Vol. 161, No. 2JOURNAL OF BACTERIOLOGY, Feb. 1985, p. 636-6400021-9193/85/020636-05$02.00/0Copyright © 1985, American Society for Microbiology

Molecular Analysis of Transferable Tetracycline Resistance Plasmidsfrom Clostridium perfringensLAWRENCE J. ABRAHAM* AND JULIAN I. ROOD

Division of Veterinary Biology, School of Veterinary Studies, Murdoch University, Murdoch, Western Australia 6150,Australia

Received 21 March 1984/Accepted 9 October 1984

Conjugative tetracycline resistance plasmids from 15 Clostridium perfringens isolates from piggeries wereanalyzed by restriction endonuclease digestion and agarose gel electrophoresis. Seven isolates from one farmwere found to carry a 47-kilobase pair (kb) plasmid, pJIR5, which had EcoRI, XbaI, and ClaI profiles thatwere identical to those of a previously characterized plasmid, pCW3. An isolate from a second farm was foundto carry a plasmid, pJIR6, which also was indistinguishable from pCW3. Five additional isolates from a thirdfarm carried a 67-kb plasmid, pJIR2, which had at least 29 kb of DNA in common with pCW3. Finally, twoisolates from a fourth farm were found to carry a 50-kb plasmid pJIR4, which appeared to consist of an entirepCW3 molecule with a 3-kb insertion. Comparative restriction maps of pCW3, pJIR2, and pJIR4 thatidentified the regions of homology among these plasmids were constructed. We suggest that many conjugativetetracycline resistance plasmids in C. perfiringens may contain a pCW3-like core.

Clostridium perfringens is an anaerobe that forms a part ofthe normal intestinal flora of humans and animals. Thisorganism is the causative agent of such diseases as gasgangrene in humans, hemorrhagic enteritis of calves, andenterotoxemia of sheep. It is also a secondary pathogen invarious disease conditions, such as necrotic enteritis ofchickens (6). In recent years there has been renewed interestin the clostridia as they have been implicated in a number ofdiseases in humans. For instance, Clostridium difficile isknown to cause antibiotic-associated pseudomembranouscolitis (4). Consequently, there is a need to understand thegenetic mechanisms whereby these anaerobes can interact,especially with regard to the transfer of drug resistancedeterminants.

Resistance transfer was first demonstrated in C. perfrin-gens by Sebald and Brdfort (12). These workers identified a

conjugative R-plasmid, pIP401, which carries the tetracy-cline and chloramphenicol resistance genes (1). Subse-quently, Rood et al. (11) characterized a C. perfringensstrain (strain CW92) that was isolated from a specimen ofhuman origin in California. This strain was chloramphenicoland tetracycline resistant, but was able to transfer only itstetracycline resistance. A conjugative tetracycline resistanceplasmid was identified in strain CW92 and designated pCW3(11).Rood et al. (10) isolated many multiply antibiotic-resistant

strains of C. perfringens from porcine feces that wereobtained from several farms in Wisconsin. Recently, Rood(8) reported that a significant proportion of the antibiotic-re-sistant strains obtained in that study have the ability totransfer their tetracycline resistance to appropriate recipientstrains. In this paper we describe the plasmids responsiblefor this property and report studies on the comparativemolecular analysis of these plasmids.(Some of the results have been reported previously [Abra-

ham and Rood, Aust. Microbiol. 4:85, 1983].)

* Corresponding author.

MATERIALS AND METHODS

Bacterial strains, media, and chemicals. Plasmid-free strainsCW504 and JIR39 are derivatives of strain CW362 (7, 8).Wild-type strains were isolated from porcine and humanfeces obtained from four farms in Wisconsin (10). All otherstrains were tetracycline-resistant transconjugants of strainCW504 or JIR39. All strains were maintained and propagatedas described previously (8). A list of the plasmids identifiedduring this study is presented in Table 1. Nutrient agar andTrypticase-peptone-glucose broth were prepared and used asdescribed previously (10). Tetracycline was a generous giftfrom Upjohn Pty. Ltd., Sydney, Australia.Growth of cells and isolation of plasmid DNA. Cells were

cultured overnight on nutrient agar containing 5 ,ug oftetracycline per ml and then subcultured in 100 ml ofTrypticase-peptone-glucose broth. Mid-log phase cells werewashed and suspended in 2 ml of25% sucrose in 30 mM TES(Tris-hydrochloride buffer [pH 8.0] containing 5 mM EDTAand 50 mM NaCl). After treatment with 0.4 ml of a lysozymesolution (10 mg/ml in TES) for 15 min at 37°C and with 0.8 mlof 250 mM EDTA (pH 8.0) for an additional 5 min, lysis wasachieved by adding 0.5 ml of 10% (wt/vol) sodium dodecylsulfate and incubating the preparation at 37°C for 5 min. Thelysate was then treated with NaCl by the method of Guerryet al. (2). The resultant lysates were subjected to cesiumchloride-ethidium bromide density gradient centrifugationfor 20 h at 49,000 rpm (type 70.1Ti rotor; Beckman modelL-8 ultracentrifuge). Plasmid bands were extracted throughthe top of the tube, and ethidium bromide was extracted withNaCl-saturated propan-2-ol. The preparations were dialyzedagainst 0.1 x TES, concentrated by pervaporation, and thendialyzed again before storage at 4°C.

Restriction endonuclease digestion and agarose gel elec-trophoresis. All restriction endonucleases were obtainedfrom Boehringer-Mannheim or Bethesda Research Labora-tories and were used according to the instructions of thesuppliers. Electrophoresis of DNA samples was carried outin 0.6 to 1.2% agarose in a horizontal gel apparatus by usinga Tris-acetate electrode buffer system, as described by

636

C. PERFRINGENS TETRACYCLINE RESISTANCE PLASMIDS

TABLE 1. Plasmids and their originsPlasmid Source Size (kb) Phenotype

pCW3 Strain CW92' 47 Tra+ TCrbpJIR1 Farm A (porcine) ca. 12 CrypticpJIR2 Farm G (porcine) 67 Tra+ TcrpJIR3 Farm A (human) ca. 4.5 CrypticpJIR4 Farm I (porcine) 50 Tra+ TcrpJIR5 Farm A (porcine and human) 47 Tra+ TcrpJIR6 Farm H (porcine) 47 Tra+ Tcr

a See reference 11.bTra+, Conjugation; Tcr, tetracycline resistance.' Farm designations are explained in reference 10.

Young et al. (16). The sizes of DNA fragments were esti-mated by comparing their mobilities with those of HindIII-cleaved XcI857, using a computer program (9).DNA hybridization studies. DNA fragments in agarose gels

were denatured and transferred to nitrocellulose filters (15).The filters were baked for 2 h at 80°C under a vacuum. DNAderived from pCW3 was labeled by nick translation with32P-labeled dCTP (Amersham Corp.). Heat-denatured probeDNA was then hybridized to the DNA present on thenitrocellulose filters as described previously (14). Hybrid-ized probe DNA was detected by autoradiography, usingintensifying screens.

RESULTSExamination of wild-type plasmid profiles. Previous studies

(8) showed that 15 of the 89 tetracycline-resistant wild-typeC. perfringens strains that were available from the originalWisconsin study (10) could transfer their antibiotic resist-ance to suitable recipient strains. In the present study,plasmid DNA was prepared from each of the 15 wild-typeisolates, and the plasmids were examined by agarose gelelectrophoresis. The profiles that were obtained could becorrelated readily with the farm of origin (data not shown).The common feature of all 15 profiles was the presence of atleast one high-molecular-weight plasmid. However, sincemany different low-molecular-weight plasmids also werepresent, it was not possible to make any absolute correla-tions between the transferable tetracycline resistance phe-notype and the presence of a specific plasmid. Therefore, wedecided to examine the transconjugants that were availablefrom the previous study (8).

Examination of transconjugants. For each wild-type donor,four independently derived transconjugants were examinedinitially; three of these were from interstrain matings of thewild type with strain CW504, and one was from an intra-strain mating of a strain CW504-derived transconjugant withstrain JIR39 (8). Agarose gel electrophoresis indicated that ahigh-molecular-weight plasmid was present in all 60 of thetransconjugants that were examined (data not shown). Fourstrain CW504-derived transconjugants also were found tocontain an additional low-molecular-weight plasmid. One ofthese transconjugants, which was from a farm A porcineisolate, harbored a plasmid (pJIR1; ca. 12 kilobase pairs[kb]) that was present in all of the farm A wild types.Although this transconjugant could in turn donate its tetra-cycline resistance, plasmid pJIR1 was not detected in thestrain JIR39 derivative that was produced. The remainingthree strain CW504-derived transconjugants that also carrieda low-molecular-weight plasmid were all derived from mat-ings with a single human farm A isolate. This plasmid(pJIR3; ca. 4.5 kb) also was present in the wild-type parent.Similarly, when one of these strains donated its tetracycline

resistance, plasmid pJIR3 was not detected in the resultingstrain JIR39-derived transconjugant. Because of the absenceof any phenotypic markers on pJIR1 or pJIR3, it was notpossible to determine whether these plasmids were mobi-lized by other plasmids present in the wild-type isolates orwhether these small plasmids were self-transmissible. Con-sidering the small sizes of pJIR1 and pJIR3, it is unlikely thatthey encode enough information to mediate their own trans-fer. A wider, more systematic study will be required toestablish whether the high-molecular-weight plasmids de-tected in this study have the ability to mobilize othernonconjugative plasmids present within the same host.

Restriction analysis of plasmids. To further characterizethe plasmids present in each of the 60 transconjugants, arestriction analysis was carried out. We found that withineach group of four transconjugants, the same restrictionprofiles were obtained. We concluded that the same high-molecular-weight plasmid was present in all four transconju-gants within each group. Consequently, further studies wererestricted to one transconjugant derived from each of the 15wild-type donors.Our results also showed that all seven transconjugants

from farm A donors carried plasmids with identical restric-tion profiles. These plasmids were all designated pJIR5. Anadditional plasmid, which was derived from the single farm

11.39.28.3

3.8

kbikb-

2.1 kb-1.9 kb-

1. 2

FIG. 1. Agarose gel electrophoresis of EcoRI-digested plasmidDNA derived from transconjugants. Plasmid DNA was digestedwith EcoRI, and the resulting fragments were subjected to elec-trophoresis in a 0.8% (wt/vol) agarose gel at 75 V for 5 h. The sizesof the pCW3 fragments are indicated. The lane labeled lambdacontains fragments of known size produced when bacteriophageXcI857 was digested with HindIll.

VOL. 161, 1985 637

638 ABRAHAM AND ROOD

r 2 v " coa(C) 4~

a a a QI

7 kb

FIG. 2. Agarose gel electrophoresis of XbaI-digested plasmidDNA derived from transconjugants. Plasmid DNA was digestedwith XbaI, and the resulting fragments were subjected to electropho-resis in a 0.8% (wt/vol) agarose gel at 35 V for 16 h. The gel was thentreated as described in the legend to Fig. 1. The sizes of theHindIlI-digested XcI857 fragments are indicated.

H isolate, was designated pJIR6. Addition of the sizes of theEcoRI and ClaI fragments established a molecular size of 47kb for pJIR5 and pJIR6. The EcoRI (Fig. 1), XbaI (Fig. 2),and ClaI (data not shown) profiles of pJIR5 were comparedwith the restriction profiles of a previously characterizedtetracycline resistance plasmid, pCW3 (11). pJIR5, pJIR6,and pCW3 had identical restriction profiles for all threeenzymes.The five transconjugants from farm G isolates also carried

plasmids with a common restriction profile. The 67-kbplasmid in these transconjugants was designated pJIR2. Acomparison of the restriction profiles of pJIR2 and pCW3indicated that pJIR2 was different from pCW3, althoughboth plasmids yielded some fragments of the same size. Anexamination of the EcoRI digests of pJIR2 and pCW3 (Fig.1) showed that six EcoRI fragments (8.3, 3.8, 3.8, 2.5, 2.1,and 1.9 kb) were shared by both of these plasmids. XbaIdigestion of pJIR2 and pCW3 (Fig. 2) confirmed that pJIR2was different than pCW3 and showed that eight fragmentswith a total size of 14.5 kb were common to both plasmids.A comparison of the ClaI profiles of pJIR2 and pCW3confirmed these results.The two transconjugants from the farm I isolates had

plasmids with identical restriction patterns. These 50-kbplasmids were designated pJIR4. A comparison of theirrestriction profiles showed that pJIR4 was very similar topCW3. The EcoRI-generated fragments of pJIR4 were iden-

tical to those of pCW3, except that one of the 3.8-kbfragments present in pCW3 was absent and three newfragments (3.5, 2.2, and 0.6 kb) were present in pJIR4 (Fig.1). The XbaI-generated fragments of pJIR4 were the same asthose of pCW3, except that pJIR4 had a 3.1-kb fragment thatwas not present in the equivalent pCW3 digest (Fig. 2). TheClaI results confirmed that pJIR4 was very similar to pCW3.DNA hybridization studies were used to confirm the

results. Plasmid DNA derived from pCW3 was nick trans-lated by using 32P-labeled dCTP and hybridized to EcoRI-digested pCW3, pJIR2, and pJIR4. The results showed thatall of the EcoRI fragments that were present in both pCW3and pJIR4 or in both pCW3 and pJIR2 hybridized with DNAderived from pCW3 (Fig. 3).Comparative analysis of pJIR2, pJIR4, and pCW3. As

pJIR2 and pJIR4 were found to be different from pCW3, wedecided to define the relationship among these three plas-mids. A complete restriction map of pCW3 was generated(Abraham and Rood, manuscript in preparation), and thenpartial restriction maps of pJIR2 and pJIR4 were deduced bycomparing restriction digests of these two plasmids withequivalent pCW3 digests. In this way we were able to mapthe regions of pJIR2 and pJIR4 that were the same as thosein pCW3.

Plasmid pJIR4 was missing the 11.7-kb ClaI fragment ofpCW3 that extended from 28.4 to 40.1 kb on the linear pCW3map (Fig. 4), but a new 14.5-kb ClaI fragment was present.The EcoRI profiles of pJIR4 and pCW3 were identical,except that one of the 3.8-kb fragments of pCW3 wasmissing in pJIR4. Both of these fragments are within the11.7-kb ClaI fragment. By carrying out EcoRI-HpaI doubledigestions (data not shown), it was possible to identify whichof the two fragments was absent in pJIR4 and thus identify

" '"'

11.39.28.3

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2.62.52.11.9

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1.2 kb-

FIG. 3. Southern blot analysis of pJIR2 and pJIR4. Hybridiza-tion and Southern blot analysis were carried out as described in thetext. DNA derived from pCW3 was labeled with 32P and used toprobe EcoRI-digested plasmid DNAs from pCW3, pJIR2, andpJIR4. The sizes of the pCW3-derived EcoRI fragments are indi-cated.

J. BACTERIOL.

C. PERFRINGENS TETRACYCLINE RESISTANCE PLASMIDS

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the region of pJIR4 that was different from pCW3. Theresults showed that the first 3.8-kb EcoRI fragment of pCW3(29.6 to 33.4 kb) (Fig. 4) was replaced by 3.5-, 2.2-, and0.6-kb EcoRI fragments in pJIR4, which accounts for the3-kb increase in size of this plasmid compared with pCW3.Comparative restriction data also indicated that pJIR2

shared a substantial region of homology with pCW3. Delin-eation of the regions common to pJIR2 and pCW3 wasachieved by comparing double digests, using ClaI and one ofa variety of other restriction enzymes. From these studieswe concluded that pJIR2 contains two regions that are nothomologous with pCW3. The location of these regions isindicated in Fig. 4.

DISCUSSIONThe study reported by Rood (8) was the first in which a

large number of wild-type antibiotic-resistant strains of C.perfringens were examined for their ability to transfer theirresistance. He showed that 15 of the 89 tetracycline-resistantisolates from the Wisconsin porcine study (10) donated theirtetracycline resistance to recipient strains. The results pre-sented here show that a plasmid molecule was present in allof the tetracycline-resistant strain CW504-derived trans-conjugants that were from the 15 wild-type isolates. Ananalysis of plasmids from the strain JIR39-derived trans-conjugants demonstrated that the plasmids were stable onsubsequent transfer. From the results we concluded that theplasmids involved in transfer carried the genes responsiblefor both conjugal transfer and tetracycline resistance.

Restriction analysis has frequently been employed toexamine the molecular relationships among various R-plas-mids (3, 13). These studies are often carried out to determinewhether R-plasmids from different sources are related orwhether they have a common evolutionary origin. Thisreport represents the first study in which this approach hasbeen used for the analysis of conjugative R-plasmids from C.perfringens. The most significant finding from these experi-ments is that conjugative R-plasmids isolated from C. perfrin-gens strains of porcine origin in Wisconsin appear to be thesame as pCW3, which was found in a human hospital isolatefrom California. Although these pCW3-like plasmids all havethe same restriction profiles, small DNA sequence differ-ences cannot be excluded, and so each plasmid was namedindividually. The fact that pJIR6 was found in the conjuga-tive tetracycline-resistant isolate from farm H and the factthat pJIR5 was found in all of the conjugative tetracycline-resistant isolates from farm A, regardless of whether theisolate was of porcine or human origin, suggest that there isfree genetic exchange between porcine and human C. per-

fringens strains. This conclusion is supported by the appar-ent identity of both pJIR5 and pJIR6 with the human hospitalplasmid, pCW3.

It is also significant that pJIR2 and pJIR4 showed substan-tial homology with pCW3. In fact, pJIR2 appears to consistof a pCW3-like plasmid with two non-homologous additionalregions of at most 38 kb of DNA. The regions of nonhomol-ogy are smaller than indicated, as we have not determinedexactly which segments of the individual restriction frag-ments that are unique to pJIR2, are the same as in pCW3.Plasmid pJIR4 also was found to be similar to pCW3.Restriction analysis revealed that pJIR4 is a pCW3-likeplasmid with only 3 kb of additional DNA that is unique topJIR4. Although we have not mapped the precise location ofthe additional DNA, restriction analysis indicates that itconsists of a single insertion of 3 kb of DNA into a 3.8-kbEcoRI fragment of pCW3.These findings have significant implications for our under-

standing of the molecular evolution of transferable tetracy-cline resistance in C. perfringens. The ubiquity of a pCW3-like element in transferable tetracycline-resistant strains inthis study raises the possibility that pCW3 or a pCW3-likeplasmid forms the basis of all transferable R-plasmids in C.perfringens. It appears that this pCW3-like core is able toacquire additional DNA (as with pJIR2 and pJIR4) by amechanism that is as yet undetermined and is able to transferthis DNA to other members of the population by a conjuga-tion-like process. It would be of great interest to study theregions surrounding the sites of heterologous insertion withinpJIR2 and pJIR4 to possibly determine how this DNA wasinserted. Although transposable elements have not beendemonstrated in this species. they have been shown to beinvolved in the acquisition of additional genetic informationby plasmids in other species (5). Current studies in ourlaboratory are aimed at the characterization of transferabledrug resistance plasmids from a wider geographical range toestablish whether the pCW3-like plasmid is truly ubiquitous.

ACKNOWLEDGMENTS

This work was supported by grants from the Australian ResearchGrants Scheme and Murdoch University. L.J.A. is the recipient ofa Commonwealth Postgraduate Research Award.We are pleased to acknowledge Lyn M. Rowland for meticulous

and cheerful technical assistance. Thanks are also extended to J.Shine for a generous gift of restriction enzymes.

LITERATURE CITED1. Brefort, G., M. Magot, H. lonesco, and M. Sebald. 1977.

Characterization and transferability of Clostridium perfringensplasmids. Plasmid. 1:52-66

pCW347 kb

- i -- -iA o 00 . 000 oUt- a U

0'1A St,II i

0 10

-X o) -A o o0 . C040. 0uU-q0U0XL u 0, c fC6

X,,T I 'I IpJIR267 kb

AUX 00000. ,, , _ u

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640 ABRAHAM AND ROOD

2. Guerry, P., D. J. Le Blanc, and S. Falkow. 1973. Generalmethod for the isolation of a plasmid deoxyribonucleic acid. J.Bacteriol. 116:1064-1066.

3. Haas, M. J., and J. Davies. 1980. Characterisation of theplasmids comprising the "R factor" R5 and their relationshipsto other R-plasmids. Plasmid 3:260-277.

4. Larson, E. H. 1979. Pseudomembranous colitis is an infection.J. Infect. 1:221-226.

5. Mitsuhashi, S., H. Hashimoto, S. lyobe, and M. lonue. 1977.Formation of conjugative drug resistance (R) plasmids, p.139-146. In A. I. Bukhari, J. A. Shapiro, and S. L. Adhya (ed.),DNA insertion elements, plasmids and episomes. Cold SpringHarbor Laboratory, Cold Spring Harbor, N.Y.

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7. Rokos, E. A., J. I. Rood, and C. L. Duncan. 1978. Multipleplasmids in different toxigenic types of Clostridium perfringens.FEMS Lett. 4:323-326.

8. Rood, J. I. 1983. Transferable tetracycline resistance in Clos-tridium perfringens strains of porcine origin. Can. J. Microbiol.29:1241-1246.

9. Rood, J. I., and J. M. Gawthorne. 1984. Apple software foranalysis of the size of restriction fragments. Nucleic Acids Res.

12:689-694.10. Rood, J. I., E. A. Maher, E. B. Somers, E. Campos, and C. L.

Duncan. 1978. Isolation and characterization of multiply antibi-otic-resistant Clostridium perfringens strains from porcine fe-ces. Antimicrob. Agents Chemother. 13:871-880.

11. Rood, J. I., V. N. Scott, and C. L. Duncan. 1978. Identificationof a transferable tetracycline resistance plasmid (pCW3) fromClostridium perfringens. Plasmid 1:563-570.

12. Sebald, M., and G. Brifort. 1975. Transfert du plasmide tetra-cycline-chloramphenicol chez Clostridium perfringens. C. R.Acad. Sci. Ser. D 281:317-319.

13. Shalita, Z., E. Murphy, and R. P. Novick. 1980. Penicillinaseplasmids of Staphylococcus aureus: structural and evolutionaryrelationships. Plasmid 3:291-311.

14. Sherman, F., G. R. Fink, and C. W. Lawrence. 1979. Methods inyeast genetics. Cold Spring Harbor Laboratory, Cold SpringHarbor, N.Y.

15. Southern, E. M. 1975. Detection of specific sequences amongDNA fragments separated by gel electrophoresis. J. Mol. Biol.98:503-517.

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