Bactericidal activity of metronidazole against Bacteroides fragilis

Metronidazole was found to be active against Bacteroides fragilis strains isolated from human lesions. The minimal inhibitory concentrations (MIC) were from 0.16 to 2.5 mug/ml and the minimal bactericidal concentrations (MBC) were from 0.16 to 2.5 mug/ml; usually the MIC and MBC figures were equivalent. These levels are easily attainable in the serum following normal therapeutic doses. The drug is not toxic and side effects are rare and it would therefore seem highly suitable for treating Bacteroides infections and also may be considered prophylactically in certain situations that are described.     

The capsular polysaccharide complex of Bacteroides fragilis induces cytokine production from human and murine phagocytic cells.

To stimulate early-phase immunologic events following Bacteroides fragilis infection in the peritoneal cavity, we examined the cytokine response of several cell types to purified capsular polysaccharide complex (CPC) and lipopolysaccharide (LPS) of this organism. Cytokines were produced from murine resident peritoneal (MRP) cells as well as human peripheral blood leukocytes. MRP cells cocultured with either B. fragilis CPC of LPS in vitro produced tumor necrosis factor alpha and interleukin-1alpha (IL-1alpha). In addition, MRP cells challenged with CPC produced IL-10. Human peripheral blood monocytes and polymorphonuclear leukocytes secreted IL-8 when cultured in the presence of CPC.     

Genetic diversity of the capsular polysaccharide C biosynthesis region of Bacteroides fragilis

A genetic approach was used to assess the heterogeneity of the capsular polysaccharide C (PS C) biosynthesis locus of Bacteroides fragilis and to determine whether distinct loci contain genes whose products are likely to be involved in conferring charged groups that enable the B. fragilis capsular polysaccharides to induce abscesses. A collection of 50 B. fragilis strains was examined. PCR analysis demonstrated that the genes flanking the PS C biosynthesis region are conserved, whereas the genes within the loci are heterogeneous. Only cfiA(+) B. fragilis strains, which represent 3% of the clinical isolates of B. fragilis, displayed heterogeneity in the regions flanking the polysaccharide biosynthesis genes. Primers were designed in the conserved regions upstream and downstream of the PS C locus and were used to amplify the region from 45 of the 50 B. fragilis strains studied. Fourteen PS C genetic loci could be differentiated by a combination of PCR and extended PCR. These loci ranged in size from 14 to 26 kb. Hybridization analysis with genes from the PS C loci of strains 9343 and 638R revealed that the majority of strains contain homologs of wcgC (N-acetylmannosamine dehydrogenase), wcfF (putative dehydrogenase), and wcgP (putative aminotransferase). The data suggest that the synthesis of polysaccharides that have zwitterionic characteristics rendering them able to induce abscesses is common in B. fragilis.     

Characterization of fimbriae from Bacteroides fragilis

Fimbriae derived from Bacteroides fragilis strain BE1 (BE1 fimbriae) appeared to be composed of subunits with a molecular weight of 40,000. Under the electron microscope the fimbriae could be visualized as straight filaments with a diameter of 4 nm. It appeared that production of the BE1 fimbriae is repressed under conditions of iron limitation, and at a growth temperature of 20 degrees C. Antibodies raised against the 40,000 dalton polypeptide, purified by means of preparative SDS-polyacrylamide gelelectrophoresis, recognized the native fimbriae, as was shown by immunogold labelling of intact bacterial cells, and by immunoprecipitation. Immunoblot experiments showed that other strains of B fragilis tested produced polypeptides, ranging in molecular weight from 40,000 to 42,000, that are antigenically related to the BE1 fimbrial subunit. No haemagglutination activity could be associated with the BE1 fimbriae.     

Oxygen-independent killing of Bacteroides fragilis by granule extracts from human polymorphonuclear leukocytes.

Granule proteins from human neutrophils were prepared by extraction with acetate, and their antibacterial activity against Bacteroides fragilis was determined. Activity was highly dependent on pH; greatest killing occurred at the most acid pH tested (pH 5.0). Optimum activity was observed at physiological ionic strength and low bacterial numbers. Killing was inhibited by incubation temperatures of less than 37 degrees C. Eight times more extract was required to kill 50% of stationary-phase bacteria, compared with those growing in logarithmic phase. The antibacterial effect of granule extract was destroyed by boiling, but some activity was retained after heating to 56 degrees C and 80 degrees C. Granule extract activity was tested under conditions in which oxygen-dependent antibacterial systems were inhibited. The rate and extent of killing was not affected by anaerobiosis, sodium azide, or cysteine hydrochloride. These results suggest that the activity of granule extract is independent of oxidative antibacterial systems, and therefore, under conditions that occur in anaerobic infections, potent leukocyte granule-associated mechanisms exist for the destruction of B. fragilis.     

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.     

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.     

Utilization of haem from the haptoglobin-haemoglobin complex by Bacteroides fragilis

Possession of specialized iron acquisition systems is a prerequisite for the survival of pathogenic bacteria in their host. The purpose of this study was to determine whether Bacteroides fragilis, a clinically important Gram-negative anaerobic bacterium, possesses a specific haem-uptake system. Growth studies indicated that this microorganism can utilize haem from either haemoglobin or haptoglobin-haemoglobin as its sole source of iron. Iron-repressible haem-binding protein complexes (HBP complexes), involved in the uptake of haem from haptoglobin-haemoglobin were detected by means of lithium dodecyl sulfate polyacrylamide gel electrophoresis (LDS-PAGE). Four polypeptides of approximately 60, 58, 49 and 35 kDa, which are part of these HBP complexes, were identified as haem-binding proteins. A 44 kDa iron-repressible outer-membrane protein is needed for a functional HBP complex, but the exact role of this protein in the uptake of haem is still unknown.     

Non-toxigenic Bacteroides fragilis (NTBF) administration reduces bacteria-driven chronic colitis and tumor development independent of polysaccharide A

Polysaccharide A (PSA), an immunogenic capsular component of non-toxigenic Bacteroides fragilis (NTBF) strain NCTC 9343, is reported to promote mucosal immune development and suppress colitis. Contrastingly, enterotoxigenic Bacteroides fragilis (ETBF) is highly associated with inflammatory bowel disease (IBD) and colorectal cancer (CRC), rapidly inducing IL-17-dependent murine colitis and tumorigenesis. In specific-pathogen-free (SPF) C57BL/6 wild-type (WT) and multiple intestinal neoplasia (Min^Apc716+/−) mice, we show that sequential treatment of the NTBF strain, 9343, followed by the ETBF strain, 86-5443-2-2 (86), diminished colitis and tumorigenesis. Mice treated simultaneously with 9343 and 86 exhibited both severe colitis and tumorigenesis. Abrogated disease severity in sequentially treated mice was attributed to 9343 strain dominance and decreased IL-17A, but 86 colonization prior to or simultaneous with 9343 mitigated the anti-inflammatory effect of 9343. Remarkably, 9343-mediated protection was independent of PSA, as sequentially treated mice receiving ΔPSA 9343 exhibited similar protection. Further, SPF WT and Min mice colonized with PSA-competent or PSA-deficient 9343 exhibited similar IL-10, IL-17, and IFN-γ responses. Treatment of 86-colonized mice with 9343 failed to disrupt 86 pathogenesis. Our findings demonstrate that 9343 colonization, independent of PSA, offers prophylaxis against colitis-inducing 86 but may not be a valid therapy once colitis is established.     

Diarrheal disease caused by enterotoxigenic Bacteroides fragilis in infant rabbits.

Enterotoxigenic Bacteroides fragilis caused severe, nonhemorrhagic, watery diarrhea when 10(9) CFU of a porcine or human isolate was administered orogastrically to 3-day-old rabbits. The bacterium colonized the intestinal tract with a predilection for the large intestine (10(9) CFU/g of cecal contents). Diarrhea occurred at an average of 4.6 days postinoculation, and 84% of rabbits were dead or moribund at an average of 8.8 days postinoculation. The disease was characterized by watery diarrhea and dehydration. Severe histologic lesions including inflammation, exfoliation of epithelial cells, and crypt hyperplasia were observed throughout the colon. There was no indication of bacteremia or of bacterial adherence to or invasion of intestinal epithelial cells. Rabbits inoculated with nonenterotoxigenic B. fragilis were colonized with B. fragilis but did not develop clinical disease or intestinal lesions. While the pathogenesis of this disease is undefined, clinical signs of disease and histologic changes were consistent with a mechanism of net secretion of fluid into the small intestine and decreased absorption of fluid from the large intestine. Enteric disease caused by enterotoxigenic B. fragilis in infant rabbits was similar to naturally occurring enteric disease associated with the bacterium in humans and livestock. This study established that enterotoxigenic B. fragilis is enteropathogenic in intact infant rabbits.     

Serum antibodies against central nervous system proteins in human demyelinating disease

An immunoblotting technique has been used to screen serum samples from patients with demyelinating disease for antibody directed against central nervous system proteins. Antibodies of the IgM, IgG and IgA class directed against one or more of the particulate fraction proteins tubulin, myelin basic protein, 69 K neurofilament protein, glial fibrillary acidic protein, myelin associated glycoprotein or Wolfgram protein were present in 94, 54 and 47%, respectively, of multiple sclerosis sera examined. IgM antibodies against tubulin and myelin basic protein predominated. A similar antibody spectrum was seen in a significant proportion of sera from patients with optic neuritis, subacute sclerosing panencephalitis and motor neurone disease, in which primary or secondary demyelination occurs. Antibodies of all three classes directed against the 169 K and 220 K neurofilament proteins and against some unidentified proteins of human peripheral nerve, kidney, liver, spleen and skeletal muscle were detected in sera from healthy subjects and patients with neurological disease.     

The effect of capsular polysaccharide and lipopolysaccharide of Bacteroides fragilis on polymorph function and serum killing

The determinant responsible for the ability of Bacteroides spp. to inhibit polymorph phagocytic killing of aerobic organisms has not yet been identified. Therefore, the roles of lipopolysaccharide and capsular polysaccharide of B. fragilis were investigated. Serum-resistant and serum-sensitive strains of Proteus mirabilis were used to indicate inhibition of phagocytic killing and serum killing of aerobes. Whole organisms of B. fragilis, purified lipopolysaccharide and capsular polysaccharide were added to an in-vitro phagocytosis system. Results showed that greater than 10(7) bacteroides/ml inhibited both serum and phagocytic killing. Concentrations below 10(7)/ml had little effect on either process. Purified capsular polysaccharide (10 or 100 micrograms/ml), either alone in the system or in combination with sub-inhibitory concentrations of B. fragilis also markedly inhibited serum and phagocytic killing. Lipopolysaccharide (9 micrograms/ml) appeared relatively inert. B. ovatus, reputedly non-capsulated, produced identical results to those obtained with B. fragilis, but an encapsulated strain of Streptococcus pneumoniae did not inhibit serum or phagocytic killing.     

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.     

Purification and characterization of an enterotoxin from Bacteroides fragilis.

An enterotoxin produced by Bacteroides fragilis was purified to homogeneity and characterized as to its biological activity and basic molecular properties. Toxin preparations were prepared by growing B. fragilis VPI 13784 in brain heart infusion broth to early stationary phase, immediately precipitating the culture supernatant fluid with 70% ammonium sulfate, and stabilizing the precipitate with the protease inhibitor TPCK (tolylsulfonyl phenylalanyl chloromethyl ketone). The toxin was sequentially purified by anion-exchange chromatography on Q-Sepharose, hydrophobic interaction chromatography on phenyl-agarose, and high-resolution ion-exchange chromatography on Mono Q. The toxin appeared homogeneous as judged by polyacrylamide gel electrophoresis. The estimated molecular weight of the highly purified toxin as determined by gel filtration chromatography on Superose-12 and sodium dodecyl sulfate-polyacrylamide gel electrophoresis is 19,000. It has an isoelectric point of approximately 4.5 and is stable at pHs 5 to 10. The purified toxin is stable at -20 and 4 degrees C and upon freeze-drying, but it is unstable at temperatures above 55 degrees C. It is sensitive to proteinase K and Streptomyces protease but is resistant to trypsin and chymotrypsin. The activity of the purified toxin is neutralized by antiserum to a toxigenic strain of B. fragilis but not by antiserum to nontoxigenic strains. N-terminal amino acid analysis reveal an unambiguous sequence of Ala-Val-Pro-Ser-Glu-Pro-Lys-Thr-Val-Tyr-Val-Ile-Xxx-Leu-Arg-Glu-Asn-Gly- Ser-Thr . The highly purified toxin induced a strong fluid accumulation response in the lamb ileal-loop assay as well as a cytotoxic response (cell rounding) on HT-29 colon carcinoma cells. Thus, the purified toxin can cause both enterotoxic and cytotoxic activities.     

Isolation of enterotoxigenic Bacteroides fragilis from humans with diarrhea.

Enterotoxigenic Bacteroides fragilis was isolated from stool specimens of 8 of 44 diarrheic individuals (ages, 4 months to 69 years). The individuals had watery diarrhea and intestinal cramping; and infants had hyperthermia, vomiting, and blood in the stools. No recognized enteric pathogens were detected in seven of the eight diarrheic individuals positive for enterotoxigenic B. fragilis. The bacterium produced an enterotoxin detectable in concentrated broth that supported bacterial growth. Fifteen adult rabbits with ligated ceca developed fatal enteric disease following intraileal injection with 5 x 10(9) CFU of enterotoxigenic B. fragilis. Conversely, eight control rabbits injected with nonenterotoxigenic B. fragilis remained clinically normal. As few as 5 x 10(3) CFU of enterotoxigenic B. fragilis caused fatal enteric disease in the rabbit model. Disease in rabbits was characterized by mucoid, often hemorrhagic, diarrhea. The bacterium colonized the caudal small intestine and the colon of the rabbits and caused moderate to severe necrotizing colitis. Enterotoxigenic B. fragilis is widespread in the intestinal tract of diarrheic humans and is enteropathogenic in adult rabbits with ligated ceca. Its possible role in the enteric disease complex merits further study.     

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.     

A serogrouping scheme for the study of the epidemiology of Bacteroides fragilis

To study the hospital epidemiology of Bacteroides fragilis, 343 isolates from infected hospitalised patients (112), infected out-patients (102), and from the faeces of uninfected hospitalised patients (47) and normal subjects in the community (82), were examined by an immunofluorescence technique. In tests with antisera against strains of 20 distinct serotypes of the fragilis group of Bacteroides, 271 (79%) strains were typable. Similarity between strains was estimated by the Jaccard similarity measure and strains were then serogrouped by cluster analysis; 88.1% of hospital strains were typable but only 71.2% of community strains (p less than 0.001). Three serogroups were prevalent among both faecal and infection isolates of hospital strains (p less than 0.01). However, no particular serogroup was prevalent among community strains and no difference was found in the distribution of serogroups between strains from faeces and those from infected lesions. One serogroup showed a significant increase (p less than 0.05) within the period of study. These findings suggest that strains of B. fragilis infecting hospitalised patients may be acquired in hospital and that they may spread to other patients.     

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.