0095-1137/89/020321-06$02.00/0
CopyrightC 1989,American Society for Microbiology
Identification
and
Classification of Campylobacter Strains by
Using
Nonradioactive
DNA
Probes
DANIELE CHEVRIER,' DANIEL LARZUL,' FRANCIS MEGRAUD,2 ANDJEAN-LUCGUESDON1* Laboratoire des Sondes Froides, Institut Pasteur, 25 rueduDr Roux, 75724 ParisCedex 15,1 and Laboratoire de
Bactériologie, Hôpital des Enfants, Bordeaux, 33077 Bordeaux Cedex,2France Received 25 May1988/Accepted 3 November 1988
Acetylaminofluorene-labeled genomic DNA probes were used for the identification and classification of
Campylobacter strains. Relationships among 17 well-known strains ofCampylobacter species and subspecies werestudied bycomparing acetylaminofluorene- or32P-labeled probes. Results obtained with bothmethods wereclosely correlated and wereinagreement with those alreadyreported. Inan identification experiment,
hybridization with nonradioactive probes was performed on 60 strains isolated from stool samples after
subculturing and quick DNAextraction; conventional biochemical tests were conductedin parallel. A good correlationwasfound between the results obtainedby nonradioactivehybridizationandbybiochemical tests.
Thus, theacetylaminofluorene-labeled genomicDNA probemethodisaninteresting alternative for laboratories
withoutaccess toradioisotopes fortheidentification andclassification of bacteria. Campylobacter species have emerged during the past 10
yearsasmajor pathogensinthehuman gastrointestinaltract (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 some
instances, C. sputorum, C. hyointestinalis, "C. fennelliae," and "C. cinaedi" (5). C. pylori has also attracted great interest because it has been associated with gastritis and duodenal ulcer disease (21).
On a clinical basis, C. jejuni infections cannot be easily distinguished from illnesses caused by other enteropatho-gens. Identification ofCampylobacterstrains at thespecies level is based on biochemical tests, tolerance to various compounds, and different incubation temperatures. To dif-ferentiatebetweencertainspecies, onlyonetestisavailable, forexample, thehippuratetesttodifferentiate C.jejuniand C. coli (44). Some of the tests involve susceptibility to antibiotics, and the result can be erroneous ifresistance is
acquired (1). Forexample, C. coliand C. laridisaredifferent
only with regard to nalidixic acid resistance and anaerobic growth in trimethylamine N-oxide. When C. coli becomes nalidixic acid resistant, the species identification is rendered verydifficult.
Now that we are aware of the diversity of the genus
Campylobacter,theexactidentificationof these bacteria has become crucial in defining the disease spectrum of each speciesaswellasforepidemiologicalpurposes.Nucleic acid
hybridization is the reference method usedintaxonomy (8, 23). It has been applied to the identification of some
Cam-pylobacter species byusing radiolabeled total DNAprobes anddotblot hybridization techniques (9, 11, 26, 34, 39, 42, 44)andtotheclassification ofsomeof them(14). However,
thedisadvantagesofradiolabeled DNA,i.e., healthhazards and short half-life, make the use of DNA hybridization
difficultin routinediagnosis and in laboratories with limited equipment.
Tchen et al. (40) have described a method based on the chemical modification ofguanine residues by using N-ace-toxy-N-2-acetylaminofluorene (AAAF)toprepare
nonradio-active probes which can be detected by an immunological
* Correspondingauthor.
method. Inthis study,weusedacetylaminofluorene (AAF)-labeled genomic DNA probes to identify all the Campylo-bacterspecies presentlyknown,exceptC. nitrofigilis (found only in plants), and we demonstrate that these "cold" probes canbe used forclassification.
MATERIALS AND METHODS
Bacterial strains. (i) Reference strains. The reference strains are listed in Table 1. They are type strains ofthe given species except for C. concisus, "C.fennelliae," C. mucosalis, andC. pylori.
According to phenotypic results and previous hybridiza-tiontests(26, 35, 36), Campylobacterreference strainshave been divided into fivegroups: group I, thermophilic strains (C. jejuni, C. coli, C. laridis, gastric Campylobacter-like organism number2 [GCLO2],and "C. upsaliensis");group
11, "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-encestrains(W.succinogenes,W.curva,and W.recta)were included inoneexperiment ascontrols.
(ii)Wild-type strains. Specimen strainswereisolated from
human feces. They were characterized by a battery of conventional tests including tests for catalase, oxidase, nitrate reductase, and urease activities; growth at 25°C, 42°C,and inanaerobiosis; susceptibilitytonalidixicacid and cephalothin; hippurate hydrolysis; production of H2S in triple sugariron agar;andanaerobicgrowthwith trimethyl-amine N-oxide.
DNA extraction. For reference strains, DNA was
ex-tracted and purified according tothe method described by Fennell et al. (5). Briefly, thebacteriagrownunderoptimal conditions on Mueller-Hinton agar plates were harvested after incubation for1to4days, dependingonthespecies,in 1 M Tris hydrochloride-O.5 M EDTA, pH 8.0. They were
then incubated successively with lysozyme (Boehringer, Mannheim, Federal Republic ofGermany) at 1 mg/ml, 1% sodium dodecyl sulfate (SDS; Sigma Chemical Co., St. Louis, Mo.),and 50,ugofproteinase K(Boehringer).Protein
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322 CHEVRIER ET AL.
TABLE 1. Reference strains
Strain Source' Group
Campylobacterjejuni CIP 702 I
C. coli CIP 7080 I
C. laridis NCTC 11352 I
GCLO2 NCTC 11848 I
"C. upsaliensis" NCTC 11541 I
C.fetus subsp. fetus CIP 5396 Il
C.fetus subsp. venerealis CIP 6829 II
C.hyointestinalis CCUG 14169 Il
C. sputorumsubsp. sputorum CCUG9728 Ili C.sputorumsubsp. bubulus CIP53103 III
C.fecalis CIP8105 IIM
C. concisus Tanner1182569 IV
C.mucosalis NCTC11001 IV
"C.fennelliae" Fennell 441 V
"C. cinaedi" ATCC 35683 V
C.cryaerophila CCUG17801
C.pylori CIP 101260
Escherichiacoli HB 101
Wollinellasuccinogenes CCUG13145
W.curva CCUG11644
CCUG13646
W.recta CCUG 11640
aATCC, American Type Culture Collection, Rockville, Md.; CCUG,
Culture CollectionUniversity ofGoÇteborg,G0teborg, Sweden; CIP,
Collec-tion Institut Pasteur, Paris, France; NCTC, NaCollec-tional CollecCollec-tion of Type Cultures, London, England; Fennell, C. Fennell, Harborview Medical
Cen-ter, Seattle, Wash.; Tanner, A. Tanner, Forsyth Dental Center, Boston,
Mass.
wasextracted once with phenol andtwice withchloroform,
and DNAwas precipitated by using isopropanol. The sam-ples were treated with RNase (Boehringer)at 50 ,ug/ml and then with pronase (Boehringer)at50,ug/ml before the DNA
concentration was measured by
A260.
For wild-type strains, stool samples
collected
frompa-tients with diarrhea were cultured in selective medium and
subsequently subcultured. Thebacterial cells (from3 x
i09
to 10 x 109) were recovered from the blood agar plates (diameter, 90mm) in 2 mlof25 mMTrishydrochloride (pH 8.0)-10 mM EDTA-50 mM glucose, centrifuged, washed once, andsuspendedin 200 piofthe samebuffer. The cells werethenlysed in 0.2 N NaOH-1%SDS for 10 minat4°C. Proteins were removed by phenol and chloroform-isoamyl alcohol (24:1) extractions; the DNA was then ethanol
pre-cipitatedand solubilizedin 400,ul of10 mMTris
hydrochlo-ride (pH 8.0)containing 1mMEDTA. DNAconcentrations weredetermined by
A260-Preparation of nucleic acid probes. For
nonradioactive-probe preparation, the purified total genomic DNA was sonicatedand labeled withAAAFbyfollowingapreviously described procedure (15, 19, 22). Briefly, 240
p.g
of AAAF permlin 1 mMsodium citrate buffer(pH 7.0)containing 20%ethanol and0.8%dimethyl sulfoxidewasmixed with 200 ,ug of sonicated DNA per ml and incubated for 1 hat37°Cin the dark. The unreacted fluorenederivativeswere subsequently removedby five extractions with ethyl ether andone extrac-tion with chloroform, and then the modified DNA was precipitated with ethanol. The percentage of AAF-modified
bases was determined by measuring the A305 and A260 as
previouslydescribed (6,7). This valueranged from 3to9%.
Since AAAF, a potential carcinogen, should be handled
cautiously, all contaminated materials were treated with concentrated sulfuric acid.
Forradioactivelabeling, thepurified DNAwastreatedby randompriming(Multiprime DNALabeling System;
Amer-sham, Little Chalfont, United Kingdom) by using [ac-32P]dCTP. The specific activities of the different probes ranged from 1.7 x 109to2.7 x 109cpm/,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 minat 4°C and neutralized in 0.15 M NaHPO4. Samples of diluted DNA were spotted
ontonitrocellulosefilters withavacuumfiltration apparatus (SRC 096 Minifoldt; Schleicher &Schuell, Dassel, Federal Republic ofGermany). For DNA-DNA homology
determi-nation, 30, 20, 10,or5 ngofhomologousDNAand30 ng of
heterologousDNAtobetestedwerespottedontothefilters. For theidentification test, 30 ng ofreference strain DNA and 50or100 ngofwild-typeDNAwasdepositedperdot.Filters were thendriedandbakedat 80°Cfor 2 h.
Conditions ofhybridizationwere those describedby Ma-niatis et al. (18) with some modifications. The filters were prehybridizedat65°Cfor5h in amixturecontaining 6x SSC (1x SSC is 150 mM NaCI plus 15 mM sodium citrate), 5x Denhardt solution, 0.5% SDS, and 100 tg of sonicated salmon sperm DNA per ml. Hybridization was carried out overnight at 65°C with 250 ng of heat-denaturated AAF-labeled DNAprobe per ml inamixturecontaining6x SSC, 5x Denhardt solution, 0.5% SDS, 10 mM EDTA, 10% dextransulfate,and 100p.g of sonicated salmon spermDNA perml. Denhardt solution contains0.1%Ficoll(Pharmacia, Uppsala, Sweden), 0.1% polyvinylpyrrolidone, and 0.1% bovineserum albumin.
The filters werewashed threetimes at room temperature in 2 x SSCcontaining0.1% SDS for 10min,twiceat50°Cfor 30 min in the same solution, and finally once in 0.1x SSC
containing 0.1% SDS at 50°C for 30 min. The filters were then incubated for 1 h with purified anti-AAF monoclonal
antibody (17)dilutedto1,ug/mlin20 mM Trishydrochloride
(pH 7.8)-150 mM NaCl-1% bovine serum albumin-0.1% Tween 20 (Merck, Darmstadt, Federal Republic of Ger-many), washed, and furtherincubatedfor 1 h with alkaline
phosphatase-labeled sheep anti-mouse
immunoglobulin
Gantibody prepared by the method of Avrameas (2) and dilutedto1,ug/ml inthe same buffer. After anotherwashing,
filterswereincubated for30min in the dark in 100 mM Tris
hydrochloride (pH 9.5)-100 mM NaCl-50 mM MgCl2-0.33
mg of Nitro Blue Tetrazolium (Sigma) per ml-0.16 mg of
5-bromo-4-chloro-3-indolyl phosphate (Boehringer)
per ml and washed indistilled water.Positivehybridizationyielded
adark 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 to obtain 106cpm/ml. After the final
wash,
filterswere cutinto individual squares. Each squarecontaining
one DNA dot wasplacedin 3 mlof scintillationliquid (ACS II; Amersham)and then counted ina Kontron MR 300counter.
DNA-DNA strain classification. When AAF-labeled DNA
probeswereused,thewetfilterswerecompared
visuallyjust
after the enzyme reactiontodetermine theextentof
hybrid-ization; afterdrying, the blue color faded. Theamounts of spottedDNAwerechosen in the range inwhich the dot color
intensitywasproportionallyrelatedtothe
quantity
of DNA.Thus, we were able to compare the dot color
intensity
obtained byusingagivenamountof DNAto betestedwith the dot colorintensity obtained byusingthe DNA homolo-goustotheDNA
probe
used. FourDNArelatedness classes (A, B, C, and D) were defined. The limits of the classescorresponded to the amount of homologous DNA
spotted
onto thefilter. Since 30 ng of DNAtobe tested and 30, 20,
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%o
t
A
1001-70e
30h
15-B
C
D
FIG. 1. DNA-DNA strain classification with AAF-labeled C. cinaedi DNAprobe. Variousamountsof DNAwere spottedontoa
nitrocellulose filter. Column A, C. cinaedi DNA in duplicate. First
row, 30ng; secondrow, 20ng;third row, 10ng;fourthrow,5 ng.
Column B, C. fennelliae DNA (30 ng) in triplicate. Column C, C. pyloriDNA(30ng)intriplicate. Column D, E.coliHB101 DNA (30 ng) in triplicate. From the results shown here, wededuced that C. fennelliae belongstoclass C and thatC.pylori andE.coli belongto
class D.
10,or5 ngof reference DNAwere spottedontoeach filter, the limits of the classes could be estimated at 70 to 100% (class A), 30to70% (class B), 15to30%(class C),and <15% (class D).
When 32P-labeled DNA probes were used, the degree of relatednesswas calculated by using the following equation:
(countsperminuteofDNAtobetested/countsperminuteof
homologous DNA) x 100.
Identification of Campylobacter species by using AAF-la-beled DNA probes. To identify Campylobacter strains iso-latedfromstool samples, thesevenstrainscommonly found
in humanfeces(C.jejuni, C. coli, C. fetus subsp. fetus, C. hyointestinalis, C. sputorum subsp. bubulus, "C. upsalien-sis,"and C.laridis)wereusedtoprepareAAF-labeled DNA probes. Some samplesgavepositive hybridizationswithtwo different probes due to cross-hybridization. In order to
eliminateall ambiguity, the sevenfilterswerecomparedand
sampleidentificationwasbasedontheprobe whichgavethe
darkest staining.
RESULTS
DNA relatedness determination. DNA relatedness among
17 Campylobacter reference strains, including the seven
genomic probes used in the identification experiment, was
determined by using AAF-labeled DNA. An example is given in Fig. 1, for which C. fennelliae, C. pylori, and Escherichia coli DNAwere testedwiththe AAF-labeled C. cinaediprobe. The three strains wererespectivelyclassified as C, D, and D as defined in Material and Methods. The
results were compared with those obtained by using 32p_ labeled probes andwithpreviously published values.
The interstrain relatedness values were determined by usingtheAAF-labeled DNAprobe (Fig. 2).Thecontrolwas
performed by hybridizingE. coli DNAto every probe; the binding of AAF-labeled DNA probes to the DNA control
was not detectable. The DNA-DNA hybridization data in
Fig. 2show a high degree of relatedness (classes Aand B) between C. jejuniand GCLO2 in group I; between C.fetus subsp.fetusand C.
feuts
subsp. venerealis in group Il;and among C. sputorum subsp. sputorum, C. sputorum subsp.bubulus, and C. fecalis in group III. A lower degree of relatedness(classC)wasobserved between C.jejuniandC. coli, C.
jejuni
and C.laridis,C. coli andGCLO2,GCLO2and "C.upsaliensis," C.fetus subsp. fetusand C. hyointestina-lis, C. fetus subsp. venerealis and C. hyointestinalis, and "C.fennelliae"
and "C. cinaedi." Moreover, strainsre-ported in the literature to have nonsignificantDNA 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- or AAF-labeledprobes. The resultsobtained byboth methods were closely correlated, since for 44 determinations (86%)
the sameclass was found with radioactive and
nonradioac-tive methods, and for the remaining7
(14%)
the two DNA relatedness values werein consecutive classes.Correlation between phenotypic identification and DNA
hybridizationidentification.Ablind, two-laboratory
compar-ative identification of
Campylobacter
strainsby
DNAhy-bridization with seven AAF-labeled DNA probes and phe-notypictests was performed with 60 Campylobacter strains isolated fromhuman feces and 4 Wollinellareferencestrains. Theresults(Table 2)wereinagreementwith both methods
for62 ofthe 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 were tested again by conventional biochemical tests, which con-firmed the validity ofthe AAF-DNAprobe result. The first strain was a nalidixic-acid resistant C. coli strain withdoubtful results for growth in
trimethylamine-N-oxide;
the second was acephalothin-susceptible
C.jejuni strain with a weakhippuratereaction. An exampleof dot blot hybridiza-tion is shown in Fig. 3, for which the seven AAF-labeled DNAprobe were usedto identify 21unknown strains.DISCUSSION
DNAprobeshavebecomeamajortool inmicrobiologyin recentyears.Their mainapplicationhasbeen invirology,in
which they are used to detect virus genomes directly in
clinical material. In the field ofbacteriology, fewer applica-tions have been established. One ofthefirstwas the detec-tionofthe DNA sequencecoding forenterotoxins in orderto
identify enterotoxigenic E. coli (24). Radiolabeled DNA
probes
have beenproposedfor identification ofstrainsatthespecies
level, such as total DNA for Mobiluncus (31) and Bacteroides (32) species, commercialized probesfor Myco-bacterium (13) and Legionella (4)species,
cloned chromo-somal DNA for C. jejuni (30), and oligonucleotides for Proteussubspecies
(10). Because of thespecific problems
linked to radioisotopes, some authors have used nonradio-active probes. Total DNA probes labeled with biotin have been
applied
toidentify
Leptospirasubspecies (41).
The DNA sulfonation method has been applied with total DNA for the detection ofChlamydia
trachomatis (3) and with cloned DNA for the identification and detection of Myco-plasma subspecies (12). In addition,oligonucleotides
cova-lently linked to an enzyme were used for identification of
enterotoxigenic
E. coli (25, 37).AAAFis abletobindonC-8 ofguanine.This substance is known to be a
modifier
ofDNA structure and an agent ofmutagenesis.
Ithasbeenused since 1984tolabel DNAtobe1
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324 CHEVRIER ET AL.
C. jejuni
C.coli C. aridis GCLO2 C. upsaliensis C. fetus ssp fetus C. fetus sspvenerealis 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. coliHB101
FIG. 2. DNArelatednessamongreference strains. Rowsarereference strains usedtoprepare DNAtargets, and columnsarereference
strains usedtoprepareDNAprobes. Four DNA relatedness classesweredefined: A (70%<R< 100%), B (30%<Rs 70%), C (15%<R 30%), and D (R c15%), where R is the degree of relatedness. Shadedsquaresindicate results obtained with bothhybridization techniques (32p_ and AAF-labeled probes). Dark shading represents homologous hybridization; when results were different between 32P- and AAF-labeledprobes, small letters inside brackets indicate results obtained with the radioactive technique.
used as probes. AAF is a large hapten and allows the
production of antibodies with high affinity (17). The existing applications in infectious disease include the detection of cytomegalovirus in human lungs (15) and the detection of hepatitis B virus in sera (16). We are not aware of an
applicationinbacteriology. When used byus,the
hybridiza-tion test using AAF-labeled DNA probes was sensitive
enoughtoallow the detection ofasfewas105
Campylobac-tercells (datanotshown).AAF ispreferredtobiotin fortwo
reasons: it isnotanaturally occurring substanceand itdoes notgive background reactions, ashave been reported with
biotin (45). However, it has the disadvantage of being
TABLE 2. Comparative identification by phenotypic and hybridizationtests
No. of strains identifiedby: Identified strains Phenotypic Hybridization
tests test
C.jejuni 27 28
C.coli 15 16
C.fetus 3 3
C.hyointestinalis 1 1
C.sputorum 1 1
"C. upsaliensis" 8 7
C.laridis 5 4
Non-Campylobacter strains 4 4
carcinogenic; thus, thelabelingof DNA mustbe performed cautiously. Incontrast,manipulationof AAF-labeledprobes does not require special precautions.
ThegenusCampylobacterisrelativelydistinct from other bacterialgenera, asshownbythe study ofRNAhomologies
(28). Moreover, within the genus, the genetic relationships
are relatively weak, exceptforafew species. These
partic-ular conditions allow theuseof total DNA in àhybridization
methodto identifythe bacteria.
Tottenetal. (44)haveproposedadifferentialspotblottest for the classification ofthermophilic C impylobacterstrains.
In thistest,organismstobe testedaresuspendedinbrothto
compare turbidity with a reference standard. Samples are
spotted onto nitrocellulose filters and heated to lyse the bacteria and denature the DNA. Using radiolabeled C. jejuni, C.coli,and C. laridis DNAsasprobes,these authors
demonstrated that Campylobacter species classification could be done as well with the spot blot test as with
quantitative whole-cellDNAhybridization. Inourstudy,we
developed a nonradioactive dot blothybridization testable
to differentiate Campylobacter species; in contrast to the spotblot testpreviously described (44), andto increase the
accuracy andavoid the nonspecific probe binding generally
encountered innonradioactivehybridizationtestswith crude biological samples, we used known amounts ofextracted DNA for spotting. The results obtained by using this test
werecomparedwith thoseobtainedby using the radioactive 3 O
.- .: D D D D D D D D D D D D
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0 _O PCiK :D D D D D D D D D D DO
D D D D D X ] C D D D D D D D D D
D D D D D À C D D D D D D D D D
D D D D D C C D D D D D D D D D
D D D D D D D D D D D D D
D D D D D D D D A ] D D D D D D
D D D D D D D D A D D D D D D
O D D D D D D D D D D O D D D D
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C. hilvointestinalis (H)
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C. faridis 'L)
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'C. upsaleunsis" (U)
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FIG. 3. Example of dot blot hybridization with AAF-labeled total genomic DNA probes. Nitrocellulose filters after dot blot hybridizationof DNA extracted from bacteriaareshown. At the left of every filter, 30 ng of reference strain DNA was spotted in duplicate 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 of controls andtested strainsonthe filtersareindicatedatlowerright.) Atthe rightofthe filters,50 (upperdot)and 100(lower dot)ngof 21 DNAstobe testedwasspotted. Each filterwashybridizedwithone
ofthesevenprobes. Strains1, 3,9,16, and 18wereidentifiedasC. jejuni; strains2,4,8,10, 11, and 17wereidentifiedasC.coli;strains
5and 7wereidentifiedasC.Iaridis;strains6, 12, 13, 19, and 20were
identified as "C. upsaliensis"; strain 15wasidentified asC.fetus, andstrains 14 and 21wereidentifiedasnon-Campylobacterstrains. hybridization test. A satisfying correlation was observed.
Moreover, the results obtained in the present work by the cross-hybridization experimentbetween the 17 DNAprobes andthecorresponding17 DNAs demonstrated thespecificity of themethod. AAF-labeled probe resultsarein agreement
withthe taxonomic datapresently available in the literature. Ingroup t, theGCLO2 strain was foundto be very closely
relatedtoC.jejuni;this observation is inagreementwith the results of Owen and Dawson (27). For DNA relatedness determination between C. jejuni and C. coli, dot color
intensity
wasestimatedtobe veryclosetothe30%spotlimit of classC,
inagreement with the34% observedby
Hébertet al. (11). In groups Il andIII,
we could notdistinguish
between C.
fetus
and C. sputorumsubspecies by
genomic
DNA
hybridization,
asthey
were tooclosely
relatedatthesubspecies
level. Theseobservationsare in agreement with the results ofHarvey
and Greenwood(9)
andRoop
et al.(35). We found a strong homology between C. sputorum
subspecies
and C.fecalis,
as wasreported by
Roop et al.(35). Although C. mucosalis and C. concisusbothhave the
phenotypic
characteristics ofgroupIV,
wefoundalow levelofDNA
relatedness,
asdidRoop
etal.(36).
In group V("C.
fennelliae"
and "C.cinaedi"),
dot colorintensity
was estimatedtobeveryclosetothe15%referenceDNAspot of classC,
thatis, slightly
higher
than the 10% observedby
Tottenet al. (43).
C.
pylori
and C.cryaerophila
are notincludedinagroup, unlike the otherspecies. Indeed,
using
AAF-labeledprobes,
we found, as didRomaniuk etal. (33)and Roopet al. (34),
that C.
pylori
and C.cryaerophila
havenorelationship
tothe otherCampylobacter
species.
Some authors have
pointed
outthedifficulty
ofobtaining
precise
identification ofCampylobacter
species
and theneed forgenetic
identification(29).
We agree with thisopinion,
especially
since our results show that 2of60 strains wereincorrectly
identifiedby
conventional tests. Ourtechnique
has the
advantage
ofbeing
nonisotopic,
anditcanbe used in anylaboratory
forboth the identification and the classifica-tionofCampylobacter
strains.LITERATURECITED
1. Altwegg, M., A. Burnens, J. Zollinger-Iten, and J. L. Penner. 1987. Problemsin identification ofCampylobacterjejuni
asso-ciated with acquisitionofresistanceto nalidixic acid. J. Clin. Microbiol.25:1807-1808.
2. Avrameas, S. 1969. Coupling of enzymes to proteins with glutaraldehyde. 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 situ
hybridizationwith sulphonatedtotal DNA. Ann. Inst.Pasteur. Microbiol. 139:115-127.
4. Edelstein,P. H.1986.Evaluation oftheGen-ProbeDNAforthe detection of legionellae in culture. J. Clin. Microbiol. 23: 481-484.
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