INFECTION AND IMMUNITY, JUlY 1985, p. 197-2010019-9567/85/070197-05$02.00/0Copyright © 1985, American Society for Microbiology

Chemical and Immunochemical Analyses of Bacteroides fragilisLipopolysaccharides

ANDREJ WEINTRAUB, BERT E. LARSSON, AND ALF A. LINDBERG*

Department of Clinical Bacteriology, Karolinska Institute, Huddinge University Hospital, S-141 86 Huddinge, Sweden

Received 11 February 1985/Accepted 7 April 1985

Lipopolysaccharides (LPSs) from 17 different Bacteroides fragilis strains were extracted in a two-stepprocedure. 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 petroleummixture. The resulting LPSs, which were essentially free from contaminating nucleic acid, proteins, andcapsular polysaccharide, were investigated for their (i) qualitative and quantitative sugar and fatty acidcomposition, (ii) immunochemical specificity by enzyme-linked immunosorbent inhibition assays, and (iii)particle weight by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Of the 17 strains, 13 had LPSswhich 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 thesame fatty acids, although in slightly varying proportions, were found in all LPSs. The 13 strains also showedthe same specificity in inhibition studies by enzyme immunoassay with rabbit anti-LPS antisera and LPSantigen. The LPS particle weights were also very similar, in the range of what is found for LPSs from roughmutant strains of enterobacteria. Our results suggest that most strains of B. fragiis have similar, if notidentical, LPSs with relatively short polysaccharide chains.

The cell envelope of Bacteroidesfragilis bacteria has beenshown to contain two major saccharide containing compo-nents, a lipopolysaccharide (LPS) and a capsular polysac-charide (13). The chemical composition and immunochemi-cal properties of B. fragilis LPS have been subject toinvestigations, but the results have varied (7, 10, 11). A mainreason for the observed variations has probably been thedifficulty in obtaining pure LPS preparations. Most extrac-tion procedures, such as phenol-water extractions or outermembrane extraction, result in preparations which containboth LPS and capsular polysaccharide (13). A further pos-sibility for variation is the observation that upon several invitro passages of the B. fragilis strain ATCC 23745, thecapsule was replaced by a glycan which was coextractedwith the LPS (12).We recently showed that B. fragilis LPS can be isolated in

pure form by first making a phenol-water extraction of thebacteria, followed by a phenol-chloroform-light petroleum(PCP) extraction of the lyophilized aqueous phase (13).Although the results from chemical analyses of the B.

fragilis LPS have varied, it is clear that this antigen differs inits composition and biological activity from most aerobicgram-negative bacteria (7). The polysaccharide part of theLPS appears to lack both a conventional 3-deoxy-D-manno-octulosonic acid and L-glycero-D-mannoheptose (5). Thefatty acid composition of the lipid part of B. fragilis is alsodifferent from that of lipid A of enterobacterial LPS since itdoes not contain 3-hydroxytetradecanoic acid, a major fattyacid in aerobic gram-negative bacteria (25).The finding that an antiserum elicited by a purified B.

fragilis LPS could identify approximately 90% of clinicalisolates of B. fragilis, raised the question of common anti-genic determinants (one or more) in the LPS (23). In thepresent study, we describe the carbohydrate and fatty acidcomposition as well as particle weight of LPS purified byPCP extraction from 17 different B. fragilis strains, most of

* Corresponding author.

which have previously been analyzed in DNA homologystudies (9). Of these 17 strains, 13 (76%) had a similarmonosaccharide composition both qualitatively and quanti-tatively and were shown to be similar, if not identical, inimmunochemical studies with a rabbit antiserum-B. fragilisLPS inhibition system.

MATERIALS AND METHODS

Bacterial strains. The B. fragilis strains used are listed inTable 1.Growth conditions. All strains were grown in a 4-liter

fermentor (Ultraferm 1601; LKB Produkter, Bromma, Swe-den). The composition, preparation of the medium, and thegrowth conditions were essentially as described earlier (13).The medium in this study did not contain fetal calf serum.

Extraction of LPS. Pelleted organisms were suspended inwater and extracted with phenol-water (24). After dialysisagainst tap water for 6 days and against distilled waterovernight, the aqueous phase was lyophilized. The LPS wasobtained from the lyophilized water phase with PCP extrac-tion (3). The LPS was subsequently precipitated from thephenol phase with distilled water, and the precipitate waswashed once with 80% aqueous phenol (wt/vol) and fourtimes with acetone. After drying, the LPS was dissolved indistilled water, dialyzed for 3 days against distilled water at4°C, and lyophilized.

Chemical methods. The protein content was determined bythe method of Lowry et al. (17) with bovine serum albuminas the standard. The presence of nucleic acids was deter-mined by UV spectroscopy (16).For sugar analyses the LPS was hydrolyzed with 0.5 M

trifluoroacetic acid at 100°C for 16 h, and the monosac-charides were analyzed with gas-liquid chromatography af-ter conversion to alditol acetates essentially as described bySawardeker et al. (21). Alditol acetates were separatedisothermically with the following columns: 3% SP 2340 on100/120 Supelcoport at 220°C and 3% OV 17 on 100/120Gas-Chrom Q at 190°C (Supelco, Bellefonte, Pa.). The

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198 WEINTRAUB, LARSSON, AND LINDBERG

TABLE 1. B. fragilis strains usedStrain Source

9343 (VPI 2553)........... National Collection of TypeCultures, London, England

23745 (VPI 5383)........... American Type CultureCollection, Rockville, Md.

10584........... National Collection of TypeCultures

E323 .... ....... Institute Pasteur de Lille, Lille,France

TM 4000 ........... Francis Tally, Tufts MedicalSchool, Boston, Mass.

Ic650 .... ....... Francis Tally2556-1 .... ....... Virginia Polytechnic Institute and

State University (VPI),Blacksburg

6059........... VPI6851........... VPI6057-B........... VPI4361........... VPI2554........... VPI6815........... VPI4117........... VPI2552........... VPI4225........... VPI5631........... VPI

columns were fitted to a Varian gas chromatograph (model1400; Palo Alto, Calif.) equipped with a flame ionizationdetector and connected to a Hewlett-Packard integrator(model 3380A; Palo Alto, Calif.). The sugars were identifiedby cochromatography with authentic standards. For quanti-tative analyses L-xylose was used as an internal standard.The fatty acids were liberated and converted to methyles-

ters by methanolysis with 2 M HCl in water-free methanol at85°C for 18 h (20). The fatty acid methylesters were thenextracted twice with n-hexane, and the volume was reducedunder a stream of nitrogen. The fatty acid methylesters wereseparated isothermically (170°C) on a WCOT OV 101 glasscapillary column (25 m; Chrompack, Middleburg, Nether-lands). The column was fitted to a Varian gas chromatograph(model 3700) which was equipped with a flame ionizationdetector and connected to a Hewlett-Packard integrator(model 3380A). For quantitative analyses heptadecanoicacid (methylester) and 3-hydroxydecanoic acid (methylester)were used as internal standards.

SDS-polyacrylamide gel electrophoresis. The discontinuousgel system used was essentially as described previously (14).The separation gel (142 by 120 by 1.5 mm) contained 20%acrylamide and 0.45% bisacrylamide. The LPS and capsular

polysaccharide samples in water were mixed with an equalvolume of 0.1 M Tris-hydrochloride buffer (pH 6.8) contain-ing 2% (wt/vol) sodium dodecyl sulfate, 10% (vol/vol) glyc-erol, 2% (vol/vol) 2-mercaptoethanol, 0.1% (wt/vol) EDTA,and 0.001% (wt/vol) phenol red. The mixtures were heated at100°C for 5 min, and 10- to 20-,ul samples containing 5 p.g ofLPS or 20 ,ug of capsular polysaccharide were applied to thesample wells. The gels were stained by the silver method(22).Immunochemical methods. Rabbit antisera against B. fra-

gilis NCTC 9343 and B. fragilis ATCC 23745 LPSs wereprepared as described earlier (13). The enzyme-linked im-munosorbent assay (ELISA) was performed as describedpreviously (16). In ELISA inhibition experiments, serumwas preincubated for 1 h with different concentrations of theLPS assayed. The 50% inhibitory value was recorded as theconcentration of inhibitor needed to obtain a 50% loweringof the optical density at 400 nm as compared to control tubeswith no inhibitor added.

RESULTSIsolation of LPSs. All B. fragilis strains were grown in a

4-liter fermentor to late logarithmic phase. The yield of thebacteria was about 2 g/liter (dry weight). The yield of crudeaqueous phase after phenol-water extraction correspondedto about 5% of the bacterial mass dry weight (wt/wt). TheLPS was subsequently isolated from the lyophilized crudeaqueous phase after phenol-water extraction with PCP ex-traction. The yield of PCP-extractable LPS was about 0.5%of the bacterial mass (wt/wt). All LPS preparations werefound to be virtually free of nucleic acids (<5 ,ug/mg of LPS)as assayed by UV spectroscopy. Furthermore, no proteinswere present (<5 ,ug/mg of LPS) as determined by themethod of Lowry et al. (17), with bovine serum albumin asstandard.Carbohydrate analyses of LPSs. After hydrolysis of the

LPS preparations (0.5 M TFA, 16 h, 100°C), the resultingmonosaccharides were reduced with NaBH4, and afteracetylation they were analyzed as alditol acetates by gas-liquid chromatography (Table 2).The total amounts of carbohydrates in the different B.

fragilis LPSs varied between 26 and 66%. In the quantitativeand qualitative sugar analysis, five groups could be seen, allcontaining D-glucosamine, as follows: (i) group 1 of 10strains with an L-rhamnose/D-galactose/D-glucose ratio of1:3-5:1; (ii) group 2 with three strains having the same ratiosas in group 1, but which in addition contained approximatelyone unit of D-galactosamine; (iii) group 3 with two strainswith an L-rhamnose/D-galactose/D-glucose/D-galactos-amine/D-glucosamine ratio of 1:1:1:1:2; (iv) one strain, VPI4225, with a higher D-glucose content; and (v) one strain,

TABLE 2. Sugar analysis of LPSs from 17 B. fragilis strainsSugar (mol %)'

Group StrainsL-Rhamnose D-Galactose D-Glucose D-Galactosamine D-Glucosamine Total %

1 NCTC 9343, NCTC 10584, ATCC 23745, 12-18 41-65 10-14 NDb 8-32 30-53E323, TM 4000, Ic650, VPI 2556-1,VPI 6059, VPI 6851, VPI 6057-B

2 VPI 6815, VPI 2554, VPI 4361 13-14 43-54 10-13 7-11 10-20 26-433 VPI 2552, VPI 4117 12 15-16 17-18 18 36-38 49-664 VPI 4225 17 13 33 16 21 505 VPI 5631 32 ND 38 ND 30 36a Determined by gas-liquid chromatography as their alditol acetates.b ND, Not detectable.

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BACTEROIDES FRAGILIS LIPOPOLYSACCHARIDES 199

VPI 5631, lacking both D-galactose and D-galactosamine(Table 2).

Fatty acids in LPSs. The total amounts of fatty acids in thevarious LPS preparations varied from 24% (VPI 2552) to52% (VPI 5631). The 9343 LPS preparation contained thefollowing fatty acids: 13-methyltetradecanoic (15%), 3-hydroxypentadecanoic (16%), 3-hydroxyhexadecanoic(35%), 3-hydroxy-15-methylhexadecanoic (21%), and 3-hydroxyheptadecanoic (6%). The quantitative analysis of theLPSs revealed that the ratios between individual fatty acidswere comparable (data not shown). The predominating fattyacids were identified as 3-hydroxyhexadecanoic and 3-hydroxy-15-methylhexadecanoic acids.

SDS-polyacrylamide gel electrophoresis of different B. fra-giis LPSs. The LPS preparations from all 17 B. fragilisstrains were examined together with capsular polysac-charides from B. fragilis NCTC 9343 and ATCC 23745 byusing SDS-polyacrylamide gel electrophoresis. LPSs fromShigella flexneri strains 3a and 4bR were used as internalstandards. The LPS isolated from strain 3a is of the smoothtype, with an 0 polysaccharide chain that contains severalrepeating units. The 4bR LPS is of the rough type; it lacksthe 0 polysaccharide chain but contains the entire corestructure (8). The S. flexneri 3a LPS was resolved into manybands, each representing a different number of repeatingunits in the LPS molecule, whereas the 4bR LPS gave onebroad band only (Fig. 1).

All of the 17 B. fragilis LPS preparations were of about thesame size as the S. flexneri 4bR LPS. Some of the LPSpreparations were not homogeneous. The LPS from B.fragilis strains NCTC 9343, ATCC 23745, E323, Ic650, VPI2554, VPI 4361, VPI 6815, and TM 4000 showed one majorand one minor band. The pure capsular polysaccharidesfrom B. fragilis NCTC 9343 and ATCC 23745 were includedas controls (Fig. 1C). None of the two preparations con-tained any detectable LPS, and only the NCTC 9343 capsulewas visible. As reported earlier (13), these two capsularpolysaccharides differ both in chemistry and immunochemi-cal specificity. The difference in the migration in the SDS-polyacrylamide gel electrophoresis can be caused by differ-ence in molecular size or difference in charge or both.Immunochemical analyses. The immunochemical speci-

ficity of the 17 LPS preparations was studied in ELISAinhibition experiments with rabbit antisera elicited against B.fragilis NCTC 9343 and ATCC 23745 LPSs. All investiga-tions were done with the homologous LPS as coating anti-gen, and the concentration of inhibitors required for 50%inhibition was determined (Fig. 2). Only results with the B.fragilis NCTC 9343 LPS-antiserum system are shown sincestudies with the ATCC 23745 LPS-antiserum system gaveessentially identical results. LPS from 12 of the strainsinhibited in the same range as the NCTC 9343 LPS; 0.05 to0.3 jig of inhibitor was required for 50% inhibition. Allstrains belong to groups 1 and 2 in terms of sugar composi-tion (Table 2). The LPSs from VPI 4117 and VPI 2552 wereless potent; the concentrations required for 50% inhibitionwere 5 and 75 p,g, respectively. When the LPS from VPI4225 and VPI 5631 were used as inhibitors, none reached a50% inhibition even with 100 ,ug (the highest concentrationused).

DISCUSSIONThe present investigation of LPSs extracted from 17

different characterized B. fragilis strains revealed moresimilarities than differences. Below we discuss the LPSswith respect to (i) their molecular size, (ii) their fatty acid

A~~

AX 2 34 5 6 79 10g,,.,tS., Si..'

12 3 4 5 6 12 3 4 56 7

FIG. 1. Migration pattern, in 20% polyacrylamide gels stainedwith silver, of S p.g LPS isolated from 17 different B. fragilis strainscompared with S. flexneri 3a and 4bR LPS and with 20 p.g ofcapsular polysaccharides from two B. fragilis strains. (A) Lanes: 1,ATC-C 23745 LPS; 2, NCTC 9343 LPS; 3, E323 LPS; 4, 1c650 LPS;5, S. flexneri 3a LPS; 6, VPI 2554 LPS; 7, VPI 4117 LPS; 8, VPI4225 LPS; 9, VPI 2552 LPS; and 10, VPI 5631 LPS. (B) Lanes: 1,VPI 6057-B LPS; 2, NCTC 10584 LPS; 3, VPI 4361 LPS; 4, VPI2556-1 LPS; 5, VPI 6815 LPS; and 6, S.flexneri 3a LPS. (C) Lanes:1, VPI 6059 LPS; 2, VPI 6851 LPS; 3, TM 4000 LPS; 4, NCTC 9343capsular polysaccharide; 5, ATCC 23745 capsular polysaccharide;6, S. flexneri 4bR LPS; and 7, S. flexneri 3a LPS.

composition, (iii) their monosaccharide composition, and(iv) their immunochemical specificity.

All B. fragilis LPSs moved extensively in the SDS-polyacrylamide gel electrophoresis (Fig. 1). They resolvedinto one, or at most two, band(s) which were seen on the gelin the same region as the LPS from S. flexneri 4bR. This is arough mutant strain which contains the enterobacterial lipidA and a polysaccharide composed of approximately 10 sugarresidues, including 3-deoxy-D-manno-octulosonic acid (8).Since the B. fragilis LPS sugar/lipid ratio is approximately1:1 and thus similar to that found in LPSs from roughmutants of enterobacterial strains like S. flexneri 4bR, weexpect the B. fragilis saccharide to be composed of ap-proximately 10 sugar residues. It is therefore unlikely thatwe would find a polysaccharide chain composed of a varyingnumber of repeating units in B. fragilis as found in the S.flexneri 3a LPS (Fig. 1) and in LPSs from smooth Esch-erichia coli (4) and Salmonella typhimurium (4) strains. The".rough" character of the B. fragilis LPS is further demon-

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200 WEINTRAUB, LARSSON, AND LINDBERG

9343,23745,10584,323,4000,6502556-1,6059,6851,6057-B,4361,

2554,6815

4117

'2552

* 4225

loo 563110010

FIG. 2. ELISA inhibition study on 17 different B. fragilis LPSs. LPS from B. fragilis NCTC 9343 was used as a coating antigen. Theantiserum used was directed against B. fragilis NCTC 9343 LPS.

strated by its ability to be extracted by the PCP mixturewhich preferentially extracts the more hydrophobic LPSfound in rough enterobacterial bacteria (3). We assume thatthe inability of the PCP mixture to extract the B. fragilisLPSs from intact bacteria probably reflects physicochemicalproperties of the B. fragilis cell envelope; the LPS may bemasked by the capsular polysaccharide.

Fatty acid analyses of the 17 different B. fragilis LPSpreparations showed that all have a similar lipid composition(data not shown). The main fatty acids are isobranchedpentadecanoic acid, 3-hydroxy-pentadecanoic acid, 3-hydroxyhexadecanoic acid, isobranched 3-hydroxyhep-tadecanoic acid, and 3-hydroxyheptadecanoic acid. Therange of 3-hydroxy fatty acids found in the B. fragilis LPS isalso quite unique, as found in an earlier study (25). The3-hydroxytetradecanoic acid, which is the major fatty acid inenterobacterial LPS, is present in B. fragilis LPS only intrace amounts. Thus, the fatty acid composition as found inthis study on 17 different LPSs is in agreement with what ourearlier collaborative studies on a few strains have shown (13,25). Although different from what has been found for entero-bacterial LPSs both in fatty acid composition and in lack ofcommon endotoxic activities (7), the large similarities infatty acids, both qualitatively and quantitatively suggeststhat B. fragilis in this respect is a fairly homogenous bacte-rial species.The qualitative and quantitative sugar analyses revealed

that 16 of 17 strains analyzed contained L-rhamnose, D-galactose, D-glucose, and D-glucosamine (Table 2). For 10 of

these strains, the ratios of these sugars were very similar(Table 2, group 1). Also, the LPSs from the three strains ofgroup 2 had the same ratio but contained, in addition,D-galactosamine. All these 13 LPSs were in addition tosimilarities in saccharide composition shown to have thesame immunochemical specificity (Fig. 2; see below). Fourstrains (VPI 4117, 2552, 4225, and 5631; Table 2) differed intheir sugar composition by having either significantly re-duced or no D-galactose in their LPSs. This coincided with areduced or no inhibitory activity in the immunochemicalassays (Table 2; Fig. 2).

Studies of the polysaccharide chain from the LPS of strain9343 using methylation analysis have shown that it has thefollowing structure (Weintraub et al., submitted forpublication):

P-D-Galpl 6-p-D-Galpl -- 6-p-D-Galpl6-P-D-Galpl 4/6-a-D-Glcpl -- 2-a-L-Rhapl

4/6t 1

P-D-Galp

This structure has an L-rhamnose/D-galactose/D-glucose ra-tio of 1:5:1. The data in Table 2 show L-rhamnose/D-galac-tose/D-glucose ratios of 1:3 to 5:1. The difference in theseresults may, however, be explained by the fact that thePCP-extracted LPS from the 9343 and other strains was amixture of two LPS populations (Fig. 1). We showed that theminor band which can be seen on the SDS-polyacrylamidegel electrophoresis corresponded to an LPS fraction in

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BACTEROIDES FRAGILIS LIPOPOLYSACCHARIDES 201

which the P1,6-linked galactose chain was lacking (submit-ted). This explains the observed variation in D-galactosecontent.The chemical and immunochemical data do suggest that

the common immunochemical specificity demonstrated for13 of 17 B. fragilis LPS is a consequence of the galactosechain and that the D-galactosamine in group 2 strain LPSsdoes not interfere with this specificity.

All LPSs studied contained D-glucosamine (Table 2). Thedemonstration of amide-linked fatty acids in the B. fragilisLPS (25) indicates that this linkage occurs between the fattyacids and the amino sugar. This would be in accordance withthe findings in enterobacterial lipid A in which the backboneis a P-1,6-linked D-glucosamine disaccharide (19).The results of the ELISA inhibition experiments are in

agreement with our own data showing that approximately90% of more than 100 clinical isolates of B. fragilis could beidentified by using the antiserum elicited by the PCP-extracted LPS from the strain NCTC 9343 LPS (23). Earlierstudies had suggested that the capsular polysaccharide was a

common antigen for the B. fragilis species (18). This invest-igation unequivocally shows that instead the LPS is thecommon antigen, a hypothesis suggested in an earlier study(13). Our results also suggest that the various serotypingsystems proposed for B. fragilis (2, 6, 15) have been largelybased on different antigenic specificities in capsular polysac-charides rather than somatic 0 antigens (i.e., LPSs).Most of the B. fragilis strains used in this study (except

NCTC 10584, TM 4000, andIc650) have earlier been dividedinto two DNA homology groups (9), and the relationshipbetween the DNA group and antigenic specificity of the cellenvelope was studied (1). It is interesting to note that withone exception (the LPS from strain VPI 5631) we found allstrains of DNA homology group I to have LPSs with thesame immunochemical specificity. The exceptional strain,VPI 5631, which lacked D-galactose in its LPS, may well bea mutant which has lost the immunochemically active sac-

charide. Our immunochemical data are not entirely in agree-ment with those of Babb and Cummins (1). This may beexplained by the fact that these investigators used sera

elicited by whole bacteria in their serological tests and theantigens were either whole cells or crude cell wall fractions.Consequently antigens and antibodies representative of boththe LPS and the capsular polysaccharide of the cell envelopewere studied simultaneously.

ACKNOWLEDGMENTS

This study was supported by grant 16X-04975 from the SwedishMedical Research Council.

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serological groups and deoxyribonucleic acid homology groupsin Bacteroides fragilis and related species. J. Clin. Microbiol.13:369-379.

2. Elhag, K. M., K. A. Bettelheim, and S. Tabaqchalli. 1977.Serological studies of Bacteroides fragilis. J. Hyg. 79:233-241.

3. Galanos, C., 0. Luderitz, and 0. Westphal. 1969. A new methodfor the extraction of R-lipopolysaccharides. Eur. J. Biochem.9:245-249.

4. Goldman, R. C., and L. Leivy. 1980. Heterogeneity of antigenic-side-chain length in lipopolysaccharide from Escherichia coliand Salmonella typhimurium LT2. Eur. J. Biochem.107:145-153.

5. Hofstad, T. 1974. The distribution of heptose and 2-keto-3-

deoxy-octonate in Bacteroidaceae. J. Gen. Microbiol.85:314-320.

6. Hofstad, T. 1977. Cross reactivity of Bacteroides fragilis 0-antigens. Acta Pathol. Microbiol. Scand. Sect. B 85:9-13.

7. Hofstad, T., K. Sveen, and G. Dahlen. 1977. Chemical composi-tion, serological reactivity and endotoxicity of lipopolysac-charides extracted in different ways from Bacteroides fragilis,Bacteroides melaninogenicus and Bacteroides oralis. ActaPathol. Microbiol. Scand. Sect. B 85:262-270.

8. Jansson, P. E., B. Lindberg, A. A. Lindberg, and R. Wollin.1979. Structural studies on the hexose region of the Enterobac-teriaceae type R3 core polysaccharide. Carbohydr. Res.68:385-389.

9. Johnsson, J. L. 1978. Taxonomy of Bacteroides. I. Deoxyribo-nucleic acid homologies among Bacteroides fragilis and othersaccharolytic Bacteroides species. Int. J. Syst. Bacteriol.28:245-256.

10. Kasper, D. L. 1976. The polysaccharide capsule of Bacteroidesfragilis subspecies fragilis: immunochemical and morphologicaldefinition. J. Infect. Dis. 133:79-87.

11. Kasper, D. L. 1976. Chemical and biological characterization ofthe lipopolysaccharide of Bacteroides fragilis subspecies fra-gilis. J. Infect. Dis. 134:59-66.

12. Kasper, D. L., A. B. Onderdonk, B. G. Reinap, and A. A.Lindberg. 1980. Variations of Bacteroides fragilis with in vitropassage: presence of an outer membrane-associated glycan andloss of capsular antigen. J. Infect. Dis. 142:750-756.

13. Kasper, D. L., A. Weintraub, A. A. Lindberg, and J. Lonngren.1983. Capsular polysaccharides and lipopolysaccharides fromtwo Bacteroides fragilis reference strains: chemical and im-munochemical characterization. J. Bacteriol. 153:991-997.

14. Laemmli, U. K. 1970. Cleavage of structural protein duringassembly of the head of the bacteriophage T4. Nature (London)227:680-685.

15. Lambe, D. W., Jr., and D. A. Moroz. 1976. Serogrouping ofBacteroides fragilis subsp. fragilis by the agglutination test. J.Clin. Microbiol. 3:586-592.

16. Lindberg, A. A., A. Weintraub, and C. E. Nord. 1979. Thehumoral antibody response to Bacteroides fragilis infections inhumans. Scand. J. Infect. Dis. Suppl. 19:46-51.

17. Lowry, 0. H., N. J. Rosebrough, A. L. Farr, and R. J. Randall.1951. Protein measurement with the Folin phenol reagent. J.Biol. Chem. 193:265-275.

18. Polk, B. F., and D. L. Kasper. 1977. Bacteroides fragilissubspecies in clinical isolates. Ann. Intern. Med. 86:569-571.

19. Rietschel, E. T., C. Galanos, 0. Luderitz, and 0. Westphal.1982. Chemical structure, physiological function and biologicalactivity of lipopolysaccharides and their lipid A component, p.183-229. In D. Webb (ed.), Immunopharmacology and theregulation of leucocyte function. Marcel Dekker, New York.

20. Rietschel, E. T., S. Hase, M. T. King, J. Redmond, and V.Lehman. 1977. Chemical structure of lipid A, p. 262-268 In D.Schlessinger (ed.), Microbiology-1977. American Society forMicrobiology, Washington, D.C.

21. Sawardeker, J. S., J. H. Sloneker, and A. Jeanes. 1965. Quanti-tative determination of monosaccharides as their alditol acetatesby gas-liquid chromatography. Anal. Chem. 37:1602-1604.

22. Tsai, C.-M., and C. E. Frasch. 1982. A sensitive silver stain fordetecting lipopolysaccharides in polyacrylamide gels. Anal.Biochem. 119:115-119.

23. Weintraub, A., A. A. Lindberg, and D. L. Kasper. 1983. Char-acterization of Bacteroides fragilis based on antigen-specificimmunofluorescence. J. Infect. Dis. 147:780.

24. Westphal, O., 0. Luderitz, and F. Bister. 1958. Uber dieExtraktion von Bakterien mit Phenol-Wasser. Z. Naturforsch.7B:148-155.

25. Wollenweber, H. W., E. T. Rietschel, T. Hofstad, A. Weintraub,and A. A. Lindberg. 1980. Nature, type of linkage, quantity, andabsolute configuration of (3-hydroxy) fatty acids in lipopolysac-charides from B. fragilis NCTC 9343 and related species. J.Bacteriol. 144:898-903.

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