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.     

Haemagglutination by the Bacteroides fragilis group

Adhesive properties of five species of Bacteroides were compared by direct haemagglutination with erythrocytes of different origin. Only strains of Bacteroides fragilis agglutinated erythrocytes and different patterns of haemagglutination were observed. None of eight carbohydrates tested inhibited haemagglutination. The activity was destroyed by heat and by periodate treatment, but not by three proteases tested.     

Post-antibiotic effect in Bacteroides fragilis group

Post-antibiotic effect (PAE) is the transient suppression of bacterial growth after brief antimicrobial exposure. While numerous reports have described PAE with aerobic and facultative microorganisms, virtually no studies have been conducted with anaerobic isolates. Intraabdominal isolates of the Bacteroides fragilis group were exposed for one hour to antibiotic (cefoxitin, cefotetan, and imipenem) concentrations two to four times the minimal inhibitory concentration (MIC). Post-antibiotic effect was described as the difference between the time required for microbial growth in the test versus the control to increase one Log10 above the quantitation observed immediately after drug removal. Bacteroides fragilis, B. ovatus, B. thetaiotaomicron and B. vulgatus exhibit PAEs for all test compounds. The time intervals for the PAEs were strain variable and ranged from six to 50 hours. No PAE was demonstrated with B. distasonis strains by the broth dilution technique. The results suggest that brief high dose exposure of some members of the B. fragilis group to anaerobe active beta-lactams produces a prolonged suppression in growth. In theory, a prolonged PAE could influence the dosage regimentation of selective antibiotics.     

Detection of beta-lactamases in the Bacteroides fragilis group

One hundred and thirty three strains of Bacteroides fragilis group isolated from clinical specimen were tested for beta-lactamase activity against five beta-lactam antibiotics, ampicillin, cefaloridin, cefuroxime, cefotaxime and cefoxitin. The Minimal Inhibitory Concentration of antibiotics was determined using the agar dilution method. Beta-lactamase production was detected by a microbiological method in 128 of the 133 (96%) strains. The detected beta-lactamases had a broad-spectrum activity, hydrolyzing both penicillins and cephalosporins (101 strains). Some strains had a wide activity against beta-lactam antibiotics, including cefoxitin (7 strains); among these strains, 3 were found hydrolyzing imipenem.     

Lectinlike adhesins in the Bacteroides fragilis group.

Lectinlike adhesins were identified in the Bacteroides fragilis group by using sugars immobilized on agarose beads either with whole bacteria by direct microscopic examination or with soluble extracts by immunoaffinoelectrophoresis. These two methods allowed the identification of two sugars reacting with whole bacteria and with the corresponding extracts: alpha-D-glucosamine and D-galactosamine. Among eight strains tested representing seven species, the two strains of B. fragilis were equally adhesive and showed the greatest adhesions. The lectinlike adhesin was purified by affinity chromatography on glucosamine-agarose or galactosamine-agarose and showed one band at 70,000 daltons in sodium dodecyl sulfate-polyacrylamide gel electrophoresis. This lectinlike adhesin may help to elucidate the roles of the B. fragilis group in the colonization of intestinal surfaces and in the predominance of B. fragilis in infections alone and in synergy with other bacteria.     

Adherence of Bacteroides fragilis group species.

The ability of piliated and capsulated Bacteroides fragilis and Bacteroides ovatus to adhere to intestinal cells and mucus was investigated. The adherence of piliated and capsulated strains was at least five times greater than the adherence of their nonpiliated and noncapsulated or capsulated only counterparts. These data illustrate the importance of pili as promoters of adherence of B. fragilis group species to the gastrointestinal mucosa.     

Difficulties in the serodiagnosis of infection with the fragilis group of Bacteroides

Bacteroides antibodies were studied in sera from 74 patients infected with the fragilis group of Bacteroides and 74 healthy control persons, by immunofluorescence of 26 different serotypes of the fragilis group. Antibodies were present at titres of 10-320 in 65 (88%) patients and 50 (68%) controls (p less than 0.01). Titres of greater than or equal to 80 were demonstrated in sera of 38 (51%) patients and 5 (7%) controls (p less than 0.01). Specific IgM antibodies were detected in sera of 42 (57%) patients at a geometric mean titre (GMT) of 30, and 8 (11%) controls at a GMT of 11 (p less than 0.01). High antibody titres as well as specific IgM were found in 32 (43%) patients, while none of the controls showed such a combination (p less than 0.01). The majority of positive patients' sera (57%) reacted with five or more serotypes, whereas most positive control sera (51%) reacted against only one or two serotypes (p less than 0.01). A selected combination of serotypes not reacting with the control sera showed positive reactions with 52 (70%) patients' sera. These findings may be useful in devising schemes for the serodiagnosis of infection caused by the fragilis group of Bacteroides. However, there are indications of geographic variation in prevalence of serotypes, which may prevent the development of a single universal scheme.     

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.     

Antimicrobial resistance patterns of Bacteroides fragilis group organisms in Korea

Antimicrobial resistance patterns of 913 clinical isolates of Bacteroides fragilis group organisms were monitored during an 8-year period in Korea. In general the resistance rates of the non-fragilis B. fragilis group species were higher than those of B. fragilis for all the drugs tested. The rate of resistance to clindamycin remarkably increased and those to some beta-lactam drugs such as piperacillin and cefotaxime also increased. No isolates were found to be resistant to imipenem, metronidazole, or chloramphenicol. beta-lactam and beta-lactamase inhibitor combinations and cefoxitin were more active than the other beta-lactams. Therefore, these agents may be considered when empirical selection of antimicrobial agents is required to treat severe anaerobic infections.     

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.     

Bacteriocin-like activity of Bacteroides fragilis group isolated from marmosets.

The ability of strains of the B. fragilis group, isolated from the oral cavity and intestine of marmosets, to produce bacteriorin-like substances in solid medium, in terms of auto-, iso- and heteroantagonism, was evaluated. Antagonistic activity was exhibited by 52% of the intestinal strains, 3 of which showed autoantagonistic activity. Three out of 9 oral strains isolated, tested against themselves, showed simultaneous isoantagonism to 4 indicator strains; but not autoantagonism. The same 9 oral strains, when tested against 16 reference strains, revealed interspecific activity only against 2 Gram-positive microorganisms. Higher activity, evaluated by the size of the inhibition halo, was observed in BHI-S agar, and greatest inhibition was obtained after 72 h of incubation.     

Pneumocephalus associated with Bacteroides fragilis meningitis

Gas within the intracranial cavity (pneumocephalus) commonly results from trauma or after surgery and rarely from infection by gas-forming organisms. The presence of pneumocephalus in the absence of injury or surgery should raise the suspicion of anaerobic infection of the central nervous system. I present a case of pneumocephalus associated with Bacteroides fragilis meningitis where the diagnosis was suspected after CT findings become available. Bacteroides fragilis meningitis is rare and often occurs in premature infants and neonates; only few cases are reported in adults. Pneumocephalus associated with Bacteroides fragilis meningitis is not described in the literature. This case also illustrates the absence of classic findings of meningeal irritation in the elderly. The literature is reviewed.     

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.     

Detection of imipenem-resistant and metronidazole-resistant Bacteroides fragilis group strains in fecal samples

Objective: To investigate the imipenem and metronidazole resistance profiles of Bacteroides fragilis group strains in fecal samples and to detect the resistance genes ( ccrA and nim ) coding for imipenem and metronidazole resistance in B. fragilis group strains.
Methods: In total, 925 fecal samples, 729 from consecutive diarrhea patients and 196 from healthy controls, were collected at Huddinge University Hospital in 1997. A modified disk diffusion method was employed to screen for imipenem-resistant and metronidazole-resistant B. fragilis group strains. In strains considered resistant by the modified disk diffusion method, the minimum inhibitory concentrations (MICs) were further determined by the agar dilution method. PCR assays were used to detect the carbapenem-hydrolyzing metallo-β-lactamase gene ( ccrA ) and the 5-nitroimidazole resistance genes ( nim ) in pure cultures (purePCR), directly from fecal samples through direct broth enrichment (dirPCR) and by immunomagnetic separation (imsPCR).
Results: Two imipenem-resistant B. fragilis strains, one of which was simultaneously resistant to metronidazole, and two B. fragilis group strains with MICs near the breakpoint for metronidazole resistance, were isolated from the fecal samples of diarrhea patients. The ccrA gene was identified in all the imipenem-resistant B. fragilis strains by purePCR, dirPCR and imsPCR. The nim genes were also detectable by these PCR assays.
Conclusions: The incidences of imipenem-resistant and metronidazole-resistant B. fragilis group strains were low in the investigated diarrhea patients. Simultaneous resistance to imipenem and metronidazole is of great concern in clinical medicine, and the proposed PCR assays may be useful in epidemiologic studies of distribution of resistance genes in the fecal microflora.     

Medium for selective isolation and presumptive identification of the Bacteroides fragilis group

We developed and evaluated a new medium (FRAG agar) for the selective isolation and presumptive identification of the Bacteroides fragilis group. This medium contains 1% D-glucuronic acid as a fermentable carbon source, a reduced peptone content, gentamicin, and 20% bile. Presumptive identification of the B. fragilis group was based on growth, fermentation, and typical colony morphology. A total of 75 stock culture isolates of the B. fragilis group grew well on this medium, and 69 showed evidence of fermentation. Of 90 other anaerobes, none grew well or fermented glucuronic acid. In a clinical trial of 100 specimens sent for anaerobic culture, FRAG agar inhibited 71 of 71 anaerobes not belonging to the B. fragilis group, as well as 104 of 110 facultative organisms. A total of 33 isolates of the B. fragilis group were recovered on the selective medium, whereas only 23 were recovered by routine methods. Of 23 cultures positive for the B. fragilis group on routine plates, 22 were positive on FRAG agar.     

Two cases of infections due to multidrug-resistant Bacteroides fragilis group strains

Bacteroides fragilis group strains are still considered susceptible to most antimicrobial agents used for the treatment of infections caused by anaerobic organisms. We describe two cases of infections due to isolates simultaneously resistant to clindamycin, tetracycline, cefoxitin, piperacillin-tazobactam, and imipenem and, in one of the two cases, to metronidazole. Such infections, although still rare, do exist and tend to complicate treatment.     

Adherence of Bacteroides fragilis in vivo

The ability of the encapsulated species Bacteroides fragilis to adhere to rat peritoneal mesothelium was compared to the adherence of unecapsulated strains of Bacteroides (B. distasonis, B. vulgatus, B. thetaiotaomicron, B. ovatus, and B. "other"). Adherence was assayed by attaching plexiglas plates containing 8-mm holes to the peritoneal mesothelium of anesthetized rats. Cell suspension (0.25 ml) was incubated in each well, after which the suspension was aspirated, and a 4-mm punch biopsy was removed. Viable organisms adhering to the biopsy specimen were enumerated by plate count following washing of the biopsy tissue. It was found that B. fragilis adhered significantly better to mesothelial tissue (10(3.00) colony-forming units per biopsy) than unencapsulated species of Bacteroides (10(1.07) colony-forming units per biopsy). This effect was not due to differential oxygen sensitivity of the various inocula. Immunization of rats with capsular polysaccharide did not demonstrate decrease in the adherence of B. fragilis; however, preincubation of the mesothelium with purified capsular polysaccharide resulted in a substantial reduction in adherence. These results indicate that B. fragilis adheres to rat peritoneal mesothelium better than unencapsulated species and suggests that the capsular polysaccharide of B. fragilis plays some role in this increased adherence.     

A possible role of Bacteroides fragilis enterotoxin in the aetiology of colorectal cancer

The prevalence of enterotoxigenic Bacteroides fragilis (ETBF) was investigated in stool specimens from 73 patients with colorectal cancer and from 59 control patients. Stool specimens were cultured on Bacteroides Bile Esculin agar and B. fragilis was identified by conventional methods. After DNA extraction, the enterotoxin gene (bft) was detected by PCR in 38% of the isolates from colorectal cancer patients, compared with 12% of the isolates from the control group (p 0.009). This is the first study demonstrating an increased prevalence of ETBF in colorectal cancer patients.     

Antimicrobial susceptibility of Bacteroides fragilis group isolates in Europe: 20 years of experience

The susceptibilities of 824 Bacteroides fragilis group isolates against nine antibiotics were evaluated in a Europe-wide study involving 13 countries. Species determination, by different methods, was carried out on all but one isolate. Resistance rates were evaluated according to species and geographical areas via CLSI and the European Committee on Antimicrobial Susceptibility Testing (EUCAST) breakpoints. The present data were compared with those obtained 10 and 20 years ago at a European level. High-level resistance (MIC ≥64 mg/L) to ampicillin was observed in 44.5% of the strains, which is a significant increase relative to 20 years ago (16%). Piperacillin/tazobactam was more active than amoxicillin/clavulanic acid (3.1% and 10.4% resistance, respectively), again with a resistance increase relative to earlier studies. Dramatic increases in resistance were observed for cefoxitin, clindamycin and moxifloxacin, with rates of 17.2%, 32.4% and 13.6%, respectively. The lowest resistances were found for imipenem, metronidazole and tigecycline (1.2%, <1% and 1.7%). Nonsusceptible strains to imipenem and metronidazole were more resistant to other anti-anaerobic drugs. Differences were detected between geographical areas, with higher resistance rates for moxifloxacin in Scandinavian countries (21.4%) than in Mediterranean countries (5.4%), whereas, for clindamycin, the resistance rates were higher in Mediterranean (41.8%) and lower in Scandinavian countries (22.5%). Piperacillin/tazobactam resistance was also higher in Scandinavian countries.