JOURNAL OF CLINICAL MICROBIOLOGY, Feb. 1989, p. 321-3260095-1137/89/020321-06$02.00/0Copyright C 1989, American Society for Microbiology

Identification and Classification of Campylobacter Strains by UsingNonradioactive DNA Probes

DANIELE CHEVRIER,' DANIEL LARZUL,' FRANCIS MEGRAUD,2 AND JEAN-LUC GUESDON1*Laboratoire des Sondes Froides, Institut Pasteur, 25 rue du Dr Roux, 75724 Paris Cedex 15,1 and Laboratoire de

Bactériologie, Hôpital des Enfants, Bordeaux, 33077 Bordeaux Cedex,2 France

Received 25 May 1988/Accepted 3 November 1988

Acetylaminofluorene-labeled genomic DNA probes were used for the identification and classification ofCampylobacter strains. Relationships among 17 well-known strains of Campylobacter species and subspecieswere studied by comparing acetylaminofluorene- or 32P-labeled probes. Results obtained with both methodswere closely correlated and were in agreement with those already reported. In an identification experiment,hybridization with nonradioactive probes was performed on 60 strains isolated from stool samples aftersubculturing and quick DNA extraction; conventional biochemical tests were conducted in parallel. A goodcorrelation was found between the results obtained by nonradioactive hybridization and by biochemical tests.Thus, the acetylaminofluorene-labeled genomic DNA probe method is an interesting alternative for laboratorieswithout access to radioisotopes for the identification and classification of bacteria.

Campylobacter species have emerged during the past 10years as major pathogens in the human gastrointestinal tract(38). C. jejuni is a leading cause of intestinal infection.However, other species can also be present, i.e., C. coli,"C. upsaliensis" (20), C. laridis, C. fetus, and, in someinstances, C. sputorum, C. hyointestinalis, "C. fennelliae,"and "C. cinaedi" (5). C. pylori has also attracted greatinterest because it has been associated with gastritis andduodenal ulcer disease (21).On a clinical basis, C. jejuni infections cannot be easily

distinguished from illnesses caused by other enteropatho-gens. Identification of Campylobacter strains at the specieslevel is based on biochemical tests, tolerance to variouscompounds, and different incubation temperatures. To dif-ferentiate between certain species, only one test is available,for example, the hippurate test to differentiate C. jejuni andC. coli (44). Some of the tests involve susceptibility toantibiotics, and the result can be erroneous if resistance isacquired (1). For example, C. coli and C. laridis are differentonly with regard to nalidixic acid resistance and anaerobicgrowth in trimethylamine N-oxide. When C. coli becomesnalidixic acid resistant, the species identification is renderedvery difficult.Now that we are aware of the diversity of the genus

Campylobacter, the exact identification of these bacteria hasbecome crucial in defining the disease spectrum of eachspecies as well as for epidemiological purposes. Nucleic acidhybridization is the reference method used in taxonomy (8,23). It has been applied to the identification of some Cam-pylobacter species by using radiolabeled total DNA probesand dot blot hybridization techniques (9, 11, 26, 34, 39, 42,44) and to the classification of some of them (14). However,the disadvantages of radiolabeled DNA, i.e., health hazardsand short half-life, make the use of DNA hybridizationdifficult in routine diagnosis and in laboratories with limitedequipment.Tchen et al. (40) have described a method based on the

chemical modification of guanine residues by using N-ace-toxy-N-2-acetylaminofluorene (AAAF) to prepare nonradio-active probes which can be detected by an immunological

* Corresponding author.

method. In this study, we used acetylaminofluorene (AAF)-labeled genomic DNA probes to identify all the Campylo-bacter species presently known, except C. nitrofigilis (foundonly in plants), and we demonstrate that these "cold"probes can be used for classification.

MATERIALS AND METHODSBacterial strains. (i) Reference strains. The reference

strains are listed in Table 1. They are type strains of thegiven species except for C. concisus, "C. fennelliae," C.mucosalis, and C. pylori.According to phenotypic results and previous hybridiza-

tion tests (26, 35, 36), Campylobacter reference strains havebeen divided into five groups: group I, thermophilic strains(C. jejuni, C. coli, C. laridis, gastric Campylobacter-likeorganism number 2 [GCLO2], and "C. upsaliensis"); group11, "fetus" group (C. fetus subsp. fetus, C. fetus subsp.venerealis, and C. hyointestinalis); group III, "sputorum"group (C. sputorum subsp. sputorum, C. sputorum subsp.bubulus, and C. fecalis); group IV, "concisus" group (C.concisus and C. mucosalis); and group V, "cinaedi" group("C. fennelliae" and "C. cinaedi"). Four Wolinella refer-ence strains (W. succinogenes, W. curva, and W. recta) wereincluded in one experiment as controls.

(ii) Wild-type strains. Specimen strains were isolated fromhuman feces. They were characterized by a battery ofconventional tests including tests for catalase, oxidase,nitrate reductase, and urease activities; growth at 25°C,42°C, and in anaerobiosis; susceptibility to nalidixic acid andcephalothin; hippurate hydrolysis; production of H2S intriple sugar iron agar; and anaerobic growth with trimethyl-amine N-oxide.DNA extraction. For reference strains, DNA was ex-

tracted and purified according to the method described byFennell et al. (5). Briefly, the bacteria grown under optimalconditions on Mueller-Hinton agar plates were harvestedafter incubation for 1 to 4 days, depending on the species, in1 M Tris hydrochloride-O.5 M EDTA, pH 8.0. They werethen incubated successively with lysozyme (Boehringer,Mannheim, Federal Republic of Germany) at 1 mg/ml, 1%sodium dodecyl sulfate (SDS; Sigma Chemical Co., St.Louis, Mo.), and 50 ,ug of proteinase K (Boehringer). Protein

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TABLE 1. Reference strains

Strain Source' Group

Campylobacterjejuni CIP 702 IC. coli CIP 7080 IC. laridis NCTC 11352 IGCLO2 NCTC 11848 I"C. upsaliensis" NCTC 11541 IC. fetus subsp. fetus CIP 5396 IlC. fetus subsp. venerealis CIP 6829 IIC. hyointestinalis CCUG 14169 IlC. sputorum subsp. sputorum CCUG 9728 IliC. sputorum subsp. bubulus CIP 53103 IIIC. fecalis CIP 8105 IIMC. concisus Tanner 1182569 IVC. mucosalis NCTC 11001 IV"C. fennelliae" Fennell 441 V"C. cinaedi" ATCC 35683 VC. cryaerophila CCUG 17801C. pylori CIP 101260Escherichia coli HB 101Wollinella succinogenes CCUG 13145W. curva CCUG 11644

CCUG 13646W. recta CCUG 11640

aATCC, American Type Culture Collection, Rockville, Md.; CCUG,Culture Collection University of GoÇteborg, G0teborg, Sweden; CIP, Collec-tion Institut Pasteur, Paris, France; NCTC, National Collection of TypeCultures, London, England; Fennell, C. Fennell, Harborview Medical Cen-ter, Seattle, Wash.; Tanner, A. Tanner, Forsyth Dental Center, Boston,Mass.

was extracted once with phenol and twice with chloroform,and DNA was precipitated by using isopropanol. The sam-ples were treated with RNase (Boehringer) at 50 ,ug/ml andthen with pronase (Boehringer) at 50 ,ug/ml before the DNAconcentration was measured by A260.For wild-type strains, stool samples collected from pa-

tients with diarrhea were cultured in selective medium andsubsequently subcultured. The bacterial cells (from 3 x i09to 10 x 109) were recovered from the blood agar plates(diameter, 90 mm) in 2 ml of 25 mM Tris hydrochloride (pH8.0)-10 mM EDTA-50 mM glucose, centrifuged, washedonce, and suspended in 200 pi of the same buffer. The cellswere then lysed in 0.2 N NaOH-1% SDS for 10 min at 4°C.Proteins were removed by phenol and chloroform-isoamylalcohol (24:1) extractions; the DNA was then ethanol pre-cipitated and solubilized in 400 ,ul of 10 mM Tris hydrochlo-ride (pH 8.0) containing 1 mM EDTA. DNA concentrationswere determined by A260-

Preparation of nucleic acid probes. For nonradioactive-probe preparation, the purified total genomic DNA wassonicated and labeled with AAAF by following a previouslydescribed procedure (15, 19, 22). Briefly, 240 p.g of AAAFper ml in 1 mM sodium citrate buffer (pH 7.0) containing 20%ethanol and 0.8% dimethyl sulfoxide was mixed with 200 ,ugof sonicated DNA per ml and incubated for 1 h at 37°C in thedark. The unreacted fluorene derivatives were subsequentlyremoved by five extractions with ethyl ether and one extrac-tion with chloroform, and then the modified DNA wasprecipitated with ethanol. The percentage of AAF-modifiedbases was determined by measuring the A305 and A260 aspreviously described (6, 7). This value ranged from 3 to 9%.Since AAAF, a potential carcinogen, should be handledcautiously, all contaminated materials were treated withconcentrated sulfuric acid.For radioactive labeling, the purified DNA was treated by

random priming (Multiprime DNA Labeling System; Amer-

sham, Little Chalfont, United Kingdom) by using [ac-32P]dCTP. The specific activities of the different probesranged from 1.7 x 109 to 2.7 x 109 cpm/,ug.Dot blot hybridization with AAF- or 32P-labeled DNA

probes. Different quantities of extracted DNA were dena-tured in 0.1 M NaOH for 10 min at 4°C and neutralized in0.15 M NaHPO4. Samples of diluted DNA were spottedonto nitrocellulose filters with a vacuum filtration apparatus(SRC 096 Minifold t; Schleicher & Schuell, Dassel, FederalRepublic of Germany). For DNA-DNA homology determi-nation, 30, 20, 10, or 5 ng of homologous DNA and 30 ng ofheterologous DNA to be tested were spotted onto the filters.For the identification test, 30 ng of reference strain DNA and50 or 100 ng of wild-type DNA was deposited per dot. Filterswere then dried and baked at 80°C for 2 h.

Conditions of hybridization were those described by Ma-niatis et al. (18) with some modifications. The filters wereprehybridized at 65°C for 5 h in a mixture containing 6x SSC(1x SSC is 150 mM NaCI plus 15 mM sodium citrate), 5xDenhardt solution, 0.5% SDS, and 100 tg of sonicatedsalmon sperm DNA per ml. Hybridization was carried outovernight at 65°C with 250 ng of heat-denaturated AAF-labeled DNA probe per ml in a mixture containing 6x SSC,5x Denhardt solution, 0.5% SDS, 10 mM EDTA, 10%dextran sulfate, and 100 p.g of sonicated salmon sperm DNAper ml. Denhardt solution contains 0.1% Ficoll (Pharmacia,Uppsala, Sweden), 0.1% polyvinylpyrrolidone, and 0.1%bovine serum albumin.The filters were washed three times at room temperature

in 2x SSC containing 0.1% SDS for 10 min, twice at 50°C for30 min in the same solution, and finally once in 0.1x SSCcontaining 0.1% SDS at 50°C for 30 min. The filters werethen incubated for 1 h with purified anti-AAF monoclonalantibody (17) diluted to 1 ,ug/ml in 20 mM Tris hydrochloride(pH 7.8)-150 mM NaCl-1% bovine serum albumin-0.1%Tween 20 (Merck, Darmstadt, Federal Republic of Ger-many), washed, and further incubated for 1 h with alkalinephosphatase-labeled sheep anti-mouse immunoglobulin Gantibody prepared by the method of Avrameas (2) anddiluted to 1 ,ug/ml in the same buffer. After another washing,filters were incubated for 30 min in the dark in 100 mM Trishydrochloride (pH 9.5)-100 mM NaCl-50 mM MgCl2-0.33mg of Nitro Blue Tetrazolium (Sigma) per ml-0.16 mg of5-bromo-4-chloro-3-indolyl phosphate (Boehringer) per mland washed in distilled water. Positive hybridization yieldeda dark blue precipitate.

Hybridization of filters with 32P-labeled probes was per-formed under the same conditions as indicated for AAF-labeled probes. The probe concentration was chosen toobtain 106 cpm/ml. After the final wash, filters were cut intoindividual squares. Each square containing one DNA dotwas placed in 3 ml of scintillation liquid (ACS II; Amersham)and then counted in a Kontron MR 300 counter.DNA-DNA strain classification. When AAF-labeled DNA

probes were used, the wet filters were compared visuallyjustafter the enzyme reaction to determine the extent of hybrid-ization; after drying, the blue color faded. The amounts ofspotted DNA were chosen in the range in which the dot colorintensity was proportionally related to the quantity of DNA.Thus, we were able to compare the dot color intensityobtained by using a given amount of DNA to be tested withthe dot color intensity obtained by using the DNA homolo-gous to the DNA probe used. Four DNA relatedness classes(A, B, C, and D) were defined. The limits of the classescorresponded to the amount of homologous DNA spottedonto the filter. Since 30 ng of DNA to be tested and 30, 20,

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%o tA

1001-

70e

30h

15-

B C D

FIG. 1. DNA-DNA strain classification with AAF-labeled C.cinaedi DNA probe. Various amounts of DNA were spotted onto a

nitrocellulose filter. Column A, C. cinaedi DNA in duplicate. Firstrow, 30 ng; second row, 20 ng; third row, 10 ng; fourth row, 5 ng.

Column B, C. fennelliae DNA (30 ng) in triplicate. Column C, C.pylori DNA (30 ng) in triplicate. Column D, E. coli HB101 DNA (30ng) in triplicate. From the results shown here, we deduced that C.fennelliae belongs to class C and that C. pylori and E. coli belong toclass D.

10, or 5 ng of reference DNA were spotted onto each filter,the limits of the classes could be estimated at 70 to 100%(class A), 30 to 70% (class B), 15 to 30% (class C), and <15%(class D).When 32P-labeled DNA probes were used, the degree of

relatedness was calculated by using the following equation:(counts per minute ofDNA to be tested/counts per minute ofhomologous DNA) x 100.

Identification of Campylobacter species by using AAF-la-beled DNA probes. To identify Campylobacter strains iso-lated from stool samples, the seven strains commonly foundin human feces (C. jejuni, C. coli, C. fetus subsp. fetus, C.hyointestinalis, C. sputorum subsp. bubulus, "C. upsalien-sis," and C. laridis) were used to prepare AAF-labeled DNAprobes. Some samples gave positive hybridizations with twodifferent probes due to cross-hybridization. In order toeliminate all ambiguity, the seven filters were compared andsample identification was based on the probe which gave thedarkest staining.

RESULTS

DNA relatedness determination. DNA relatedness among17 Campylobacter reference strains, including the seven

genomic probes used in the identification experiment, wasdetermined by using AAF-labeled DNA. An example isgiven in Fig. 1, for which C. fennelliae, C. pylori, andEscherichia coli DNA were tested with the AAF-labeled C.cinaedi probe. The three strains were respectively classifiedas C, D, and D as defined in Material and Methods. Theresults were compared with those obtained by using 32p_labeled probes and with previously published values.The interstrain relatedness values were determined by

using the AAF-labeled DNA probe (Fig. 2). The control wasperformed by hybridizing E. coli DNA to every probe; thebinding of AAF-labeled DNA probes to the DNA controlwas not detectable. The DNA-DNA hybridization data in

Fig. 2 show a high degree of relatedness (classes A and B)between C. jejuni and GCLO2 in group I; between C. fetussubsp. fetus and C. feuts subsp. venerealis in group Il; andamong C. sputorum subsp. sputorum, C. sputorum subsp.bubulus, and C. fecalis in group III. A lower degree ofrelatedness (class C) was observed between C. jejuni and C.coli, C.jejuni and C. laridis, C. coli and GCLO2, GCLO2 and"C. upsaliensis," C. fetus subsp. fetus and C. hyointestina-lis, C. fetus subsp. venerealis and C. hyointestinalis, and"C. fennelliae" and "C. cinaedi." Moreover, strains re-ported in the literature to have nonsignificant DNA homol-ogy were classified as being in class D by the presentnonradioactive technique.Within each group, the relatedness values were deter-

mined by DNA hybridization methods by using 32P- orAAF-labeled probes. The results obtained by both methodswere closely correlated, since for 44 determinations (86%)the same class was found with radioactive and nonradioac-tive methods, and for the remaining 7 (14%) the two DNArelatedness values were in consecutive classes.

Correlation between phenotypic identification and DNAhybridization identification. A blind, two-laboratory compar-ative identification of Campylobacter strains by DNA hy-bridization with seven AAF-labeled DNA probes and phe-notypic tests was performed with 60 Campylobacter strainsisolated from human feces and 4 Wollinella reference strains.The results (Table 2) were in agreement with both methods

for 62 of the 64 strains. Two discrepancies were noted. C.laridis was identified as C. coli with the probes, and "C.upsaliensis" was identified as C. jejuni. These strains weretested again by conventional biochemical tests, which con-firmed the validity of the AAF-DNA probe result. The firststrain was a nalidixic-acid resistant C. coli strain withdoubtful results for growth in trimethylamine-N-oxide; thesecond was a cephalothin-susceptible C. jejuni strain with aweak hippurate reaction. An example of dot blot hybridiza-tion is shown in Fig. 3, for which the seven AAF-labeledDNA probe were used to identify 21 unknown strains.

DISCUSSION

DNA probes have become a major tool in microbiology inrecent years. Their main application has been in virology, inwhich they are used to detect virus genomes directly inclinical material. In the field of bacteriology, fewer applica-tions have been established. One of the first was the detec-tion of the DNA sequence coding for enterotoxins in order toidentify enterotoxigenic E. coli (24). Radiolabeled DNAprobes have been proposed for identification of strains at thespecies level, such as total DNA for Mobiluncus (31) andBacteroides (32) species, commercialized probes for Myco-bacterium (13) and Legionella (4) species, cloned chromo-somal DNA for C. jejuni (30), and oligonucleotides forProteus subspecies (10). Because of the specific problemslinked to radioisotopes, some authors have used nonradio-active probes. Total DNA probes labeled with biotin havebeen applied to identify Leptospira subspecies (41). TheDNA sulfonation method has been applied with total DNAfor the detection of Chlamydia trachomatis (3) and withcloned DNA for the identification and detection of Myco-plasma subspecies (12). In addition, oligonucleotides cova-lently linked to an enzyme were used for identification ofenterotoxigenic E. coli (25, 37).AAAF is able to bind on C-8 of guanine. This substance is

known to be a modifier of DNA structure and an agent ofmutagenesis. It has been used since 1984 to label DNA to be

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324 CHEVRIER ET AL.

C. jejuni

C. coli

C. aridis

GCLO2

C. upsaliensis

C. fetus ssp fetus

C. fetus ssp venerealis

C. hyointestinalis

C. sputorum ssp sputorum

C. sputorum ssp bubulus

C. fecalis

C. concisus

C. mucosalis

C. fennellia"

C. cinodim

C. cryaerophila

C. pylori

E. coli HB101

FIG. 2. DNA relatedness among reference strains. Rows are reference strains used to prepare DNA targets, and columns are referencestrains used to prepare DNA probes. Four DNA relatedness classes were defined: A (70% < R < 100%), B (30% < R s 70%), C (15% < R

30%), and D (R c 15%), where R is the degree of relatedness. Shaded squares indicate results obtained with both hybridization techniques(32p_ and AAF-labeled probes). Dark shading represents homologous hybridization; when results were different between 32P- andAAF-labeled probes, small letters inside brackets indicate results obtained with the radioactive technique.

used as probes. AAF is a large hapten and allows theproduction of antibodies with high affinity (17). The existingapplications in infectious disease include the detection ofcytomegalovirus in human lungs (15) and the detection ofhepatitis B virus in sera (16). We are not aware of anapplication in bacteriology. When used by us, the hybridiza-tion test using AAF-labeled DNA probes was sensitiveenough to allow the detection of as few as 105 Campylobac-ter cells (data not shown). AAF is preferred to biotin for tworeasons: it is not a naturally occurring substance and it doesnot give background reactions, as have been reported withbiotin (45). However, it has the disadvantage of being

TABLE 2. Comparative identification by phenotypic andhybridization tests

No. of strains identified by:Identified strains Phenotypic Hybridization

tests test

C. jejuni 27 28C. coli 15 16C. fetus 3 3C. hyointestinalis 1 1C. sputorum 1 1"C. upsaliensis" 8 7C. laridis 5 4Non-Campylobacter strains 4 4

carcinogenic; thus, the labeling of DNA must be performedcautiously. In contrast, manipulation of AAF-labeled probesdoes not require special precautions.The genus Campylobacter is relatively distinct from other

bacterial genera, as shown by the study of RNA homologies(28). Moreover, within the genus, the genetic relationshipsare relatively weak, except for a few species. These partic-ular conditions allow the use of total DNA in à hybridizationmethod to identify the bacteria.

Totten et al. (44) have proposed a differential spot blot testfor the classification of thermophilic C impylobacter strains.In this test, organisms to be tested are suspended in broth tocompare turbidity with a reference standard. Samples are

spotted onto nitrocellulose filters and heated to lyse thebacteria and denature the DNA. Using radiolabeled C.jejuni, C. coli, and C. laridis DNAs as probes, these authorsdemonstrated that Campylobacter species classificationcould be done as well with the spot blot test as withquantitative whole-cell DNA hybridization. In our study, wedeveloped a nonradioactive dot blot hybridization test ableto differentiate Campylobacter species; in contrast to thespot blot test previously described (44), and to increase theaccuracy and avoid the nonspecific probe binding generallyencountered in nonradioactive hybridization tests with crudebiological samples, we used known amounts of extractedDNA for spotting. The results obtained by using this testwere compared with those obtained by using the radioactive

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FIG. 3. Example of dot blot hybridization with AAF-labeledtotal genomic DNA probes. Nitrocellulose filters after dot blothybridization ofDNA extracted from bacteria are shown. At the leftof every filter, 30 ng of reference strain DNA was spotted induplicate as controls as follows: C. jejuni (J), C. coli (C), C.hyointestinalis (H), C. fetus subsp. fetus (F), "C. upsaliensis" (U),C. laridis (L) and C. sputorum subsp. bubulus (S). (Locations ofcontrols and tested strains on the filters are indicated at lower right.)At the right of the filters, 50 (upper dot) and 100 (lower dot) ng of 21DNAs to be tested was spotted. Each filter was hybridized with oneof the seven probes. Strains 1, 3, 9, 16, and 18 were identified as C.jejuni; strains 2, 4, 8, 10, 11, and 17 were identified as C. coli; strains5 and 7 were identified as C. Iaridis; strains 6, 12, 13, 19, and 20 wereidentified as "C. upsaliensis"; strain 15 was identified as C. fetus,and strains 14 and 21 were identified as non-Campylobacter strains.

hybridization test. A satisfying correlation was observed.Moreover, the results obtained in the present work by thecross-hybridization experiment between the 17 DNA probesand the corresponding 17 DNAs demonstrated the specificityof the method. AAF-labeled probe results are in agreementwith the taxonomic data presently available in the literature.In group t, the GCLO2 strain was found to be very closelyrelated to C. jejuni; this observation is in agreement with theresults of Owen and Dawson (27). For DNA relatednessdetermination between C. jejuni and C. coli, dot color

intensity was estimated to be very close to the 30% spot limitof class C, in agreement with the 34% observed by Hébert etal. (11). In groups Il and III, we could not distinguishbetween C. fetus and C. sputorum subspecies by genomicDNA hybridization, as they were too closely related at thesubspecies level. These observations are in agreement withthe results of Harvey and Greenwood (9) and Roop et al.(35). We found a strong homology between C. sputorumsubspecies and C. fecalis, as was reported by Roop et al.(35). Although C. mucosalis and C. concisus both have thephenotypic characteristics of group IV, we found a low levelofDNA relatedness, as did Roop et al. (36). In group V ("C.fennelliae" and "C. cinaedi"), dot color intensity wasestimated to be very close to the 15% reference DNA spot ofclass C, that is, slightly higher than the 10% observed byTotten et al. (43).

C. pylori and C. cryaerophila are not included in a group,unlike the other species. Indeed, using AAF-labeled probes,we found, as did Romaniuk et al. (33) and Roop et al. (34),that C. pylori and C. cryaerophila have no relationship to theother Campylobacter species.Some authors have pointed out the difficulty of obtaining

precise identification of Campylobacter species and the needfor genetic identification (29). We agree with this opinion,especially since our results show that 2 of 60 strains wereincorrectly identified by conventional tests. Our techniquehas the advantage of being nonisotopic, and it can be used inany laboratory for both the identification and the classifica-tion of Campylobacter strains.

LITERATURE CITED1. Altwegg, M., A. Burnens, J. Zollinger-Iten, and J. L. Penner.

1987. Problems in identification of Campylobacter jejuni asso-ciated with acquisition of resistance to nalidixic acid. J. Clin.Microbiol. 25:1807-1808.

2. Avrameas, S. 1969. Coupling of enzymes to proteins withglutaraldehyde. Immunochemistry. 6:43-52.

3. Dutilh, B., C. Bébéar, D. Taylor-Robinson, and P. A. D. Gri-mont. 1988. Detection of Chlamydia trachomatis by in situhybridization with sulphonated total DNA. Ann. Inst. Pasteur.Microbiol. 139:115-127.

4. Edelstein, P. H. 1986. Evaluation of the Gen-Probe DNA for thedetection of legionellae in culture. J. Clin. Microbiol. 23:481-484.

5. Fennell, C. L., P. A. Totten, T. C. Queen, D. L. Patton, K. K.Holmes, and W. E. Stamm. 1984. Characterization of Campylo-bacter-like organisms isolated from homosexual men. J. Infect.Dis. 149:58-66.

6. Fuchs, R. P. P., and M. P. Daune. 1972. Physical studies ondeoxyribonucleic acid after covalent binding of a carcinogen.Biochemistry 11:2659-2666.

7. Fuchs, R. P. P., and M. P. Daune. 1973. Physical basis ofchemical carcinogenesis by N-2-fluorenylacetamide derivativesand analogs. FEBS Lett. 34:295-298.

8. Grimont, P. A. D. 1984. DNA/DNA hybridization in bacterialtaxonomy, p. 11-19. In A. Sanna and G. Morace (ed.), Newhorizons in microbiology. Elsevier Science Publishing, Inc.,New York.

9. Harvey, S. M., and J. R. Greenwood. 1983. Relationships amongcatalase-positive campylobacters determined by deoxyribonu-cleic acid-deoxyribonucleic acid hybridization. Int. J. Syst.Bacteriol. 33:275-284.

10. Haun, G., and U. Gobel. 1987. Oligonucleotide probes forgenus-, species-, and subspecies-specific identification of repre-sentatives of the genus Proteus. FEMS Microbiol. Lett. 43:187-193.

11. Hébert, G. A., P. Edmonds, and D. J. Brenner. 1984. DNArelatedness among strains of Campylobacter jejuni and Cam-pylobacter coli with divergent serogroup and hippurate reac-tions. J. Clin. Microbiol. 20:138-140.

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326 CHEVRIER ET AL.

12. Hyman, H. C., D. Yogen, and S. Razin. 1987. DNA probes fordetection and identification of Mycoplasma pneumonia andMycoplasma genitalium. J. Clin. Microbiol. 25:726-728.

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