JOURNAL OF CLINICAL MICROBIOLOGY, Jan. 1985, p. 24-28 Vol. 21, No. 10095-1137/85/010024-05$02.00/0Copyright C 1985, American Society for Microbiology

Bacterial Chromosomal Restriction Endonuclease Analysis of theHomology of Bacteroides Species

W. C. BRADBURY,t* R. G. E. MURRAY, C. MANCINI, AND V. L. MORRISDepartment of Microbiology and Immunology, University of Western Ontario, London, Ontario, Canada M6A SCI

Received 25 May 1984/Accepted 21 September 1984

Chromosomal DNAs of selected Bacteroides organisms whose relatedness had been previously determined by"conventional" filter-annealing studies (J. L. Johnson, Int. J. Syst. Bacteriol. 28:245, 1978) were furtheranalyzed by restriction endonuclease analysis coupled with the Southern hybridization procedure (E. M.Southern, J. Mol. Biol. 98:503, 1975). By comparing their EcoRI restriction fragment patterns in agarose gelelectrophoresis, each Bacteroides strain could be clearly differentiated. As a simple and direct means forcomparison purposes this method was particularly useful for differentiating genetically similar organisms suchas Bacteroides strains of the same species which shared >75% homology. In contrast, bacterial chromosomalrestriction endonuclease analysis in conjunction with Southern hybridizations was most effectively used todetermine the significance of low levels of homology (<24%) as this technique provided additional informationon the nature and relative distribution of that homology when the areas of homology were displayed asreproducible bands in autoradiograms.

Bacterial organisms of the genus Bacteroides are a groupof phenotypically similar gram-negative rods (15), whichwere formerly classified as a single species Bacteroidesfragilis, with five subspecies (12). Recent DNA hybridiza-tion studies have separated this group at the species level,with 10 major homologous groups now recognized (12). Wehave chosen representatives of these groups showing a widerange of homology values (<4 to 80%), as determined byJohnson (12), for further examination, using bacterial chro-mosomal restriction endonuclease analysis (REA) coupledwith hybridizations by the Southern blotting technique (23).This method displays DNA fragments in order of size andindicates the distribution and equivalence of these fragmentsin related genomes by the use of appropriate labeled probes.Therefore, REA data can be used to extend the observationsof conventional DNA filter hybridization techniques. In thepresent communication we find that Bacteroides speciesshowing similar homology by "conventional" DNA filter-annealing studies can be further distinguished by REA.

MATERIALS AND METHODSBacterial strains and media. Bacteroides strains were ob-

tained from J. L. Johnson (Anaerobe Laboratory, VirginiaPolytechnic Institute and State University, Blacksburg). Thesix strains used in our study were selected from a largercollection of 16 reference strains that Johnson (12) used tosubdivide the former "Bacteroides fragilis group" into 10distinct homology groups. These six strains are B. ovatus0038 (neotype strain), B. ovatus 3524, "3524-A," B. vulgatus4245, B. uniforms 0061, and B. fragilis 2553 (neotype strain)(Table 1). The strain identification numbers are those of theJohnson reference collection. They were cultivated in brainheart infusion broth (Difco Laboratories, Detroit, Mich.),supplemented as below, in an anaerobic tent at 37°C. Themedium for the 1-liter cultures used for DNA isolation con-tained 3.7% (wt/vol) dehydrated brain heart infusion broth,0.5% yeast extract (Difco), 0.05% cysteine-HCI, 1% heme,

* Corresponding author.t Present address: Department of Microbiology, NUW-13-102,

Toronto General Hospital, Toronto, Ontario, Canada M5G 1L7.

and 0.02% vitamin K. This medium was seeded with 30 ml ofculture grown overnight.DNA isolation. Cells were washed once with single-

strength citrate buffer (SSC; containing 0.15 M NaCI, 0.01 Mtrisodium citrate, pH 7.0) and suspended in 15 ml of STEbuffer (0.1 M NaCI, 0.02 M Tris, 0.01 M EDTA, pH 7.4).DNA samples were prepared according to the method ofCohen et al. (5) with modifications. Samples were treatedwith pronase (Sigma Chemical Co., St. Louis, Mo.) (self-di-gested for 2 h at 37°C, 1 mg/ml) and 1% sodium dodecylsulfate for 1 h or overnight at 37°C. The mixture was thentreated three separate times with equal volumes of phenol-chloroform (50:50), and the upper aqueous phases werecarefully collected and pooled. Each sample was treatedwith RNase (bovine pancreatic, type 1A; Worthington Diag-nostics, Mississauga, Ontario, Canada) (heat treated at 90°Cfor 10 min; 50 ,ug/ml) for 1 h at 37°C. Each sample wasdialyzed against TE buffer (5 mM Tris-hydrochloride, 0.1mM EDTA, pH 7.4), and the DNA concentrations weredetermined by the diphenylamine assay of Burton (4). TheDNA samples were subjected to agarose gel electrophoresisbefore restriction endonuclease digestion to test for thepresence of extrachromosomal plasmid DNA (1, 14), but noplasmid DNA was detected. Only a single 19- to 21-megadalton (Md) band, representative ofchromosomal DNA(1), was seen.

Preparation of 32P-labeled DNA probes. The DNA sampleswere labeled by nick translation (13, 21) with minor modifi-cations. Standard reactions used approximately 0.5 ,ug ofduplex DNA, 180 pmol each of [a-32P]dATP and [a-32P]dTTP (400 Ci/mmol; Amersham Radiochemicals, Oak-ville, Ontario, Canada), and 180 and 400 pmol of unlabeleddCTP and dGTP, respectively. The reaction was initiatedwith 5 to 10 U ofDNA polymerase I (New England Nuclear,Beverly, Mass.) and incubated for 1 h at 15°C in the presenceof DNase (5 ng/ml). The addition of 0.5 M EDTA, pH 7.8,terminated the reaction, and the labeled DNA was extractedtwo separate times with an equal volume of chloroform-isoamyl alcohol (24:1). The aqueous phases were pooled andloaded on a G-75 Sephadex column (10 ml) in 1 mMTris-hydrochloride (pH 8.0)-0.25 mM EDTA and eluted.

24

REA OF BACTEROIDES SPECIES HOMOLOGY 25

TABLE 1. Reciprocal interstrain homology values among DNA reference organismS used in this study"% Homology

Probe B. fragilis B. ovatus B. uniformis B. vulgatus2553 0038 3524 "3452-A' 0061 4245

B. fragilis 2553 100 15 13 10 21 10(neotype strain) 100 24 9 23 17 20

B. ovatus 0038 24 100 80 31 17 8(neotype strain) 15 100 75 37 9 8

B. vulgatus 4245 20 8 0 4 19 10010 8 8 0 14 100

a Data are reproduced from reference 12. B. fragilis 2553, B. ovatus 0038, and B. v'ulgatus 4245 were used for the nick-translated, 32P-labeled DNA probes in ourstudy.

Peak fractions were collected and precipitated overnight at-20°C. The specific activities obtained were routinely 108cpm/,ug of DNA. The labeled probes were used within 1month of preparation.REA. Approximately 5 to 10 ,ug of high-molecular-weight

bacterial DNA was completely digested in a reaction mix-ture containing a fivefold excess of EcoRI enzyme (Boehrin-ger Mannheim, St. Laurent, Quebec, Canada; or BethesdaResearch Laboratories, Bethesda, Md.) in EcoRI buffer (100mM Tris-hydrochloride, pH 7.5, 50 mM NaCI, 10 mMMgCl2) at 37°C for 16 to 18 h. Identical results were obtainedwith EcoRI from both commercial sources. The reactionvolumes were approximately 100 LI and the reactions were

monitored for complete digestion, using bacteriophage X

DNA (Miles Laboratories, Elkhart, Ind.). All reactions were

terminated by the addition of 10 ,uI of DGE buffer (0.12%bromophenol blue, 25% glycerol, 0.2 M EDTA) and heatedfor 10 min at 68°C. The DNA fragments were separated byelectrophoresis in 0.7% agarose gels in EB buffer (40 mMTris-hydrochloride, pH 8.0, 2 mM EDTA, 20 mM sodiumacetate) (22). Gels were stained with ethidium bromide (1,ug/ml) for 1 h and photographed under UV illumination onKodak High Contrast Copy 35-mm film, using a no. 25 redKodak Wratten filter. Banding patterns in all digests were

reproducible.Southern transfer and hybridization to 32P-labeled probe

DNA. Agarose gels were treated for 1 to 2 h in 1 liter of 1.5M NaCI-0.5 M NaOH at room temperature and then for 3 to4 h in 1 liter of 0.5 M Tris-hydrochloride (pH 7.0)-3 M NaCIat room temperature. The DNA was blotted onto nitrocellu-lose filters according to the method of Southern (23) as

adapted by Shank et al. (22). The blots were dried in vacuoat 80°C for 1 to 2 h and stored at room temperature in vacuountil required for the hybridization experiments.

Before hybridization, each of the 32P-labeled DNA probeswas boiled for 10 min in 3 M NaOH (16). Filters were

hybridized as previously described (6, 17, 22). The filterswere first preannealed at 41°C for 24 h with annealing buffer(3 x SSC, 50% formamide, 200 ,ug of yeast RNA per ml, 20,ug of sheared denatured salmon sperm DNA per ml, lxDenhardt buffer). Each labeled DNA probe was hybridizedseparately to a single filter at approximately 8,500 cpm/cm2of filter and incubated at 41°C for 48 h in annealing buffer.Filters were then washed in successive baths of (i) 2x SSC(1 liter) for 1 h at room temperature, (ii) 0.lx SSC (2 liters;1:10)-0.1% sodium dodecyl sulfate at 50°C for 1 h and then30 min at room temperature, and (iii) a 30-min wash in 0.lxSSC (1:10). Filters were then air dried and exposed at -70°Cto Kodak RP-Royal X-Omat film in the presence of Dupont

Cronex Lightening Plus intensifying screens (24). Eachhybridization was performed at least twice.

RESULTSThe patterns of the bacterial restriction endonuclease

EcoRI digests of chromosomal DNA extracted and purifiedfrom the six Bacteroides strains may be compared in Fig.1A. Clearly, each strain can be differentiated on the basis ofits characteristic restriction pattern in agarose gel electropho-resis. This was not surprising as it was known that theseBacteroides species were very heterogeneous by DNA ho-mology, ranging from <4 to 80%, as determined by aconventional filter competition method of hybridization (12)(Table 1).To determine the specific nature of this homology, we

compared the patterns generated by the annealing of B.fragilis 2553 labeled DNA probe with Southern blots ofEcoRI-digested DNA from B. uniformis 0061 and B. ovatus0038. It was previously established that B. fragilis 2553shared only 15 to 24% homology with the latter two organ-isms (12) (Table 1). In Fig. 2 it is clearly evident that theareas of homology visible by REA are drastically different intheir EcoRI sites and thus do not represent identical areas ineach of the Bacteroides genomes. Although their restrictionpatterns appeared similar in agarose gel electrophoresis, fewEcoRI fragments were homologous (Fig. 2, arrows). Aninteresting observation on these data was that the onlyvisible area of homology between B. fragilis 2553 and B.uniforms 0061 was in the region of a 17.5-Md EcoRIfragment.

Similar data were obtained when we annealed a B. vulga-tus 4245 labeled DNA probe with EcoRI-digested DNA fromB. uniforms 0061, "3452-A," and B. ovatus strains 0038 and3524. It was previously shown that B. vulgatus 4245 sharedvery limited homology with either of the B. ovatus strains (0to 8%) and <19% homology with B. uniforms 0061 (12)(Table 1). Again, analysis with EcoRI indicated that areas ofhomology were quite different in their EcoRI sites and couldbe clearly distinguished, although in some instances it wasapparent that the same EcoRI site was common to differentstrains (Fig. 3). This was observed for a 14.6-Md EcoRIfragment present in both the 0061 and 3524 genomes and a13.8-Md EcoRI fragment present in both the 0038 and"3452-A" genomes (Fig. 3, arrows).

In the above cases, the use of REA coupled with hybrid-ization and the Southern blotting procedure (23), using totalchromosomal nick-translated DNA probes, permitted fur-ther differentiation of Bacteroides species known to sharelimited homology (<24% [12]) (Table 1).

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26 BRADBURY ET AL.

teroides strains of the same species (e.g., B. ovatus 0038 and3524) that were genetically very similar by hybridizationanalysis (>75% homology [12]) could also be distinguishedby their characteristic restriction patterns in gels. Thus,when the genetic relationships between bacterial organismshas been established by analytical hybridization techniques,REA may be used directly as a simple and rapid method forstrain and isolate differentiation. Furthermore, this techiqueoffers considerable potential for use as an epidemiologicaltool for identifying sources and tracing routes of transmis-sion of infectious bacteria, since it permits a direct approachto differentiating organisms regardless of whether or notalternative typing methods such as serotyping, bac-teriophage typing, and bacteriocin typing are available. Forinstance, we have recently demonstrated the ability of REAto establish the epidemiological relationships between iso-lates of Campylobacterjejuni involved in cases of enteritis ina laboratory worker (20) and residents of a boarding school

FIG. 1. Agarose gel electrophoresis of EcoRI digests of Bac-teroides chromosomal DNA. (A) Approximately 1 to 2 ,ug of DNAwas used in each lane for illustration. EcoRI digests were subjectedto electrophoresis through 0.7% agarose and photographed by UVillumination after ethidium bromide staining as described in the text.In each electrophoresis run the molecular weights of the Bac-teroides DNA fragments were determined by using EcoRI fragmentsof bacteriophage X DNA of known molecular weights (MW) rangingfrom 2.15 x 106 to 13.8 x 106. (B) Enlargement of B. ovatus 0038and B. ovatus 3524 as in (A) above. Although these closely relatedstrains shared >90% homology (12), they could be differentiated bytheir patterns of EcoRI-digested DNAs (arrows).

On the other hand, the extent of EcoRI homology betweenBacteroides strains of the same species which shared >75%homology (e.g., B. ovatus 0038 and 3524, Table 1) resulted inthe hybridization of multiple restriction fragments whichyielded smeared tracks in autoradiograms when B. ovatus0038 nick-translated DNA was used as probe (shorter expo-

sure periods yielded similar results; data not shown). In thiscase discernible differences in their EcoRI restriction pat-terns in agarose gel electrophoresis made it possible todifferentiate these closely related B. ovatuis strains (Fig. 1B,arrows).

DISCUSSIONThe patterns of bacterial chromosomal DNA fragmenta-

tion or genome "fingerprints" produced by EcoRI restric-tion endonuclease in agarose gel electrophoresis clearlydifferentiated each Bacteroides organism examined. Bac-

0061

r -- mcn

Ln)C(N~

0038r.*- L- _-

175 1-, FSw

*__4

t gsae :

_t: :eN,:,`.

2 3 4 5

FIG. 2. Hybridization of B. uniforms 0061 and B. ovatus 0038EcoRI-digested chromosomal DNAs with B. fragilis 2553 probeDNA. DNA was prepared from each strain and digested withEcoRI, followed by agarose gel electrophoresis. The DNA frag-ments were then transferred to nitrocellulose filters by the blottingmethods of Southern (23) and hybridized with the B. fragilis 2553probe DNA as described in the text. (Lanes 2 to 4) EcoRI digests inagarose gel electrophoresis; (lanes 1 and 5) hybridization profilesgenerated by the 0038 probe DNA annealed with 0061 and 0038chromosomal DNAs. Many of the EcoRI DNA restriction fragmentsof 2553, 0061, and 0038 had very similar mobilities in agarose gelelectrophoresis (arrows) but few of them were homologous (openarrowheads). MW, Molecular weight.

OcX X3c ><

::.

zO

C4

'q a- > C

qnt c;3

en CN *_

> D 2C, >

o Lo 4n

13.8-

4.75-

3.75-3.50-

3.0-

2.15-

J. CLIN. MICROBIOL.

1

REA OF BACTEROIDES SPECIES HOMOLOGY 27

o

0061 0038 3524 3452-A

8 9

FIG. 3. Hybridization of 0061, 0038, 3524. and "3452-A" EcoRI-digested chromosomal DNAs with B. vulgawts 4245 probe DNA.(Lanes 1, 2, 4, 6, 8) EcoRl digests in agarose gel electrophoresis;(lanes 3, 5, 7, 9) hybridization profiles generated by the 4245 probeDNA annealed with 0061. 0038. 3524, and '3452-A' chromosomalDNAs. (open arrowheads) A 13.8-M fragment was present in 0038and '3452-A" genomes but not in others, and. similarly, a 14.6-Mfragment was present in the 0061 and 3524 genomes but not inothers. MW, Molecular weight.

(2). In the latter studies, inconsistencies in serotyping reac-

tions prompted the use of the HindIII restriction endonucle-ase, which subsequently clearly distinguished between epi-demiologically linked and unlinked C. jejuni isolates byagarose gel electrophoresis.

Bacterial chromosomal REA in conjunction with Southernhybridizations (23) can be combined very effectively withconventional hybridization data to determine the signifi-cance of low levels of homology detected by conventionalhybridization procedures (e.g., <247c; Table 1). The re-

ported homology values of O to 4% between B. i'ulgautis 4245and "3452-A" are nomalized values (12; Table 1), andtherefore it is difficult to determine if these low levels ofannealing are significant by using conventional hybridizationtechniques. In contrast, with the additional information on

the nature and relative distribution of that homology pro-vided by REA and Southern hybridizations, the significanceof the low levels of annealing between distantly relatedorganisms may be determined directly, when the areas ofhomology are displayed as reproducible bands in autora-diograms.

Nucleic acid hybridization probes and assays offer new

possibilities to clinical laboratory diagnosis to identify, lo-cate, characterize, and quantitate the genomic informationof any target organism. In this regard, REA makes itpossible to investigate the nature of shared DNA sequences.

For example, specific DNA fragments may be identified byREA forltheir homology to a specific group of organisms(7) or their association with a particular disease (18). Thesefragments may be extracted from gels, purified from otherhost cell sequences by molecular cloning (13), and labeledfor use as a probe.

It is anticipated that DNA probes prepared by nicktranslation of whole bacterial chromosomal DNA in themanner we have demonstrated have an application in taxo-nomic studies and assessment of the genetic relationshipsbetween bacterial organisms when DNA-DNA homologydata indicate separation at the species level. We are pres-ently studying the usefulness of this type of DNA probe indot-blot assays (25) for rapid identification of clinicallyimportant anaerobes such as Clostridium diffi(ile (19) andBacteroides species (3, 9).

ACKNOWLEDGMENTSWe are grateful to the Medical Research Council of Canada and to

the National Cancer Institute of Canada for the support given to ourresearch. One of us (W.C.B.) was supported as a Post-DoctoralFellow 1979-80 by funds from the Medical Research Council ofCanada.We are particularly appreciative of the encouragement, growth

facilities, and helpful discussions by R. H. Behme of the Microbiol-ogy Department, University Hospital, London, Ontario. The tech-nical assistance of E. C. M. LeeChan and M. Hall is gratefullyacknowledged.

LITERATURE CITED1. Bradbury, W. C., M. A. Marko, J. N. Hennessy, and J. L.

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2. Bradbury, W. C., A. D. Pearson, M. A. Marko, R. V. Congi,and J. L. Penner. 1984. Investigation of a Canmpylobacterjejunioutbreak by serotyping and chromosomal restriction endonu-clease analysis. J. Clin. Microbiol. 19:342-346.

3. Brook, I. 1984. Anaerobic infections in childhood. Rev. Infect.Dis. 6:S187-S192.

4. Burton, K. 1968. Determination of DNA concentration withdiphenylamine. Methods Enzymol. 12B:163-166.

5. Cohen, J., J. E. Majors, and H. E. Varmus. 1979. Organizationof mouse mammary tumor virus-specific DNA endogenous toBALB/c mice. J. Virol. 32:483-496.

6. Cohen, J. C., P. R. Shank, V. L. Morris, R. Cardiff, and H. E.Varmus. 1979. Integration of the DNA of mouse mammarytumor virus in virus-infected normal and neoplastic tissues ofthe mouse. Cell 16:333-345.

7. Fitts, R., M. Diamond, C. Hamilton, and M. Neri. 1983. DNA-DNA hybridization assay for detection of Salmonella spp. infoods. Apple. Environ. Microbiol. 46:1146-1151.

8. Grunstein, M., and D. S. Hogness. 1975. Colony hybridization: amethod for the isolation of cloned DNA's that contain a specificgene. Proc. Natl. Acad. Sci. U.S.A. 72:3961-3965.

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10. Jeffrey, A., and D. Kleid. 1975. Nucleotide sequence of therightward operator of phage. Proc. Natl. Acad. Sci. U.S.A.72:1184-1188.

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12. Johnson, J. L. 1978. Taxonomy of Bacteroides. I. Deoxyri-bonucleic acid homologies among Bacieroidesfragilis and othersaccharolytic Bacteroides species. tnt. J. Syst. Bacteriol.28:245-256.

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cloning-a laboratory manual. Cold Spring Harbor Laboratory,Cold Spring Harbor, N.Y.

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15. Moore, W. E. C., and L. V. Holdeman. 1973. New names andcombinations ini the genera Bacteroides Catellani and Chalmers,Fusobacterium Knorr, Eubacterium Prevot, PropionibacteriumDelwich, and Lactobacillus Orla-Jensen. Int. J. Syst. Bacteriol.23:69-74.

16. Morris, V. L., E. Madeiros, G. M. Ringold, J. M. Bishop, andH. E. Varmus. 1977. Comparison of mouse mammary tumorvirus-specific DNA in inbred, wild and Asian mice, and intumors and normal organs from inbred mice. J. Mol. Biol.114:73-92.

17. Morris, V. L., J. E. Vlasschaert, C. L. Beard, M. F. Milazzo,and W. C. Bradbury. 1980. Mammary tumors from BALB/cmice with a reported high mammary tumor incidence haveacquired new mammary tumor virus DNA sequences. Virology100:101-109.

18. Moseley, S. C., I. Huq, A. R. M. A. Alin, M. So, M. Samadpour-Motelebi, and S. Falkow. 1980. Detection of enterotoxigenicEscherichia coli by DNA colony hybridization. J. Infect. Dis.142:892-898.

19. Mulligan, M. E. 1984. Epidemiology of Clostridium difficile-in-

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21. Rigby, P. W. J., M. Dieckmann, C. Rhodes, and P. Berg. 1977.Labeling deoxyribonucleic acid to high specific activity in vitroby nick translation with DNA polymerase I. J. Mol. Biol.113:237-251.

22. Shank, P. R., J. C. Cohen, H. E. Varmus, K. R. Yamamoto, andG. M. Ringold. 1978. Mapping of linear and circular form ofmouse mammary tumor virus DNA with restriction endonucle-ases: evidence for a large specific deletion occurring at highfrequency during circularization. Proc. Natl. Acad. Sci. U.S.A.75:2112-2116.

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24. Swanstrom, R., and P. R. Shank. 1978. X-ray intensifyingscreens greatly enhance the detection by autoradiography of theradioactive isotopes 32P and 1251. Anal. Biochem. 86:184-192.

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