS) prepared from Bacteroides intermedius (Prevotella intermedia) and Bacteroides (Porphyromonas) gingivalis by hot phenol-water extraction induced interleukin-8 (IL-8) mRNA in normal human gingival fibroblast cultures, as demonstrated by Northern (RNA) blot analysis. IL-8 mRNA levels began to increase after a 2-h exposure, reached a maximum after 12 h, and then dropped to the unstimulated level at 48 h. IL-8 mRNA levels were also enhanced in a dose-dependent manner. By contrast, LPS specimens from various Salmonella species with S and R chemotypes and bacterial [corrected] and synthetic lipid A preparations did not increase IL-8 mRNA levels in fibroblasts. Although recombinant human IL-1 alpha induced IL-8 mRNA expression in fibroblast cultures, an antiserum to recombinant human IL-1 alpha did not decrease the IL-8 mRNA accumulation induced by B. intermedius LPS. Fibroblasts primed with natural human gamma interferon (IFN-gamma) expressed higher IL-8 mRNA levels upon stimulation with B. intermedius LPS, but not with Salmonella LPS, compared with nontreated cells. Natural human IFN-beta exhibited a similar priming effect on the fibroblasts, and antiserum to IFN-beta added to the cultures together with B. intermedius LPS decreased the IL-8 mRNA levels. Therefore, endogenous IFN-beta enhanced IL-8 mRNA production in response to B. intermedius LPS in fibroblasts.     

Evaluation of xylan fermentation for the identification of Bacteroides ovatus and Bacteroides thetaiotaomicron.

The reliability of xylan fermentation in distinguishing Bacteroides ovatus from Bacteroides thetaiotaomicron was examined with strains previously identified by DNA homology studies or bacteriophage sensitivity testing or both. All of the 12 B. ovatus strains produced acid ranging from pH 5.0 to 5.6 in peptone-yeast-xylan broth; none of the 19 B. thetaiotaomicron strains produced acid, and the final pH was 6.0 or 6.1. Xylan fermentation and bacteriophage sensitivity-DNA homology appear to be equivalent in their ability to differentiate these two Bacteroides species.     

Bacteriologic and clinical study of Bacteroides oris and Bacteroides buccae

We characterized clinical isolates previously identified in our laboratory as Bacteroides ruminicola, the human strains of which are now classified as Bacteroides oris and Bacteroides buccae. A total of 72 isolates (55 B. buccae isolates and 17 B. oris isolates) recovered over a 10-year period were studied. They were differentiated from each other by special-potency antibiotic disks and the RapID-ANA system. The two organisms were associated with a variety of infections, the majority being pleuropulmonary (29.2%) and infections of the head and neck region (27.8%). The infections were always polymicrobial, usually with more than five organisms per specimen. A total of 44% of the B. oris strains and 27% of the B. buccae strains were resistant to penicillin G (breakpoint, 2 U/ml), and this correlated with the presence of beta-lactamase. Although B. oris and B. buccae are found with some frequency in human infections, they are present primarily as components of a mixed flora.