Immunochemical characterization of two surface polysaccharides of Bacteroides fragilis.

Immunochemical analysis of the capsular polysaccharide from Bacteroides fragilis NCTC 9343 revealed a novel structure composed of two distinct polysaccharides. Immunoelectrophoresis of an extract of purified surface polysaccharide from fermenter-grown organisms showed a complex precipitin profile with varying anodal mobility. DEAE-Sephacel anion-exchange chromatography of the polysaccharide extract failed to separate the majority of this aggregate. Disaggregation of this complex was accomplished by very mild acid treatment; purification was achieved by DEAE-Sephacel anion-exchange chromatography. Polysaccharide A had a neutral charge at pH 7.3, a net negative charge at pH 8.6, and an average Mr = 110,000; chemical analysis showed it to contain galactose, galactosamine, and an unidentified amino sugar. Polysaccharide B eluted from the anion-exchange column with increased salt concentration; it had a net negative charge and an average Mr = 200,000, and contained fucose, galactose, quinovosamine, galacturonic acid, and glucosamine. Neither of these polysaccharides contained detectable 3-deoxy-D-manno-octolusonic acid, and both were recognized as distinct antigens on the basis of their reactivity with monoclonal antibodies CE3 and F10, which reacted with the complex before acid treatment. These data indicate that the capsule of B. fragilis NCTC 9343 comprises two discrete, surface-exposed polysaccharides with differing physiochemical properties that are distinct from the lipopolysaccharide of this organism. The finding of two surface polysaccharides has not been described for other bacteria pathogenic to humans.

Rapid differentiation of the species of the genus Bacteroides sensu stricto by capillary gas chromatography of cellular carbohydrates.

Whole-cell hydrolysates of Bacteroides fragilis, the type species of the genus Bacteroides Castellani and Chalmers 1919, and the genetical closely related species B. vulgatus, B. ovatus, B. eggerthii, B. distasonis, B. uniformis, B. thetaiotaomicron, B. stercoris, B. merdae, and B. caccae were used to determine characteristic carbohydrate patterns by capillary gas chromatography. On the basis of the chemical derivatization of the carbohydrates seven characteristic peaks for peracetylated aldononitriles and nine characteristic peaks for peracetylated o-methyloximes were selected from the carbohydrate fingerprints of the reference strains to prepare a dichotomous identification key. The classification of an unknown strain supposed to belong to the formerly called 'Bacteroides fragilis group' is possible with this key. Some of the advantages of the technique were that the identification of Bacteroides fragilis-like strains requires only 4-5 h after primary isolation and that the bacteria can be exposed to oxygen because viability of the organisms is not necessary. Sophisticated anaerobic techniques can therefore be avoided for identification.

Outer membrane proteins of Bacteroides fragilis grown in vivo.

The outer membrane protein (OMP) profiles of four different strains of Bacteroides fragilis, as determined by Coomassie blue stained polyacrylamide gels, were compared after growth in broth culture and in the mouse peritoneal cavity. There was no induction of the expression of large quantities of novel OMP after growth in vivo. Mouse immunoglobulin G and albumin were associated with the bacterial OMP, but could be removed by washing.

The cell-surface antigens of Bacteroides thetaiotaomicron.

Three strains of B. thetaiotaomicron of different origin were investigated. Lipopolysaccharides were extracted from the studied strains using a phenol-water method. The best purification of LPS was achieved by digestion with nuclease and subsequent ultracentrifugation. Capsular material (CPS) was obtained from the most heavily encapsulated strain. The preparation were analyzed chemically, and their serological activity was determined. All antigens were active with homologous antibacterial sera in immunodiffusion, crossed immunoelectrophoresis, and passive hemagglutination tests. In the CPS equal amounts of saccharides and proteins were detected. All antigens were analyzed by polyacrylamide gel electrophoresis with SDS. Capsular antigen slowly migrated in the gel in the form of a single band. Migration pattern of lipopolysaccharides of the studied B. thetaiotaomicron strain was characteristic for S-type LPS.

Structural characterization of the lipid A component of Bacteroides fragilis strain NCTC 9343 lipopolysaccharide.

The chemical structure of Bacteroides fragilis NCTC 9343 lipid A was characterized by using conventional chemical procedures, methylation analysis, and laser desorption mass spectrometry. It was found that B. fragilis lipid A consists of a beta-D-glucosaminyl-(1-6)-D-glucosaminyl-1-O-phosphate backbone whose hydroxyl groups in positions 4, 4' and 6' are free, the latter serving as the attachment site for the polysaccharide component in lipopolysaccharide. This backbone molecule carries up to of five molecules of ester- and amide-bound long chain non-hydroxylated and (R)-3-hydroxy fatty acids. With regard to the distribution on the fatty acids on the lipid A backbone, a considerable heterogeneity was revealed by laser desorption mass spectrometry. Despite this heterogeneity, a major species of B. fragilis lipid A could be defined in which the hydroxyl group at position 3' of the distal GlcN carries (R)-3-hydroxyhexadecanoic acid and the hydroxyl group at position 3 of the reducing GlcN is acylated by (R)-3-hydroxypentadecanoic acid. The amino group of the distal GlcN residue carries (R)-3-(13-methyltetradecanoyloxy)-15-methylhexadecanoic acid and that of the reducing GlcN group (R)-3-hydroxyhexadecanoic acid. The absence of ester-bound phosphate and ester-linked 3-acyloxyacyl groups, the presence of not more than five acyl residues and the predominance of fatty acids possessing 15-17 carbon atoms are unique features of B. fragilis lipid A which differentiate it from enterobacterial and other lipids A and which are likely to be related to its low endotoxic activity.

Incidence of hemagglutination activity among pathogenic and non-pathogenic Bacteroides fragilis strains and role of capsule and pili in HA and adherence.

We analyzed the ability of 120 encapsulated strains of B. fragilis to agglutinate guinea pig and human red blood cells. Sixteen strains showed a strong hemagglutination (HA) ability, 21 strains a moderate HA ability, 7 strains a weak HA ability and 74 strains did not agglutinate the tested red blood cells. Six strains tested from each HA group were able to adhere to cheek epithelial cells and to a cultured human intestinal cell line. Hemagglutinating strains were the most adhesive. By electron microscopy, pilus-like structures were found in three of the encapsulated adhesive strains. Treatment of the bacterial cells with pronase E reduced both HA ability and adherence of piliated encapsulated, and of piliated non-encapsulated strains. Glucosidase treatment of cells reduced HA activity and adherence of piliated encapsulated and of non-piliated encapsulated strains. Finally, it was found that hemagglutinating strains are more frequently isolated from clinical specimens (55%) than from feces of healthy donors (20%).

Detection of 2-keto-3-deoxyoctonate in endotoxins isolated from six reference strains of the Bacteroides fragilis group.

1. Endotoxins isolated from six serotype specific reference strains of the Bacteroides fragilis group were dephosphorylated by treatment with aqueous 50% hydrofluoric acid. 2. Mild acidic hydrolysis of the dephosphorylated endotoxins released 2-keto-3-deoxyaldonic acid, the presence of which was demonstrated by the colorimetric thiobarbituric acid assay (TBA). 3. Thin layer chromatography of the dephosphorylated lipopolysaccharide of B. fragilis IPL E 323 (serotype E2), after acidic hydrolysis, revealed a TBA-positive substance with the same Rf-value as authentical 2-keto-3-deoxyoctolusonic acid (KDO). 4. Quantification of 2-keto-3-deoxyoctonate-in the lipopolysaccharide of B. fragilis IPL E 323 by means of the TBA resulted in a KDO content of 15 nM mg-1 lipopolysaccharide.

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.

Bacteraemia and seeding of capsulate Bacteroides spp. and anaerobic cocci.

The effect of capsulation on the ability of Bacteroides fragilis, B. asaccharolyticus and anaerobic gram-positive cocci to induce bacteraemia and seeding to various organs was investigated. The test species were injected into mice subcutaneously alone, or mixed with other aerobic or facultative organisms. Capsulate anaerobes were isolated more frequently from the blood, spleen, liver, and kidneys of infected animals than were non-capsulate organisms. After injection of single anaerobic strains, capsulate organisms were recovered from 163 (39%) of 420 animals; non-capsulate anaerobes were recovered from only 14 (3%) of 420 animals. After injection of B. fragilis mixed with aerobic or facultative organisms, the capsulate B. fragilis strain was isolated more often and for longer periods than the non-capsulate strain. Capsulate B. fragilis was also recovered more often 5 days after injection with other organisms, than when injected alone. These data demonstrate that capsulate Bacteroides spp. and anaerobic gram-positive cocci are more virulent than non-capsulate strains in single and mixed infections.

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.

High homology of membrane proteins electrophoretic pattern in Bacteroides fragilis as confirmed by various solubilizing methods.

Membrane proteins from 25 strains of B. fragilis isolated in different laboratories in Northern Italy were examined by SDS-PAGE and isoelectrofocusing. The electrophoretic patterns of inner and outer membrane after Sarkosyl and SDS solubilization of all the isolates were consistently similar to that of the reference strain. The protein profiles of the different species belonging to the B. fragilis group are clearly distinguishable with negligible similarities. Our data clearly show that this approach is extremely helpful and reliable in providing additional verification of the identity of strains recognized by conventional tests. In this connection PAGIF analysis of triton solubilized isolated envelopes reduces technical time and difficulties, thus improving analytical accuracy.

Structural studies of the polysaccharide part of the cell wall lipopolysaccharide from Bacteroides fragilis NCTC 9343.

The structure of the polysaccharide part of the lipopolysaccharide from Bacteroides fragilis NCTC 9343 has been determined using sugar and methylation analysis as the principal tools. Phenol--water extraction followed by a phenol--chloroform--light petroleum extraction yielded a lipopolysaccharide suitable for structural analysis. Analysis of sugars using alditol acetates showed that the polysaccharide contained L-rhamnose, D-galactose and D-glucose in the approximate molar ratios of 1:5:1. After weak acid hydrolysis, two polysaccharide fractions were isolated by gel permeation chromatography: PSI and PSII with the sugar molar ratios 1:5:1 and 1:2:1 respectively. Chromium trioxide oxidation revealed that all galactosyl residues have the beta configuration, and that the rhamnosyl and glucosyl residues have the alpha configuration. From methylation analysis of lipopolysaccharide and the PS I and PS II fractions the following structures could be deduced.

Chemical and immunochemical analyses of Bacteroides fragilis lipopolysaccharides.

Lipopolysaccharides (LPSs) from 17 different Bacteroides fragilis strains were extracted in a two-step procedure. The first step was a hot phenol-water extraction of whole bacteria, resulting in a crude aqueous phase, which after lyophilization in a second step was extracted with a phenol-chloroform-light petroleum mixture. The resulting LPSs, which were essentially free from contaminating nucleic acid, proteins, and capsular polysaccharide, were investigated for their qualitative and quantitative sugar and fatty acid composition, immunochemical specificity by enzyme-linked immunosorbent inhibition assays, and particle weight by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Of the 17 strains, 13 had LPSs which all contained L-rhamnose, D-glucose, D-galactose, and D-glucosamine in approximately the same ratio. Three of these LPSs also contained D-galactosamine. The fatty acid composition was also similar in that the same fatty acids, although in slightly varying proportions, were found in all LPSs. The 13 strains also showed the same specificity in inhibition studies by enzyme immunoassay with rabbit anti-LPS antisera and LPS antigen. The LPS particle weights were also very similar, in the range of what is found for LPSs from rough mutant strains of enterobacteria. Our results suggest that most strains of B. fragilis have similar, if not identical, LPSs with relatively short polysaccharide chains.

Characteristics of the outer membrane of selected oral Bacteroides species.

The outer membranes from selected oral Bacteroides species were isolated and characterized morphologically, chemically, and physically. Both sucrose density gradient centrifugation and sodium dodecyl sulfate--polyacrylamide gel electrophoresis (SDS-PAGE) revealed outer membranes which varied slightly with the species, as well as showing complex polypeptide patterns after SDS--PAGE. The polypeptide distribution revealed a species-specific pattern; however, there was often a variation within a given species in many minor polypeptide bands, with heat-modifiable minor peptides occurring in nearly all species. Protein-associated carbohydrates occurred in several species. Outer membrane fragments (blebs) recovered by ultracentrifugation of the growth supernatants were either nearly identical to the outer membrane polypeptide patterns, or contained reduced amounts of specific polypeptides, again varying with the species. 125I-labelling of whole cells indicated possible surface exposure for several of the major polypeptides.

The growth and survival of capsulate and non-capsulate Bacteroides fragilis in vivo and in vitro.

The growth of capsulate and non- capsulate Bacteroides fragilis in chambers implanted in the mouse peritoneal cavity was compared. Capsulate and essentially non- capsulate (less than 1% capsulate ) populations of B. fragilis strains NCTC9343 and NCTC10584 consistently grew exponentially to greater than 10(9) cfu/ml within 24 h in vivo, and low numbers of capsulate bacteria were maintained in the essentially non- capsulate population; however, the degree of capsulation of the capsulate population decreased by more than 60%. B. fragilis ATCC23745 differed from strains NCTC9343 and NCTC10584 in that growth was unpredictable and only occurred in some of the implanted chambers. Capsule production by cells of strain ATCC23745 varied from chamber to chamber: sometimes the proportion of capsulate cells increased after prolonged implantation. This could occur with either an increase or decrease in viable numbers in vivo and also after in-vitro incubation of this strain in chambers. The survival of capsulate and non- capsulate B. fragilis strains NCTC9343 and ATCC23745 was compared in aerobic and anaerobic conditions in vitro. In anaerobic conditions, capsulate and non- capsulate strain NCTC9343 survived equally well, whereas capsulate ATCC23745 survived better than its non- capsulate variants. Capsulate populations of both strains survived better than non- capsulate in aerobic conditions.

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.

[Typing of Bacteroides fragilis by bacteriocin production].

Sixty-four strains of Bacteroides fragilis isolated from clinical specimens were tested for their bacteriocin production. It showed that 65.6% of them produced bacteriocin(s) and gave different patterns of inhibition. Among them 9 with clear inhibition zones were chosen as the indicator strains. With this indicator s,et 75.2% of 109 clinically isolated B. fragilis strains could be typed, with type 482 as the predominant type. The colonial dissociants gave the same bacteriocin type, but small and opaque colonies tend to lose their bactericidal ability.

Capsular polysaccharides and lipopolysaccharides from two strains of Bacteroides fragilis.

The capsular polysaccharide from Bacteroides fragilis strain NCTC 9343 contained six sugars: L-fucose, D-galactose, D- and L-quinovosamine, D-glucosamine, and galacturonic acid. The capsule of B. fragilis strain ATCC 23745 in addition contained D-glucose, L-fucosamine, L-rhamnosamine, and a 3-amino-3,6-dideoxyhexose, but lacked D-quinovosamine. The latter capsule also contained alanine (4%). The lipopolysaccharide of both strains contained the same sugars (L-rhamnose, D-glucose, D-galactose, and D-glucosamine) and fatty acids (13-methyltetradecanoic, 3-hydroxypentadecanoic as major constituents). The capsular polysaccharide of both strains promoted abscess formation, whereas the lipopolysaccharide failed to do so.

Purification and properties of a cell-bound bacteriocin from a Bacteroides fragilis strain.

A cell-bound bacteriocin was extracted from a Bacteroides fragilis BF-11 strain by treating the cells with a low-molarity buffer (0.01 M Tris-hydrochloride, pH 8.0). Sucrose osmotic shock experiments and ultrasonic lysis of whole cells indicated that the majority of the bacteriocin was located at the cell surface. Culture supernatants contained no significant bacteriocin activity. The bacteriocin was purified by DEAE-cellulose and Sephacryl S200 chromatography and had an apparent molecular weight of approximately 7,000. It was relatively heat stable and was inactivated by proteases. There was a delay of approximately 3.5 h before DNA, RNA, and protein synthesis were inhibited by the bacteriocin. Inhibition of macromolecular synthesis coincided with lysis of the susceptible indicator strain.

Purification and characterization of a bacteriocin from Bacteroides fragilis.

A bacteriocin produced by Bacteroides fragilis 1356 was purified from culture medium and characterized. The spectrum of the inhibitory activity of this bacteriocin was species specific. The bacteriocin was recovered from the initial stages of purification as a complex, greater than 2 X 10(7) daltons in mass, containing protein, lipid, and carbohydrate. The dissociation of this complex by 6.0 M guanidine hydrochloride permitted further purification of the bacteriocin by removal of lipid and carbohydrate. Analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis indicated that the purified bacteriocin was homogeneous, with a relative molecular weight of 5,000. The activity of the purified bacteriocin was not affected by RNase, DNase, phospholipase A, pancreatic lipase, or dextranase, but was destroyed by trypsin, proteinase K, heat (80 degrees C, 30 min), or a pH below 5 or above 8. Amino acid analysis indicated a predominance of acidic and polar amino acids.