JOURNAL OF CLINICAL MICROBIOLOGY, Mar. 1988, p. 448-4520095-1137/88/030448-05$02.00/0Copyright «D 1988, American Society for Microbiology

Detection of Bacteroides fragilis, Bacteroides thetaiotaomicron, andBacteroides ovatus in Clinical Specimens by Immunofluorescencewith a Monoclonal Antibody to B. fragilis Lipopolysaccharide

MATTI K. VILJANEN,* LINNEA LINKO, AND OLLI-PEKKA LEHTONEN

Department of Medical Microbiology, University of Turku, Kiinamyllynkatu 13, SF-20520, Turku, Finland

Received 28 August 1987/Accepted 11 December 1987

A total of 1,897 clinical specimens (1,019 aspirates and 876 swabs) were studied by indirect immunofluo-rescence (IF) with a mouse monoclonal antibody (MAb) against a D-galactose oligomer of Bacteroidesfragilislipopolysaccharide. The MAb has been shown to react with 96% of clinical B. fragilis isolates and with about50% of Bacteroides ovatus and Bacteroides thetaiotaomicron isolates but not with other aerobic or anaerobicorganisms tested. The sensitivity of IF in comparison with culturing was 78.9% for all three species. Of the 32strains originating from culture-positive, IF-negative specimens, 13 lacked the target determinant for the MAb.Sensitivity was highest with specimens taken from the perineal area (87.1%) and lowest with those taken fromundefined sites (56.6%). Sensitivity was better with aspirates (86.8%) than with swabs (72.6%). The specificityof IF was 95.6% for all of the material. Positive and negative predictive values were 51.1 and 98.0%,respectively. Neither long transportation times of specimens nor antimicrobial therapy seemed to correlate withthe occurrence of IF-positive, culture-negative specimens. This study shows that a single MAb can be used toestablish an IF assay that can complement isolation in the detection of these three members of the B. fragilisgroup.

Bacteroides fragilis is the anaerobic species most oftenisolated in clinically important mixed anaerobic infections(2). New antimicrobial drugs have greatly improved theoutcome of infections with B. fragilis. Thus, it is importantfor clinicians to obtain rapid information on the possibleinvolvement of this organism in an infection. Culturing is tooslow a method even under optimal conditions, and rapidmethods have to be based on the detection of either meta-bolic or antigenic substances of the bacterium. Only a fewreports have indicated that the direct analysis of clinicalspecimens by gas-liquid chromatography can provide areliable presumptive identification of anaerobes (3). In con-trast, there are numerous reports on the use of immunoflu-orescence (IF) techniques in the diagnosis of infections withBacteroides species (1, 4, 6, 8, 11, 15, 16, 18, 19), andcommercial kits are even available for this purpose. How-ever, despite promising preliminary results, IF techniqueshave not gained any wide use. All earlier IF techniques forB. fragilis detection have used polyclonal antibodies, andcross-reactions have been difficult to eliminate.We previously reported on the production and character-

ization of a monoclonal antibody (MAb) directed against thelipopolysaccharide (LPS) of B. fragilis (13). The targetdeterminant is composed of a D-galactose oligomer in thecore of LPS (12). The MAb has been shown to react with96% of clinical B. fragilis isolates and with about 50% ofBacteroides ovatus and Bacteroides thetaiotaomicron iso-lates but not with other aerobic or anaerobic organismstested (13). The purpose of this study was to investigate thesensitivity and specificity of an IF method with this MAb inthe direct detection of the three members of the B. fragilisgroup (BFG) in clinical specimens.

* Corresponding author.

MATERIALS AND METHODS

MAb. The production and characterization of the MAbhave been described earlier (12, 13). Briefly, B. fragilisATCC 23745 was grown on brucella blood agar platessupplemented with vitamin K (10 ,ug/ml) and hemin (1 ,g/ml)in anaerobic jars. Outer membranes were removed by treat-ing the bacteria with heat and shearing in the presence ofEDTA (9). Outer membranes were separated by ultracentrif-ugation and treated with Triton X-100 (no. 8603; E. MerckAG, Darmstadt, Federal Republic of Germany) at roomtemperature for 20 min. Insoluble material was separated byultracentrifugation, and the supernatant was used for immu-nizations of BALB/c mice. The method of Kohler andMilstein (10) was used for the production of hybridomas.Culture fluids of the hybridomas obtained were tested for B.fragilis-specific antibodies by an enzyme immunoassay withbacterial sonic extract or LPS as the antigen (13). Nineteenstable clones reacting with both of the antigens were estab-lished, and one of them (designated BF5) was selected forthis study. The selected MAb reacted with 96% of clinical B.fragilis isolates, with the type strain (NCTC 9343), and withone reference strain (ATCC 23745). Positive reactions werealso obtained with five of nine B. thetaiotaomicron and twoof five B. ovatus isolates (13).Only in vitro-produced MAbs were used throughout the

study, since the preliminary experiments showed that asciticfluids contained large numbers of polyclonal antibodiesagainst various organisms of the normal flora, e.g., Esche-richia coli and staphylococci.

IF staining. IF staining was carried out concomitantly withthe primary Gram staining of the specimens. Clinical mate-rial was smeared directly on slides; isolated colonies weresuspended in saline solution before being smeared. Speci-mens were dried in the open air and fixed with cacodylate-formaldehyde solution (Sigma Chemical Co., St. Louis,Mo.) at 4°C for 15 min. The slides were washed with distilled

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TABLE 1. Origins of specimens and comparison of isolationand IF in relation to the specimen groups

No. of positiveOrigin of results in: Sensitivity

specimen (n) Isolation IF (%)bonly only Both

Peritoneal cavity (369) il 39 56 83.6Perineal area (104) 4 18 27 87.1Urogenital area (95) 1 4 80.0Abdominal wall (104) 2 7 9 81.8Thoracic wall (73) 1 3 0Lower extremities (293) 1 12 10 76.9Upper extremities (88) 2 2 2Head and neck (96) 0 4 1Pleurae and lungs (110) 1 0 1Bronchi (23) 0 1 0Sinuses (49) 0 2 0Peritonsillar abscess (31) 0 5 0Brain abscess (115) 0Cerebrospinal fluid (10) 0 0Joint fluid (185) 0 1 0Bone (22) 0 0 1Miscellaneous or unknown (230) 9 17 9 56.3

a MAb to B. fragilis LPS was used in the IF assay.b Calculated only in groups with over five specimens positive in both tests.

The overall sensitivity was 78.9%.

water and allowed to dry for 5 min. The MAb (undilutedculture medium) was incubated on the slides in a humidchamber at 23°C for 30 min. After the slides were washedand dried, fluorescein-conjugated rabbit antibodies [F(ab)2fraction] to mouse immunoglobulin (Dakopatts a/s, Glo-strup, Denmark) diluted 1:40 with phosphate-buffered salinesupplemented with 1% normal sheep serum were added andincubated at 23°C for 30 min. After the slides were washed,cover slips were placed on the slides; buffered glycerol wasused as the mounting solution. An SM-Lux UV microscope(H. E. Leitz Wetzlar GmbH, Wetzlar, Federal Republic ofGermany) was used in the inspection of the preparation(magnification, 630x). A preparation was interpreted asnegative if fewer than four fluorescing organisms per slidewere found. The fluorescence should be an apple greenoutlining of the bacteria. Even if the fluorescing materialmainly consisted of phagocytized or cell-associated bacterialparticles, a prerequisite for positivity was the occurrence ofsome intact organisms in the specimen. If a specimen waspositive by IF but did not yield any of the three BFG species,the organisms that were isolated were tested by IF forcross-reactivity. Conversely, if a BFG species was isolatedbut the specimen was negative by IF, the isolated strain wastested by IF to find out if it belonged to the strains notcontaining the target determinant for the MAb.During the present work it was found that the MAb also

reacts with some group B streptococci (GBS). However,streptococci could be morphologically easily differentiatedfrom bacteroides. The one specimen containing positiveGBS was not included in the number of IF-positive speci-mens.

Cultivation and identification of bacteria. The specimenswere plated on chocolate agar (Columbia agar base; OxoidLtd., London, England) and blood agar (blood agar base,with 5% bovine blood; Oxoid) for aerobic culturing. Theplates were examined after 24 and 48 h of incubation, and theisolates were identified by standard procedures. Anaerobicculturing was carried out on brucella blood agar platessupplemented with menadione and L-cysteine (MC plates),

MC plates supplemented with neomycin (0.2 mg/ml), andbacteroides bile esculin plates (14). In addition, the speci-mens were inoculated into 10 ml of thioglycolate brothsupplemented with Fildes enrichment. The anaerobic plateswere incubated in jars with a gas mixture containing 80% N2,10%CO2, and 10% H2 at 35°C for 48 h. The API 20A system(API International, S.A., Geneva, Switzerland) supple-mented with some tests listed in the Anaerobe LaboratoryManual (5) and the Wadsworth Anaerobic BacteriologyManual (17) was used in the identification of anaerobicbacteria. Test tube modifications of trehalose, xylose, rham-nose, and mannitol utilization tests were used to confirm thedifferentiation of B. thetaiotaomicron and B. ovatus and inthe differentiation of these species from the other indole-positive bacteroides, Bacteroides uniforms, Bacteroidesasaccharolyticus, and Bacteroides eggerthii.

Clinical specimens. A total of 1,897 random clinical spec-imens were tested. All specimens were from patients hospi-talized in the Turku University Central Hospital or theTurku Municipal Hospital. The main categories of samplingsites are listed in Table 1. Of the specimens, 53.6% were pusaspirates or body fluids, and 46.4% were swabs. The aspi-rates were taken into syringes, capped tubes, or anaerobictransportation lagenae (Portagerm; BioMerieux, Charbon-nieres-les-Bains, France).

RESULTSOf the 1,897 specimens studied, 108 yielded B. fragilis, 41

yielded B. thetaiotaomicron, and 3 yielded B. ovatus incultivation. Of the B. fragilis-positive specimens, 90 werepositive by IF. The corresponding figure for the specimensgrowing either of the two other species was 30 (Table 2).Thus, the sensitivities of IF by comparison with culturingwere 83.3% for B. fragilis, 68.2% for B. thetaiotaomicronand B. ovatus, and 78.9% for all three species (Table 2). IFof smears of BFG colonies isolated from the 32 IF-negativespecimens revealed that 13 (40.6%) of these isolates werenegative and, evidently, lacked the determinant recognizedby the MAb (Table 2).A rough semiquantitative comparison showed that the

sensitivity of IF correlated with the number of BFG colonieson the primary culture plate (Table 3). A majority of speci-mens strongly positive by IF yielded more than 50 BFGcolonies on the primary culture plate. However, there were53 specimens strongly positive by IF but negative for thethree BFG species. Conversely, 21 specimens growing morethan 50 BFG colonies on the primary culture plate proved IFnegative. Nine of them proved negative in the staining ofcolonies (Table 3).The proportions of swabs in all of the material and among

the specimens positive by IF were 46.2 and 48.9%, respec-

TABLE 2. Comparison of isolation and IF with MAb toB. fragilis LPS in the detection of the three BFG

species in 1,897 random specimens

No. of specimens withOrganism indicated no. of organisms Sensitivity Specificityisolated positive by IF: (%) (%)

<4a 4 to 10 >10

B. fragilis 18 (7) 14 76 83.3B. thetaiotaomicron 14 (6) 2 28 68.2

or B. ov'atusNone or other 1,630 62 53 93.4

a IF was interpreted as negative. Numbers in parenthesis are numbers ofstrains without the target determinant for the MAb.

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TABLE 3. Semiquantitative comparison of isolation and directdetection of B. fragilis, B. thetaiotaomicron, and B. ovatus

by IF with MAb to B. fragilis LPS

No. of specimens with indicated no. ofNo. of organisms bacteroides colonies on the primary culture plateau:positive by IF

0 <10 10 to 50 >50

<4b 1,630 7 (4) 4 (0) 21 (9)3 to 10 62 6 19>10 53 9 86

a The primary culture plate was a bacteroides bile esculin plate. Numbers inparenthesis are numbers of strains without the target determinant for theMAb. The sensitivities for the categories <10, 10 to 50, and >50 were 68.1,71.4, and 81.9t%, respectively.

b IF was interpreted as negative.

tively (Table 4). The sensitivity of IF was higher with theaspirates (86.8%) than with the swabs (72.6%) (Table 4). Aslightly higher sensitivity was also obtained with abscess andbody fluid specimens (both 81.5%) than with wound speci-mens (74.1%) (Table 4).The overall specificity of IF was 93.4% (Table 2). There

were 115 specimens among the 235 IF-positive specimensfrom which none of the three BFG species could be isolated.On the other hand, 1,630 specimens of the 1,662 IF-negativespecimens also were negative in isolation. The positive andnegative predictive values of IF were thus 51.1% and 98.0%,respectively.Of the 115 IF-positive, isolation-negative specimens, 53

were interpreted strongly positive in IF, since more than 10fluorescing organisms were seen per slide. A total of 59.1%of the 115 specimens were from anatomical locations poten-tially infected with gastrointestinal microbial flora (i.e., theperitoneal cavity, perineal area, abdominal wall, and uroge-nital area); the corresponding figures were 75.0% for BFGisolation-positive specimens and 35.4% for all of the material(Table 1). The mean transportation time for the 115 speci-mens did not differ significantly from that for the otherspecimens (data not shown). Based on information on thesubmission form, the incidence of ongoing antimicrobialtherapy was even lower among isolation-negative patients(50.4%) than among isolation-positive patients (61.2%). Toget more reliable information on therapy, we inspectedpatient records for 57 of the 115 patients. In only 16 casescould the therapy be considered effective against bacte-roides.The discordance between isolation and IF was particularly

prominent with the specimens taken from peritonsillar ab-

TABLE 4. Effects of the sampling method and infection type onthe detection of B. fragilis, B. thetaiotaomicron, and B. ovatus

by isolation and IF with MAb to B. fragilis LPS

No. of positive results in:Parameter Sensitivity of

(no. of specimens) Isolation IF only Both IF (%)only

Sampling methodAspiration (1,019) 9 61 59 86.8Swabbing (876) 23 54 61 72.6

Infection typeAbscess (342) 5 32 22 81.5Wound infection (674) 14 37 40 74.1Infectious exudate (767) 12 44 53 81.5

or body fluidUndefined (106) 1 2 5 83.3

TABLE 5. Microbiological findings for the 115 specimenspositive in IF with MAb but negative in isolation for

B. fragilis, B. thetaiotaomicron, or B. ovatus

Microbiological result No. ofspecimens

Gram-negative rods in primary staining but not inisolation ........................................... 3

Totally isolation negative ........................................... 25At least one aerobe ........................................... 76At least one anaerobe........................................... 58Mixed infection .................. ......................... 44Isolated microbes

Escherichia coli.......................................... 18Streptococcus faecalis .......................................... 12Klebsiella sp.......................................... 7Viridans group streptococcus ................................... 7Beta-hemolytic GBS ........................................... 3Staphylococcus aureus .......................................... 2Coagulase-negative staphylococcus ........................... 3Bacteroides vulgatus .......................................... 3Bacteroides distasonis .......................................... 2Unidentified bacteroides ......................................... 7Fusobacterium sp.......................................... 4Streptococcus intermedius ...................................... 3Anaerobic gram-negative rod .......................... ......... 7Anaerobic gram-positive coccus .................. ............. 3Anaerobic gram-positive rod.................................... 3Candida albicans .......................................... 7

a Only microbes isolated from more than one specimen are shown.

scesses or the head and neck area (Table 1). None of theseven IF-positive peritonsillar abscess specimens and onlyone of the five IF-positive head and neck specimens yieldedany of the three BFG species in isolation. All of the sevenperitonsillar abscess specimens yielded one or more otherbacterial species in isolation, and only one specimen fromthe head and neck area was totally isolation negative.

Table 5 shows the microbiological findings for the 115IF-positive specimens not yielding any of the three BFGspecies in isolation. In only three cases was a gram-negativerod found in the primary staining but not in isolation. In 44cases, both aerobic and anaerobic organisms were isolated.E. coli was the most frequently isolated bacterial species.Staphylococcus aureus was isolated only from two speci-mens, and coagulase-negative staphylococci were isolatedfrom three specimens. IF of colonies taken from purecultures revealed that none of the organisms isolated fromthese specimens reacted with the MAb.Two main microscopic results were observed in the posi-

tive cases. If a positive specimen did not contain leukocytes,the bacteria remained intact and were easy to identify (Fig.1A). When leukocytes were abundant, both intact organismsand phagocytized or leukocyte-associated fluorescing mate-rial were seen (Fig. 1B). As mentioned in Materials andMethods, a prerequisite for positivity by IF was the occur-rence of intact bacteria, even in the leukocyte-rich speci-mens. No fluorescing large organisms resembling L-phasebacterial cells, tissue cells, or leukocytes were present in thedirect smears.

DISCUSSION

In this study, an in vitro-produced MAb with a well-characterized target determinant in the LPS of B. fragiliswas used in an IF technique for detecting this microbe andtwo other BFG species, B. thetaiotaomicron and B. ovatus,

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DETECTION OF B. FRAGILIS 451

FIG. 1. IF staining with the MAb against the D-galactose oligo-mer in B. fragiles LPS. (A) Intact B. fragilis organisms with typicalfluorescent outlining. (B) Leukocyte-associated bacterial fragmentsin an isolation-positive specimen with abundant leukocytosis.

directly in 1,897 clinical specimens. By comparison withcultivation, the sensitivity of the assay varied from 87.1 to56.3%, depending on the anatomical location from which thespecimen was taken. Earlier reported sensitivities of IF withpolyclonal antibodies in the direct detection ofB.fragilis andrelated species varied between 80.9 (15) and 100% (8). Evenwith the same commercial reagents very different resultshave been obtained (80.9 to 98.5%) (1, 15). These resultshave been based mostly on relatively limited and selectedmaterials. Perhaps the lowest sensitivity figure occurredwith the largest amount of material (15). Conclusions aboutthe performance of a diagnostic test must therefore be basedon relatively long experience with defined material of repre-sentative clinical specimens. The differences may also reflectthe efficacy of B. fragilis isolation in different laboratories.

The sensitivity of the assay was better with the aspiratesthan with the swabs. This result evidently was due to largernumbers of bacteria in aspirates, which could be taken onlyfrom infections with abundant pus production, and not to thesuperiority of aspiration as a sampling method.When considering the sensitivity figures obtained, it must

be kept in mind that relatively strict positivity criteria wereused. In specimens with abundant leukocytosis, largeamounts of fluorescing material, both leukocyte associatedand free, was a common finding. However, a prerequisite forpositivity by IF was the occurrence of intact bacteria with afluorescent outlining.Some specimens remained negative by IF since they

yielded a BFG strain that did not contain the target deter-minant for the MAb. This was the case with 7 of the 18 B.fragilis strains and 6 of 14 B. thetaiotaomicron and B. ovatusstrains isolated from IF-negative specimens. The sensitivityshould be improved by developing MAbs against thesestrains and using antibody mixtures. However, our prelimi-nary experiments indicate that the strains not reacting withthe MAb used in this study do not contain a common epitopethat could yield MAbs with sufficient coverage. MAbs ob-tained by immunization with some species not having theD-galactose oligomer have been strictly strain specific, eventhough some of them have been directed against LPS.The specificity of the present method, 93.4% in all of the

material, is on the same level as in the reports with polyclo-nal antisera. However, almost as many IF-positive speci-mens (115 of 120) were found among both negative andpositive specimens in isolation for the three BFG species,lowering the positive predictive value of the assay to 51.1%.In our opinion, this value does not do justice to IF. Thespecificity of IF was ascertained by many means. The MAbhas proven specific for the three BFG species (13). The onlyexception is the reactivity with GBS strains observed in thisstudy. However, this reactivity caused only one false-posi-tive result and was not taken into account. The bacteriaisolated from the IF-positive, BFG-negative specimens weretested for their reactivity with the MAb, and none of themproved reactive. Furthermore, several of these specimenswere totally isolation negative. This result strongly suggeststhat the discrepancy between isolation and IF could not becaused by the poor specificity of IF. A more plausibleexplanation is that the specimens really contained killedBFG bacteria or bacteria with a low ability to grow onartificial media, making the use of isolation as a referencemethod questionable in the assessment of positive predictivevalues of IF and other antigen detection methods.

In addition to antimicrobial therapy, inappropriate collec-tion and transportation of specimens may affect adverselyculture results. However, neither transportation time norantimicrobial therapy could explain the discrepancy in thisstudy. Overgrowth by other bacterial species and the result-ing failure to recognize bacteroides colonies has been sug-gested as one possible explanation for negative cultures (1).In the present study, aerobic bacterial species were isolatedfrom 76 and anaerobic species were isolated from 58 of the115 specimens positive by IF but negative for the three BFGspecies in cultures. In these specimens, interference byother microbes may have been a factor impairing the recog-nition of bacteroides, although the selective bacteroides bileesculin plate largely overcame this problem. However, 25 ofthe specimens were totally isolation negative. Thus, over-growth cannot be held as a general explanation for thediscrepant results.With specimens taken from peritonsillar abscesses or the

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head and neck area, the discordance between IF and isola-tion was most prominent. None of the seven IF-positiveperitonsillar abscess specimens yielded any of the three BFGspecies in isolation; only one specimen from the head andneck area was positive in both tests. With these specimensinterference by other microbes may have taken place, sincealmost all of them were polymicrobial in nature. Suchinterference may occur in the infectious focus and, withsome`local factors, may impair the viability of bacteroides.This problem clearly needs further exploration.DeGirolami and Mepani (1) have reported that GBS may

cause a false-positive reaction in a commercial bacteroidesIF test with polyclonal antibodies. Since the specimen inquestion in their study was not incubated anaerobically, thepresence of B. fragilis could not be outruled. Our resultssuggest that this cross-reaction may be a true one, since 1 ofthe 12 GBS isolated in this study proved reactive with theMAb. Our preliminary studies have shown that ca. 12% ofStreptococcus agalactiae strains isolated in our laboratoryare positive. The staining of the streptococci was bright andresembled the membranous fluorescence of B. fragilis. Thisresult suggests that the determinant is located on the surfaceof the organism. Studies on the nature of the cross-reactiveantigen and its relation to type-specific antigens of GBS areunder way.Leukocytes heavily loaded with fluorescing bacterial frag-

ments were commonly found in leukocyte-rich specimensyielding B. fragilis in cultivation. Although phagocytizedbacterial fragments could not be discriminated from thoseattached to the surfaces of cells, this phenomenon may be anexpression of the low toxicity of B. fragilis LPS (7). On theother hand, we did not find fluorescing L forms or bacteriaresembling eucaryotic cells, both of which have been re-ported to occur in some clinical specimens (16).An attempt to use the antibody in ascitic fluid led to

cross-reactions with several organisms of the normal flora.Since absorption of ascitic fluids was not considered reason-able, only an in vitro-produced MAb was used throughoutthe study; such an MAb is generally recommended fordiagnostic assays in bacteriology. Based on our experience,the use of undiluted culture medium was not any majordrawback, since the hybridoma could be easily grown inlarge volumes of medium.

This study shows that with a single MAb it is possible toestablish an IF assay that detects ca. 79% of isolation-positive B. fragilis infections. The best sensitivities wereobtained with specimens potentially infected with intestinalmicrobial flora. The specificity of IF was carefully ascer-tained, and the isolation-negative specimens that were pos-itive by IF could not be considered false-positive, since IFcan detect nonviable bacteria.

ACKNOWLEDGMENTS

This study was supported by grants from the Academy of Finland,the Emil Aaltonen Foundation, and the Research and ScienceFoundation of Laake Oy.We thank Maija Sarasniemi and Tarja Laine for technical assis-

tance and Simo Merne for revising the language of the manuscript.

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