JOURNAL OF CLINICAL MICROBIOLOGY, Mar. 1994, p. 679-683 Vol. 32, No. 30095-1 137/94/$04.00+0Copyright © 1994, American Society for Microbiology

Detection of Bacteroides fragilis in Clinical Specimens by PCRYUKO YAMASHITA,* SHIGERU KOHNO, HIRONOBU KOGA, KAZUNORI TOMONO,

AND MITSUO KAKUThe Second Department of Internal Medicine, Nagasaki University School

of Medicine, 1-7-1 Sakamoto, Nagasaki, 852, Japan

Received 25 August 1993/Returned for modification 28 September 1993/Accepted 2 December 1993

The direct detection of Bacteroidesfragilis from clinical specimens was examined by using the PCR methodfor amplifying a specific fragment of the glutamine synthetase gene from B. fragilis. By this method, all five B.fragilis strains tested were detected, but DNAs from anaerobic bacteria of 24 other species tested, from aerobicbacteria of 12 species tested, and from human leukocytes were not amplified. Using the nested PCR method,we were able to detect as little as one bacterial cell or 100 fg of chromosomal DNA of B. fragilis. A total of 39clinical specimens, which consisted of 19 bronchial aspirates, 10 percutaneous lung aspirates, 2 transtrachealaspirates, 6 pleural fluid specimens, and 2 pus specimens, were tested. All four culture-positive samples, ofwhich two were bronchial aspirates, one was pleural fluid, and one was pus, were positive by PCR. Among 35culture-negative samples, 2 bronchial aspirates were positive by PCR. One was from a patient whose twoprevious samples were positive by both culture and PCR. It had been submitted for culture several hours aftercollection, and clindamycin had been administered to the patient before collection of the specimen. The otherbronchial aspirate positive by PCR was from a pneumonia patient who had also been administeredclindamycin. We believe that B. firagilis was present in these two specimens but that either it was dead, it wasbelow the level detectable by culture, or the process of anaerobic culture was unsuccessful. Thus, the PCRmethod may be considered useful for the sensitive and rapid detection of anaerobes in clinical specimens.

Anaerobes are known to be causative organisms of variousinfectious diseases in humans (4, 8), but the need for specialtechniques and equipment to culture these organisms hasdelayed improvement in detection of anaerobes from clinicalspecimens (13). It is more difficult to isolate anaerobes fromlower respiratory tract infections, in particular, because anaer-obes exist within the oral cavity as normal flora (13). There-fore, appropriate specimens for anaerobic culture need to betaken by special techniques, such as transtracheal aspiration,percutaneous lung aspiration, bronchoscopy aspiration with aprotected catheter brush (PCB), and the like, which weredeveloped in order to avoid oropharyngeal contamination(1-4). Since anaerobic conditions are difficult to maintainwhen a small quantity of specimen is obtained and since thelidocaine used for local anesthesia in bronchoscopic examina-tion has antibacterial effects (1, 2, 4), the successful culture ofanaerobes is difficult. Additionally, conventional methods foridentification of anaerobes by biochemical tests or gas-liquidchromatography are troublesome and time-consuming, andcommercial kits for rapid identification are less accurate thanconventional methods (13).The anaerobes frequently isolated from human clinical

specimens are Bacteroides fragilis, Prevotella melaninogenica,Fusobacterium nucleatum, and Peptostreptococcus spp. (Weak-ly saccharolytic Bacteroides spp. have now been reclassified inthe genus Prevotella [8].) B. fragilis is particularly importantbecause of its pathogenicity, frequency, and resistance to manycommonly used antimicrobial agents (8).

In order to rapidly detect and/or identify B. fragilis and otherBacteroides spp., the use of various methods, such as immuno-fluorescence (6, 21), DNA probes (10, 15), and rRNA restric-

* Corresponding author. Mailing address: The Second Departmentof Internal Medicine, Nagasaki University School of Medicine, 1-7-1Sakamoto, Nagasaki, 852, Japan. Phone: 0958-47-2111. Fax: 0958-49-5236.

tion fragment length polymorphisms (19), has been described.However, each of these methods has disadvantages in speci-ficity or sensitivity. In recent years, the PCR method (16),which can amplify a specific gene fragment rapidly in vitro, hasbeen developed and come into wide use. In the field of clinicalbacteriology, this method is very useful to detect organismswhich are difficult to culture, for example, Mycobacteriumtuberculosis (12, 14, 18), Legionella pneumophila (7, 20), andthe like. Furthermore, a nested PCR method which is com-posed of a two-step PCR can enhance sensitivity more than thesingle-step PCR (11, 12, 14). Thus, the PCR method can detecta target organism directly from clinical specimens withoutculture. In this study, we used the nested PCR method todetect B. fragilis in clinical specimens obtained from patientswith anaerobic infections, mainly lower respiratory tract infec-tions.

MATERMILS AND METHODS

Bacterial strains. The organisms used to evaluate the spec-ificity of the PCR method for detection of B. fragilis are asfollows. For anaerobic bacteria, the following 29 strains, mostof which were kindly supplied by the Institute of AnaerobicBacteriology, Gifu University School of Medicine, were tested:B. fragilis (ATCC 25285 and four clinical isolates), Bacteroidescaccae (GAI-973173), Bacteroides distasonis (GAI-5462), Bac-teroides eggerthii (GAI-5478), Bacteroides merdae (GAI-93174),Bacteroides ovatus (GAI-5630), Bacteroides stercoris (GAI-93175), Bacteroides thetaiotaomicron (ATCC 29741), Bacte-roides uniformis (GAI-5466), Bacteroides vulgatus (GAI-5460),Bacteroides ureolyticus (GAI-5544), Prevotella bivia (GAl-5518), Prevotella intermedia (GAI-5592), Prevotella oralis(GAI-7801), Prevotella oris (GAI-7508), P. melaninogenica(clinical isolate), Porphylomonas gingivalis (GAI-7802), F.nucleatum (GAI-5464), Fusobacterium varium (GAI-5566),Fusobacterium necrophorum (GAI-5634), Peptostreptococcusmagnus (GAI-5600), Peptostreptococcus asaccharolyticus

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TABLE 1. Detection of B. fragilis in clinical specimens

No.

Specimen PCR positive PCR negativeCulture Culture Culture Culture Totalpositive negative positive negative

Bronchial aspirateCannula 2 2 0 12 16"PCB 0 0 0 3 3

Percutaneous lung 0 0 0 10 1obaspirate

Transtracheal 0 0 0 2 2aspirate

Pleural fluid 1 0 0 5 6Pus 1 0 0 1 2

Total 4 2 0 33 39

a Among these specimens, nine were obtained from patients with pneumoniaand seven were from patients with lung abscess.

b Among these specimens, three were obtained from patients with pneumoniaand seven were from patients with lung abscess.

(GAI-5534), Peptostreptococcus anaerobius (GAI-5598), Pep-tostreptococcus micros (GAI-5540), and Veillonella parvula(GAI-5602). For aerobic bacteria, the following 12 strains weretested: Staphylococcus aureus (ATCC 25923), Streptococcuspneumoniae (clinical isolate), Streptococcus intermedius(ATCC 27735), Escherichia coli (ATCC 25922), Klebsiellapneumoniae (clinical isolate), Enterobacter cloacae (ATCC23355), Serratia marcescens (ATCC 8100), Proteus mirabilis(clinical isolate), Pseudomonas aeruginosa (ATCC 27853),Acinetobacter calcoaceticus (clinical isolate), Haemophilus in-fluenzae (ATCC 35036), and Moraxella catarrhalis (clinicalisolate). Additionally, DNA extracted from human leukocyteswas evaluated. All anaerobic bacterial strains and S. interme-dius were cultured under anaerobic conditions on modifiedGifu Anaerobic Medium agar (Nissui Pharmaceutical Co.,Tokyo, Japan) plates. (S. intermedius grows better underanaerobic conditions.) Other aerobic bacterial strains werecultured in Luria broth (17), except for strains of S. pneu-moniae, K pneumoniae, H. influenzae, and M. catarrhalis,which were cultured in Trypticase soy broth (BBL Microbiol-ogy Systems). To calculate the detection limit for B. fragilis bythe PCR method, serial dilutions of a suspension with B. fragiliswere prepared as follows. Several colonies of B. fragilis cul-tured on modified Gifu Anaerobic Medium agar under anaer-obic conditions for 2 days were suspended in sterile distilledwater, and this suspension was diluted 10-fold with steriledistilled water. The number of bacterial cells was determinedby inoculating 100 RIl of each dilution onto modified GifuAnaerobic Medium agar. Serial dilutions from 104 CFU to10- CFU were used for the PCR described below.

Patients and clinical specimens. Thirty-nine specimens from37 patients with infectious diseases were examined. (Threesamples of bronchial aspirate obtained with a cannula fromone patient with pneumonia were examined.) The clinicalspecimens used for detection of B. fragilis are classified inTable 1. Nine of 16 samples of bronchial aspirate obtained witha cannula, 3 of 3 samples of bronchial aspirate obtained with aPCB, 3 of 10 samples of percutaneous lung aspirate, and 2 of2 samples of transtracheal aspirate were obtained from pa-tients with pneumonia. Seven of 16 samples of bronchialaspirate obtained with a cannula and 7 of 10 samples ofpercutaneous lung aspirate were from patients with lungabscess. All six samples of pleural fluid were obtained from

patients with pyothrax. Both of two samples of pus wereobtained from patients with subcutaneous abscess. Each sam-ple was tested by Gram stain, aerobic and anaerobic culture,and nested PCR examination.

Bacteriologic processing. The specimens were plated ontothe following four kinds of media for anaerobic culture(Kyokuto Pharmaceutical Co., Tokyo, Japan): brucella HKagar with hemolyzed rabbit blood and defibrinated sheepblood, phenylethyl alcohol brucella HK agar with hemolyzedrabbit blood, paromomycin-vancomycin brucella HK agar withhemolyzed rabbit blood, and bacteroides bile esculin agar. Thespecimens generally were plated immediately after collection,and then the media were placed in GasPak Pouches (BBL) toput them under anaerobic conditions. They were incubated at37°C for more than 2 days and less than 7 days. An anaerobicchamber (Forma Scientific Inc.) or anaerobic jars (Anaer-oPack; Mitsubushi Gas Inc., Tokyo, Japan) were used forsubculture. Anaerobes isolated by this process were identifiedby using RapID-ANA (Innovative Diagnostic Systems, Inc.,Atlanta, Ga.). As media for aerobes and fungi, sheep bloodagar (BBL), chocolate agar (BBL), bromthymol blue lactoseagar (Nissui Pharmaceutical Co.), and Inhibitory Mold Agar(BBL) were used.DNA extraction from bacterial strains. A large number of

colonies obtained from each bacterial strain on agar weresuspended in 400 ,ul of TE buffer (10 mM Tris hydrochloride[pH 8], 1 mM EDTA). Each bacterial strain cultured in brothwas centrifuged at 10,000 x g for 10 min, and the pellet wasresuspended in 400 pul of TE buffer. Suspensions of gram-negative bacteria were incubated with lysozyme (to a finalconcentration of 1 mg/ml), and suspensions of gram-positivebacteria were incubated with mutanolysin (to a final concen-tration of 125 U/ml) (9) at 37°C for 60 min. Proteinase K andsodium dodecyl sulfate (SDS) were added to each suspensionto final concentrations of 1 mg/ml and 1%, respectively, andeach mixture was incubated at 60°C for 4 h. For the extractionof nucleic acids, an equal volume of phenol-chloroform-isoamyl alcohol (25:24:1) was added, each mixture was centri-fuged at 10,000 x g for 2 min, and then the supernatant wastransferred to a fresh tube. This procedure was repeated twice,and then the same procedure with an equal volume of chloro-form-isoamyl alcohol (24:1) was performed once. The DNAwas precipitated with 1/10 volume of 3 M sodium acetate and2 volumes of ice-cold absolute ethanol at - 80°C for 30 minand then centrifuged at 10,000 x g for 15 min at 0°C. Theair-dried pellet was resuspended in double-distilled water anddiluted to 10 ng/lld. The quantity of DNA in each sample wasmeasured with a fluorometer (model 450; Sequoia-TurnerCorporation). Serial dilutions containing DNA of B. fragilis inamounts from 10 ng to 10 fg were prepared to calculate thedetection limit according to the DNA quantity.DNA extraction from clinical specimens. When the speci-

mens were purulent, they were incubated with dithiothreitol(to a final concentration of 1 mg/ml) at 37°C for 30 min at thebeginning of the procedure. All samples were centrifuged at10,000 x g for 10 min, and each pellet was resuspended in 400,ul of TE buffer. They were incubated with lysozyme (1 mg/ml)at 37°C for 60 min and then with proteinase K (1 mg/ml) andSDS (1%) at 60°C for more than 4 h. Next, DNA was extractedby phenol and chloroform treatment and recovered by ethanolprecipitation as described above. The extracted DNA wasresuspended in 100 p.1 of distilled water, 10 p.1 of which wasused for PCR.

Synthetic oligonucleotides. Two sets of primers encoding theglutamine synthetase gene (reported by Hill et al. [5]) weresynthesized and used for the nested PCR. The sequence of the

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DETECTION OF BACTEROIDES FRAGILIS BY PCR 681

A582 bp ,

B582bp o-

1 2 3 4 5 6 7 8 9101112

FIG. 1. Agarose gel electrophoresis (A) and Southern blot hybrid-ization (B) of amplified DNA from Bacteroides sp. by using primersBFR-1 and BFR-2 and detection probe BFR-5. Lanes: 1, molecularsize markers; 2, negative control (distilled water); 3, B. fragilis; 4, B.caccae; 5, B. distasonis; 6, B. eggerthii; 7, B. nierdae; 8, B. ovatus; 9, B.stercoris; 10, B. tletaiotaomicroni; I1, B. uniiformis; 12, B. vulgatus.

B

- 582 bp

glutamine synthetase gene of B. fraigilis differs markedly fromthose of other prokaryotes and eukaryotes. Parts of the GSgene sequences that differ between B. fragilis and other pro-karyotes and eukaryotes were chosen. BFR-1 (5'-ACTCTlTTGTATCCCGACGATT-3' and BFR-2 (5'-GAGGTTGATGCCTGTATCGGT-3') were used for the first PCR as the outerprimers. BFR-1 and BFR-2 were located at nucleotides 484 to504 and nucleotides 1045 to 1065, respectively, from the codingstrand. BFR-3 (5'-GCTACCGAAGTATGCCAGCTT-3') andBFR-4 (5'-GTGGTCATTCGCCAGATTACA-3') were usedfor the second PCR as the inner primers. BFR-3 was located atnucleotides 580 to 600, and BFR-4 was located at nucleotides901 to 921. BFR-5 (5'-AGCCAATTCAAACTGGTTGG-3'),which was located at nucleotides 866 to 885, was used as adetection probe. These oligonucleotides were synthesized witha DNA synthesizer (model 380B; Applied Biosystems, FosterCity, Calif.).DNA amplification. For the first PCR, 10 pL. of each

extracted DNA was added to each reaction mixture, whichcontained 50 mM KCl, 10 mM Tris-HCl (pH 8.3), 1.5 mMMgCl,, 0.01% gelatin, 200 p.M each dATP, dCTP, dGTP, anddTTP, 1.0 p.M each of a set of primers (BFR-1 and BFR-2),1.25 U of Taq DNA polymerase, and distilled water, in a finalvolume of 50 pl.. Each reaction mixture was overlaid with adrop of mineral oil. After an initial denaturation step at 94°C

1 2 3 4 5 6 7 8 9 1011 12 13 14 15A

582 bp ,

B

582bp

FIG. 2. Agarose gel electrophoresis (A) and Southern blot hybrid-ization (B) of amplified DNA from anaerobic bacteria and humanleukocytes by using primers BFR-1 and BFR-2 and detection probeBFR-5. Lanes: 1, negative control; 2, B. fragilis (type strain); 3, B.thetaiotaomicron; 4, B. ureiolyticus; 5, P. bivia; 6, P. intermedia; 7. P.oralis; 8, P. oris; 9, P. melaninogenica; 10, Porphylomonas gingivalis; 1,

human leukocytes; 12 to 15, clinical isolates of B. fragilis.

FIG. 3. Agarose gel electrophoresis (A) and Southern blot hybrid-ization (B) of amplified DNA from aerobic bacteria by using primersBFR-l and BFR-2 and detection probe BFR-5. Lanes: 1, negativecontrol; 2, S. aureus; 3, S. pnieulnoniae; 4, S. inwtermedius; 5, E. coli: 6, Kpneunoniace; 7, E. cloacae; 8, S. marcescenis; 9, P. mirabilis; 10, P.aeruginiosa; llA. calcoaceticus; 12, H. inzfiuenizae; 13, M. catarrlhalis; 14,B. fragilis (positive control).

for 10 min, 35 cycles of amplification were performed. Eachcycle consisted of denaturation at 94°C for min, annealing at63°C for 1.5 min, and extension at 72°C for I min. For thesecond PCR, 10 p.l of each first PCR product was added to afreshly prepared reaction mixture containing BFR-3 andBFR-4 as primers. PCR amplification was performed underthe conditions described above.

Gel electrophoresis and Southern blot hybridization. Tenmicroliters of each PCR product was electrophoresed with 2 pl.of gel-loading buffer (0.25% bromophenol blue, 0.25% xylenecyanol FF, 15% Ficoll in water) through a 1.5% agarose gel at100 V for 30 min in TBE buffer (45 mM Tris-HCI, 45 mM boricacid, mM EDTA [pH 8.0]) containing ethidium bromide (0.5jig/ml). A mixture of pHY300PLK digested with HintdIII andHaeIII and pHY300.2PLK digested with HaeIII (Yakult Co.,Tokyo, Japan) was used as a molecular weight marker. The gelwas photographed under UV light.

For Southern blotting, the PCR products were transferred toa nylon membrane (Hybond-N+), using 0.4 N NaOH, andwere fixed to the membrane by UV irradiation. The membranewas hybridized with the detection probe, BFR-5, by using anenhanced chemiluminescence 3'-oligolabelling and detectionsystem (RPN 2131; Amersham International plc.). The speci-ficity of the PCR reaction was confirmed by the presence orabsence of the intended band.

RESULTS

Specificity of the PCR. Among 25 species (29 strains) ofanaerobes, amplification of the 582-base fragment by usingBFR-1 and BFR-2 as primers was achieved only with the DNAisolated from B. fragilis. Twelve species of aerobes and humanleukocytes were used for the study of specificity as well, but the582-base fragment was not obtained. The results for eightBacteroides species (the former B. fragilis group) are shown inFig. 1. The results for 5 strains of B. fragilis, 8 species of otheranaerobes, and human leukocytes are shown in Fig. 2, andthose for 12 species of aerobes are shown in Fig. 3. Weak bandsvery close to the 582-base fragment of B. fragilis were noted forB. caccae, B. merdae, and B. vulgatlus (Fig. IA), but the342-base fragment was not obtained by the nested PCRmethod for those species (data not shown). Furthermore, aSouthern blot hybridization assay confirmed that the bands

A

582bp

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A582bp P.

B

342bp o-

1 2 3 4 5 6 7

FIG. 4. Sensitivity of first (A) and second (B) PCR steps with a10-fold bacterial dilution. Lanes: 1, molecular size markers; 2, 104CFU; 3, 103 CFU; 4, 102 CFU; 5, 10' CFU; 6, 100 CFU; 7, 10-' CFU.

were not specific to B. fragilis (Fig. 1B). Several bands ofdifferent molecular sizes were obtained for several speciesother than B. fragilis (Fig. 1A, 2A, and 3A), but a Southern blothybridization assay subsequently confirmed that they were notspecific to B. fragilis (Fig. 1B, 2B, and 3B). The results for theremaining eight anaerobic species also indicated that thismethod was specific for B. fragilis (data not shown).

Sensitivity of the nested PCR method. The sensitivity of thenested PCR method was evaluated by using serial dilution ofbacterial cells and DNA of B. fragilis. In the bacterial cell study,103 CFU of B. fragilis was able to be detected by the first PCR,and one bacterial cell could be detected by the nested PCR(Fig. 4). Similarly, 1 ng of chromosomal DNA of B. fragilis wasdetectable by the first PCR, and 100 fg was detectable by thenested PCR (Fig. 5).

Detection of B. fragilis from clinical specimens. To evaluatethe application of this PCR method to clinical specimens, 39samples from 37 patients suspected of having anaerobic bac-terial infections were examined. Four specimens that werepositive by culture (two samples of bronchial aspirate from onepatient with pneumonia, one sample of pleural fluid from apatient with pyothorax, and one sample of pus from a patientwith a subcutaneous abscess from which B. fragilis was isolated)were all positive by both PCR and the hybridization assay withthe PCR products (Table 1; Fig. 6). Two samples of bronchialaspirate were negative by culture but positive by PCR (Table 1;Fig. 6). One of these was the third sample from a pneumoniapatient whose other two samples were positive by both cultureand PCR. The first and second specimens had been culturedanaerobically immediately after collection, but the third spec-imen had been cultured several hours after collection. A Gramstain of this third specimen revealed some gram-negativebacilli, and only P. aeruginosa was isolated by culture. Theother sample of bronchial aspirate that was negative by culturebut positive by PCR was from another pneumonia patient. Inthis specimen, many gram-positive cocci were found by Gramstain, and Streptococcus mitis and aerobic gram-positive bacilli,

A582 bp -

B

342 bp v-

1 2 3 4 5 6 7 8

12457I.FIG. 5. Sensitivity of first (A) and second (B) PCR steps on the

basis of DNA quantity. Lanes: 1, molecular size markers; 2, 10 ng; 3,1 ng; 4, 100 pg; 5, 10 pg; 6, 1 pg; 7, 100 fg; 8, 10 fg.

A

342 bp -

B

1 2 3 4 5 6 7 8 9

-t

*.s-.., im"

FIG. 6. Agarose gel electrophoresis (A) and Southern blot hybrid-ization (B) of amplified DNA from clinical specimens by the nestedPCR method with detection probe BFR-5. Lanes: 1, molecular sizemarkers; 2, positive control (B. fragilis); 3 to 5, samples of bronchialaspirate from one patient with pneumonia; 6, sample of pleural fluidfrom a patient with pyothorax; 7, sample of pus from a patient with asubcutaneous abscess; 8, sample of bronchial aspirate from a patientwith pneumonia; 9, sample of bronchial aspirate from a patient withlung abscess. Samples in lanes 3, 4, 6, and 7 were culture positive; thosein lanes 5, 8, and 9 were culture negative.

which failed to be identified because of their inactive biochem-ical reactions, were isolated by culture. Neither of the DNAsextracted from these two bacterial strains was amplified byPCR with primers specific to B. fragilis. Clindamycin had beenadministered to both of these two patients before collection ofthe specimens. The remaining 33 specimens were negative byboth culture and PCR. The gel electrophoresis and the hybrid-ization assay of all the positive clinical specimens and arepresentative negative specimen are shown in Fig. 6. Thenegative specimen was a sample of bronchial aspirate from apatient with lung abscess, from which Streptococcus constella-tus, F. nucleatum, and P. intermedia were isolated by culture.

DISCUSSION

Various methods to detect or identify B. fragilis and otherBacteroides spp. rapidly and accurately have been reported. Byimmunofluorescence, B. fragilis and members of the Bacte-roides melaninogenicus group in clinical specimens can bedetected directly and rapidly, even when they are not viable,but the method produces some false-positive results, andevaluation of unstable reactions is difficult (6, 21). DNA probesfor Bacteroides spp. are more specific because these bacteriaare differentiated at the genetic level. However, the limit ofdetection is 106 bacteria, and therefore it is possible thatfalse-negative results will be obtained. Also, culture is requiredto increase numbers of bacteria for direct detection fromclinical specimens (10). The PCR method, which can amplifythe specific gene fragment by as much as 106-fold by using TaqDNA polymerase and specific primers for the target genefragment in vitro, can detect and identify target organisms,even when they are not viable, with great specificity, sensitivity,and rapidity (16).

In this study, we developed specific primers for B. fragilis andused them to examine the PCR method. The method wasspecific for B. fragilis. No significant cross-reactivity with otheranaerobes or aerobes was observed. In regard to sensitivity, wecould detect as little as one bacterial cell of B. fragilis and 100fg of DNA by the nested PCR method.

Regarding detection of B. fragilis in clinical specimens, allfour of the culture-positive specimens were positive by PCR.Two specimens which were negative by culture were positive byPCR. The following are possible reasons for this finding:contamination of B. fragilis DNA, cross-reactivity with other

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DETECTION OF BACTEROIDES FRAGILIS BY PCR 683

organisms, reaction to dead bacteria, or failure of the anaer-obic culture process. The possibility of contamination is slight,since precautions to prevent it were taken during the process ofsample preparation and DNA amplification and since targetDNA amplified by this method was not observed in negativecontrols without template DNA, which were treated at sametime. Cross-reactivity with other organisms was ruled out bythe negative results of PCR with DNA extracted from organ-isms isolated from these specimens. Because several hours hadpassed before the collected specimen was cultured anaerobi-cally and because clindamycin had been administered to bothpatients before collection of these specimens, we regard thesepositive results as being caused by the reaction to DNA of deadB. fragilis. This ability to detect dead organisms is a greatadvantage of the PCR method for detection of anaerobes.Detection may be possible even when collection and transpor-tation of the specimen are carried out improperly or when thespecimen is obtained after the administration of antibiotics.Accordingly, the PCR method stands to become a very usefulstrategy for detection of anaerobes. However, there remainsthe problem of culture-negative but PCR-positive results.Hereafter, to establish the utility of this method, it will benecessary to examine a larger number of specimens, to con-tinue comparing the results of PCR with those of culture, andto investigate the cause in cases of discrepancy. Various kindsof clinical specimens were used in this study, but comparison ofresults between the different specimens was not possiblebecause of the small number of positive samples.

All specimens that showed positive results were highlypurulent. DNA extracted from such specimens contains toomuch human DNA, which may obstruct the PCR; nevertheless,we were able to detect target DNA from purulent specimens.On the other hand, since the volume of specimens obtained bybronchoscopy aspiration using a PCB or by percutaneous lungaspiration is too small, we had no positive results from suchspecimens in this study. From these data, we can conclude thatas large a volume of the clinical specimen as possible must betaken, especially when the method of PCB or needle aspirationis used.The PCR system investigated in this study was specific only

for B. fragilis among Bacteroides spp. B. fragilis is isolated fromclinical specimens most frequently, but other Bacteroides spp.often are also isolated, and they are as virulent and as resistantto many antimicrobial agents as B. fragilis is. If a PCR systemfor detection of all Bacteroides spp. is developed, it may bemore useful from the clinical point of view. As a furtherapplication, a PCR system for detection of the anaerobes P.melaninogenica, F. nucleatum, and Peptostreptococcus spp.,which are important in anaerobic infections of the lung andmore difficult to isolate by culture, should be developed.Additionally, this system is expected to be extended to a widervariety of samples, such as blood and biopsied tissue. Thus, thePCR method shows much promise for the detection of anaer-obes.

ACKNOWLEDGMENTSWe thank Kohei Hara for guidance and Kazue Ueno and Kunitomo

Watanabe for kindly providing some anaerobic bacterial strains.

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