Vol. 33, No. 9ANTIMICROBIAL AGENTS AND CHEMOTHERAPY, Sept. 1989, p. 1569-15740066-4804/89/091569-06$02.00/0Copyright C 1989, American Society for Microbiology

Hybridization Analysis of Three Chloramphenicol ResistanceDeterminants from Clostridium perfringens and Clostridium difficile

JULIAN I. ROOD,'* SARA JEFFERSON,1 TRUDI L. BANNAM,1 JULIE M. WILKIE,1PETER MULLANY,2 AND BRENDAN W. WREN2

Department of Microbiology, Monash University, Clayton 3168, Australia,1 and Department of Medical Microbiology,St. Bartholomew's Hospital Medical College, West Smithfield, London ECIA 7BE, United Kingdom2

Received 3 April 1989/Accepted 27 June 1989

The chloramphenicol resistance determinant from a nonconjugative strain of Clostridium perfringens wascloned and shown to be expressed in Escherichia coli. Subcloning and deletion analysis localized the resistancegene, catQ, to within a 1.25-kilobase (kb) partial Sau3A fragment. The catQ gene contained internal HindII,HaeIII, and DraI restriction sites and was distinct from the catP gene, which was originally cloned (L. J.Abraham, A. J. Wales, and J. I. Rood, Plasmid 14:37-46, 1985) from the conjugative C. perfringens Rplasmid, pIP401. Hybridization studies were carried out with a 0.35-kb DraI-PstI fragment of pJIR260 as aninternal catQ-specific probe and a 0.38-kb EcoRV-Hinfl fragment of pJIR62 as an internal catP-specific geneprobe. The results showed that the catP and catQ genes were not similar and that neither probe hybridized withcat genes from other bacterial genera. However, the catP gene was similar to the cloned catD gene fromClostridium difficile. Comparative studies with both catP and catD probes showed that these genes hadsignificant restriction identity. We therefore suggest that these genes were derived from a common source.

Chloramphenicol-resistant strains of the gram-positive,anaerobic bacterium Clostridium perfringens have been iso-lated by several workers (10, 21, 22), although they are lessfrequently encountered than tetracycline- or erythromycin-resistant isolates (18, 20, 23). Three closely related, conju-gative chloramphenicol resistance plasmids (pIP401, pJIR25,and pJIR27) have been identified in C. perfringens (6, 8).Plasmid pIP401 is indistinguishable from the prototype C.perfringens R plasmid, pCW3 (3), except that it containsTn4451, a 6.2-kilobase (kb) transposon which codes forchloramphenicol resistance (4). The wild-type strain fromwhich pCW3 was originally isolated, CW92, is also resistantto chloramphenicol, but the resistance determinant is nottransferable (21). Both CW92 and CP590 (the parent strain ofpIP401) produce chloramphenicol acetyltransferases (21,27).The chloramphenicol resistance genes from pIP401 and

pJIR27 have been cloned, are both expressed in Escherichiacoli, and share considerable restriction identity (4, 6). Anal-ysis of subclones has shown that the resistance gene islocated within a 1.5-kb region of the pIP401-derived recom-binant clone pJIR45 (6). By using a gene probe consisting ofthe 1.3-kb HindIII-EcoRV fragment, which contains most ofthe resistance gene, it was shown that the chloramphenicolresistance determinants from pIP401, pJIR25, and pJIR27are homologous (6). Subsequent studies have revealed thatthe chloramphenicol resistance genes from pIP401 andpJIR27 are located on two closely related transposons,Tn4451 and Tn4452, which can excise precisely and sponta-neously in E. coli (4, 5) and can transpose to the E. colichromosome (4). Recently, the chloramphenicol resistancedeterminant from a clinical isolate of Clostridium difficile hasbeen cloned and analyzed. This gene, which is carried onpPPM9, is expressed in only one orientation in E. coli (25).

In this paper we report the cloning and genetic mapping ofthe chloramphenicol resistance determinant from C. perfrin-gens CW531, a pCW3-cured strain derived from CW92 (21).

* Corresponding author.

In addition, the results of hybridization analyses with gene-specific probes from both C. perfringens chloramphenicolresistance determinants are described. These experimentsshow that the pIP401-derived gene is homologous to thecloned C. difficile determinant carried on pPPM9.

MATERIALS AND METHODS

Bacterial strains and plasmids. The origin and properties ofbacterial strains and plasmids are described in Tables 1 and2. E. coli JM105, C600, and DH5ao were used as recipientsfor gene-cloning manipulations. E. coli (3), C. perfringens(20), and Staphylococcus aureus (16) strains were grown aspreviously described. Bacillus subtilis was grown in R broth(3). Media were supplemented with ampicillin (100 ,ug/ml) orchloramphenicol (5 or 30 ,ug/ml), as required.DNA methods. E. coli (1, 4, 25), C. perfringens (2), and S.

aureus (16) DNAs were prepared as described previously.DNA from B. subtilis was made by using the C. perfringensmethod (2). Restriction endonucleases and other enzymeswere from Boehringer Mannheim Biochemicals, BethesdaResearch Laboratories, Inc., Pharmacia, or New EnglandBioLabs, Inc. Restriction endonuclease analysis and agarosegel electrophoresis of plasmid DNA were as describedpreviously (2, 7, 17, 25). DNA fragments were purified fromlow-melting-temperature agarose gels (SeaPlaque) by phenolextraction (17).

Southern hybridizations were done as previously de-scribed (7) with [32P]dTTP-labeled probe DNA. The probewas hybridized to single-stranded DNA immobilized onnitrocellulose filters (BA-85; Schleicher & Schuell, Inc.) asdescribed previously (17). Stringency washes (two times for30 min each time) were done in 0.16 x SSC (lx SSC is 0.15M NaCl plus 0.015 M sodium citrate)-1% sodium dodecylsulfate at 65°C.

RESULTS

Cloning of the chloramphenicol resistance determinant fromCW531. Chromosomal DNA was purified from C. perfrin-

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TABLE 1. Origin and characteristics of bacterial strains

Strain Relevant characteristicsa Source or reference

E. coliJFM43 C600 hsdR hsdM+ 19JM1O5 rpsL endA sbcB15 hspR4 26

A(lac-proAB) [F' traD36proAB lacIqZ AMI5]

DH5a F- endAl hsdRJ7 recAl Bethesda ResearchgyrA96 relAl 480dlacZ Laboratories, Inc.AM15

C. perfringensCW92 Tcr Cmr (pCW3) 21CW531 Tcs Cmr (pCW3) 21CP590 Tcr Cmr (pIP401) 6, 8CP600 Tcr Cm' (pJIR25) 6, 81329 Tc' Cmr (pJIR27) 6, 101143, 1432C Tcr Cmr 10a Tcr, Tetracycline resistance; Tc', tetracycline susceptibility; Cmr, chlor-

amphenicol resistance.

gens CW531 and was digested with EcoRI, XbaI, or Sac.Gene banks were constructed in pUC18 and were used totransform E. coli JM1OS to chloramphenicol resistance.Three chloramphenicol-resistant clones were obtained fromca. 2,400 Sacl-derived recombinants. No chloramphenicol-resistant clones were obtained from the other gene banks.

Plasmid DNA was purified from each of the three recom-binants and was digested with Sacl. Each plasmid containeda 6.5-kb Sacd insert which was in the same orientation. Oneof these plasmids, pJIR158, was chosen for further study.Digestion with-one or a number of restriction endonucleaseswas used to derive an unequivocal map of pJIR158 (Fig. 1).Transformation experiments showed that pJIR158 was sta-ble in E. coli, unlike the pIP401-derived recombinant plas-mid pJIR45 (4). Hybridization experiments revealed thepresence of a similar 6.5-kb band in SacI digests of DNAfrom CW531 and indicated that this hybridizing fragmentwas contained within the chromosomal band on an agarosegel rather than in a high-molecular-weight plasmid fraction(data not shown).

Localization of the chloramphenicol resistance gene on

pJIR158. Deletion derivatives of pJIR158 were isolated afterPstI digestion and subsequent ligation. All Qf these 5.3-kbplasmids conferred chloramphenicol resistance, which indi-cated that the resistance gene was located within the 2.65-kbPstI-SacI fragment of pJIR158 (Fig. 1). The deletion plas-mid, pJIR186, was mapped further (Fig. 1) and then analyzedby cloning partial Sau3A fragments into the BamHl site ofpUC18. The smallest recombinant plasmid which conferredchloramphenicol resistance was pJIR235, a 3.95-kb plasmidwhich contained both the 0.5- and 0.75-kb Sau3A fragmentsfrom pJIR186 in their original orientations (Fig. 1). PlasmidpJIR235 contained two Hindll sites, one within the multiplecloning site and one within the cloned segment. A deletionderivative of pJIR235 was constructed by digestion withHindII and recircularization. The resultant recombinant,pJIRZ40 (Fig. 1), did not confer chloramphenicol resistance.BAL 31 digestion was used to further define the resistance

determinant. Plasmid pJIR235 was digested sequentiallywith EcoRI, BAL 31 (for various time -periods), and thenHindlIl. The resultant fragments were then ligated toHindIII-SmaI-digested pUC18 DNA. Strain DH5a-derivedtransformants which were ampicillin resistant and chloram-phenicol susceptible and gave white colonies on X-gal me-

dium were selected and analyzed for their plasmid content.The largest plasmid which did not confer chloramphenicolresistance, pJIR241, was shown to have a deletion whichextends from the EcoRI site to within approximately 50 basepairs (bp) of the internal Dral site (Fig. 1). The resistancedeterminant was therefore shown to extend at least to thelocation of the BAL 31 deletion site in pJIRZ41. A similardeletion process was used to elucidate the other end of theresistance gene. Plasmid pJIR235 was BAL 31 treated fromthe HindIII site and cloned into the EcoRI-HindIl sites ofpUC18. The resultant deletion derivative, pJIR260, did notconfer chloramphenicol resistance and was shown to containa 350-bp deletion extending from the HindJII site. On thebasis of these experiments, we concluded that a 350-bpregion extending from just downstream of the first Hinfl site

TABLE 2. Properties of plasmids

Plasmid Relevant characteristics Source or reference

E. colipUC18 Apr lacZ' 26pACYC184 Tc' Cmr 9pJIR62 pUC18fQ(catP:HindIII/BAL31 1.4kb) 6pJIR97 pUC18CI(XbaI 8.5kb:pJIR27) 4pJIR158 pUC18Q(catQ:SacI 6.5kb) CW531-derived recombinantpJIR161 pJIR69::Tnl725(cat) Mitchison and Rood (unpublished results)pJIR186 pJIR158APstI (3.9kb) RecombinantpJIR235 pUC18QI(Sau3A:pJIR186 1.25kb) Sau3A-BamHI recombinantpJIR240 pJIR235AHindII (0.7kb) RecombinantpJIR241 pUC18Q(JIindIII/BAL31:pJIR235 0.75kb) Digestion from EcoRI site, HindlIl-Smal

recombinantpJIR260 pUC18Ql(EcoRI/BAL31:pJIR235 0.9kb) Digestion from HindIII site, EcoRI-

Hindll recombinantpPPM9 pUC13QI(catD:BamHI 1.9kb C. difficile) 25pIJ880 pBR322fQ(cat:Bcll 1.9kb Streptomryces acrimycini) 12

S. aureuspC194, cat 14pC221, pC223

Streptococcus agalactiae pCO2 cat 15B. subtilis pPL531 pUB11OQZ(cat86:EcoRI 2.2 kb Bacillus pumilus) 11

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FIG. 1. Restriction maps of pJIR158 and its derivatives. The location of the catQ gene is indicated by the hatched box. Sizes are indicatedin kilobases. Relevant restriction sites from the multiple cloning region of the vector are as indicated, but the remaining vector portions ofthe recombinant plasmids are not shown. Resistance (Cmr) and susceptibility (Cms) to chloramphenicol of strains carrying the relevantplasmid are also indicated.

to 50 bp past the Dral site was located within the resistancegene (Fig. 1).

Hybridization analysis of clostridial cat genes. Previousstudies showed that the pIP401-derived chloramphenicolresistance gene, which we now designate catP, was locatedwithin a 1.3-kb HindIII-EcoRV fragment of pJIR62 (4, 6).Analysis of the nucleotide sequence of the catP gene (24)revealed the presence of an internal, catP-specific 380-bpEcoRV-Hinfl fragment suitable for use as a gene probe. The350-bp DraI-PstI fragment of pJIR260 was used as a specificgene probe for the CW531-derived chloramphenicol resis-tance gene, which we have designated catQ.

Six chloramphenicol-resistant wild-type C. perfringensisolates were available for hybridization analysis. DNA wasprepared from each of these strains, separated by agarose gelelectrophoresis (Fig. 2A), and hybridized with each of the C.perfringens cat gene probes. The results showed that in fiveof these strains the catP probe was homologous to largeplasmids present in the DNA preparations but that nohomology was detected with DNA from CW92 or the catQ-recombinant pJIR260 (Fig. 2B). These results were con-

firmed by the observation that the catQ probe hybridizedonly to DNA preparations from CW92, CW531, and pJIR260(Fig. 3).Chloramphenicol resistance genes from a representative

selection of bacterial genera were also tested for their abilityto hybridize to the two C. perfringens-derived probes. Noneof these cat genes were similar to either the catQ-specific(data not shown) or catP-specific probe (Fig. 4). However,the catP probe did hybridize to the 1.9-kb Sau3A fragment ofrecombinant plasmid pPPM9 (25), which carried the C.difficile-derived chloramphenicol resistance determinant,designated catD (Fig. 4). No homology was observed be-tween catQ and catD, even at low stringency (Fig. 5).The relationship between pPPM9 and the catP plasmid

pJIR62 was investigated further by carrying out additionalhybridization experiments with both catP-specific (data notshown) and catD-specific (Fig. 5) probes. Each plasmid wasdigested by various combinations of restriction endonu-cleases, and Southern blots were performed. The enzymecombinations were chosen on the basis of the known se-quence of the catP gene (24). The results revealed that the

VOL. 33, 1989

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FIG. 2. Examination of C. perfringens DNA by Southern hybrid-ization analysis with a catP-specific probe. DNA preparationsconsisted of Hindlll-digested XcI857, pJIR260 digested with DraIand PstI, pJIR62 digested with Hindlll and EcoRI, undigestedchromosomal DNA from CW92, and undigested plasmid DNA fromthe remaining strains. DNA was separated by agarose gel electro-phoresis (A), transferred to nitrocellulose, and hybridized with thecatP-specific gene probe (B).

two cat genes had similar internal EcoRV-Hinfl and EcoRV-Dral fragments of identical size.

DISCUSSIONThe existence of two distinct chloramphenicol resistance

determinants in C. perfringens has been clearly demon-strated. The first of these genes, catP, has been shown to belocated on large plasmids in five of the C. perfringens

FIG. 3. Examination of C. perfringens DNA by Southern hybrid-ization analysis with a catQ-specific probe. DNA preparationsconsisted of HindIll-digested XcI857 (sizes of fragments are indi-cated in kilobases), pJIR62 and pJIR260 digested with both Hindliland EcoRI, undigested chromosomal DNA from CW92 and CW531,and undigested plasmid DNA preparations from the remainingstrains. DNA was separated by agarose gel electrophoresis, trans-ferred to nitrocellulose, and hybridized with the catQ-specific geneprobe. The resultant autoradiograph is shown.

FIG. 4. Examination of various chloramphenicol resistance de-terminants by Southern hybridization analysis with a catP-specificprobe. Plasmid DNA preparations were as follows: lane 1, XcI857(HindIII); lane 2, pJIR235 (Hindlll plus EcoRI); lane 3, pJIR62(Hinfl); lane 4, pJIR97 (HindIII); lane 5, pJIR161 (HindIII); lane 6,pC194 (HindIII); lane 7, pC221 (HindIII); lane 8, pC223 (HindIII);lane 9, pPPM9 (Sau3A); lane 10, pIJ880 (SmaI); lane 11, pPL531(PstI plus EcoRI); lane 12, pACYC184 (HaeIII); lane 13, pCO2(HindIII). The enzymes (indicated above in parentheses) werechosen so that the cat genes were located on a single DNA fragment.DNA was separated by agarose gel electrophoresis (A), transferredto nitrocellulose, and hybridized with the catP-specific gene probe(B). Note that the hybridization observed in lanes 5 and 10 was dueto slight pUC contamination of the probe. The cat genes were notlocated on these fragments.

isolates that were tested. In three such strains (CP590,CP600, and 1329), the chloramphenicol resistance plasmidshave been shown to be conjugative (6, 8). Previous resultshave shown that the catP gene is carried on transposons intwo of these plasmids (pIP401 and pJIR27) (4). No transfer ofchloramphenicol resistance was observed when strains 1143and 1432C were used as donors in mixed-plate matings.However, the chloramphenicol resistance plasmids carriedin these strains may still be conjugative because both ofthese strains produce bacteriocins which may mask conju-gative transfer by killing the recipient cells (A. J. Wales andJ. I. Rood, unpublished results).

FIG. 5. Examination of catP and catQ DNA by Southern hybrid-ization analysis with a catD-specific probe. Plasmid DNA wasdigested and separated by agarose gel electrophoresis, transferred tonitrocellulose, and hybridized with a 0.27-kb EcoRV-TaqI fragment(25). The resultant autoradiograph is shown. Relevant size markersin kilobases are indicated. Lanes 1 and 2 contained EcoRV-Hinfldigests, and lanes 3 and 4 contained EcoRV-DraI digests of pPPM9(catD) and pJIR62 (catP), respectively. Lanes 5 and 6 containedHinfl and Hinfl-DraI digests of pJIR235 (catQ), respectively.

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CLOSTRIDIAL cat GENES 1573

A second C. perfringens chloramphenicol resistance gene,catQ, has been cloned and identified as a result of theseexperiments. This gene had a restriction profile differentfrom that of the catP gene and did not hybridize with eithercatP or any of the recognized cat genes from other genera.The catQ gene appears to be chromosomally determined.Furthermore, there is no evidence that catQ is located on atransposable genetic element. The wild-type strain, CW92,which carries catQ, also harbors the conjugative tetracyclineresistance plasmid pCW3. If catQ were capable of transpos-ing to pCW3, this event should have been detected inprevious experiments (21). Strain CW92 was originally iso-lated in the United States, whereas the isolates carrying thecatP-like determinants were all from Western Europe (8,10). However, it would be necessary to examine additionalchloramphenicol-resistant isolates of C. perfringens before itcould be concluded that the catP and catQ determinantswere restricted to particular geographical locations. Bycomparison, the C. perfringens tetracycline resistance deter-minant, tetP, is universally distributed (1, 6).The chloramphenicol resistance gene from C. difficile,

catD, was previously cloned in E. coli (25). Hybridizationexperiments have shown that the catD and catP genes arehomologous. On the basis of our results and the knownrestriction maps of the catP (4, 24) and catD genes (25), it isclear that although the genes may not be absolutely indistin-guishable, they do have significant restriction identity. Pre-vious results showed that the catD gene requires a vectorpromoter for expression in E. coli (25), whereas the catPgene is expressed from its own promotor (6). We concludethat despite the homology between the structural genes,there must be functional differences in their regulatoryregions. DNA sequence and gene expression studies are inprogress to determine the basis for these differences.Although the origin of the catD and catP genes remains

unknown, the low G+C content of catP (24) is consistentwith a clostridial source. Conjugative interspecies transfer ofthese genes between C. difficile and C. perfringens mayaccount for the appearance of the gene in both species.Transfer of pIP401 from C. perfringens to C. difficile hasbeen reported (13). However, the resultant tetracycline-resistant transconjugants were unstable. It is not knownwhether the chloramphenicol resistance determinant wasalso transferred. The catD gene is chromosomally locatedand does not appear to be part of a readily identifiabletransposon. In addition, we have not been able to transfercatD in filter-mating experiments. Therefore, even if catDoriginated as a result of conjugative transfer from C. perfrin-gens, it is clear that either the C. perfringens plasmids andtransposons are not functional in C. difficile or significantrearrangements have occurred since the transfer event.Alternatively, transfer may have occurred from C. difficile toC. perfringens or independently from some other microor-ganism to each of the clostridia. In this situation, we assumethat subsequent genetic rearrangements have led to theincorporation of catP into transposons, such as Tn4451 andTn4452, which are located on conjugative tetracycline resis-tance plasmids. Recent studies (24) have shown that theamino acid sequence of the catP gene product is very similarto that of chloramphenicol acetyltransferases from otherbacterial genera.The conclusion that there are at least two hybridization

classes of clostridial chloramphenicol resistance genes isvery similar to results recently obtained for the clostridialmacrolide-lincosamide resistance determinants. Molecularstudies have shown that there is more than one hybridization

class of erythromycin resistance determinant in C. perfrin-gens and that one of these determinants, ermP, is homolo-gous to erm genes from C. difficile (7). These erm genes arealso homologous to the class B/AM erm gene from thepromiscuous streptococcal plasmid pAMP1, suggesting thatthe latter plasmid may have been the exogenous source ofthe gene in both clostridial species. In contrast, only onehybridization class of C. perfringens tetracycline resistancedeterminant has been identified (1). This ubiquitous tetPdeterminant does not hybridize with DNA from either tetra-cycline-resistant isolates of C. difficile or tet genes fromother bacterial genera (1). The basis for the apparent differ-ences in the molecular epidemiology of this determinant andthat of the cat and erm genes is not known.

ACKNOWLEDGMENTSWe acknowledge the most capable technical assistance .of Mau-

reen Barnes, Clare Phillips, and Pauline Howath. We thank J.Messing, G. Dutta, D. Hopwood, R. Skurray, P. Lovett, and R.Lutticken for the provision of strains or plasmid DNA.

LITERATURE CITED1. Abraham, L. J., D. I. Berryman, and J. I. Rood. 1988. Hybrid-

ization analysis of the class P tetracycline resistance determi-nant from the Clostridium perfringens R-plasmid, pCW3. Plas-mid 19:113-120.

2. Abraham, L. J., and J. I. Rood. 1985. Molecular analysis oftransferable tetracycline resistance plasmids from Clostridiumnperfringens. J. Bacteriol. 161:636-640.

3. Abraham, L. J., and J. I. Rood. 1985. Cloning and analysis ofthe Clostridium perfringens tetracycline resistance plasmid,pCW3. Plasmid 13:155-162.

4. Abraham, L. J., and J. I. Rood. 1987. Identification of Tn445Iand Tn4452, chloramphenicol resistance transposons from Clos-tridium perfringeiis. J. Bacteriol. 169:1579-1584.

5. Abraham, L. J., and J. I. Rood. 1988. The Clostridium perfrin-gens chloramphenicol resistance transposon Tn4451 excisesprecisely in Escherichia coli. Plasmid 19:164-168.

6. Abraham, L. J., A. J. Wales, and J. I. Rood. 1985. Worldwidedistribution of the conjugative Clostridium perfringens tetracy-cline resistance plasmid, pCW3. Plasmid 14:37-46.

7. Berryman, D. I., and J. I. Rood. 1989. Cloning and hybridizationanalysis of ermP, a macrolide-lincosamide-streptogramin B re-sistance determinant from Clostridium perfringens. Antimicrob.Agents Chemother. 33:1346-1353.

8. Brefort, G., M. Magot, H. lonesco, and M. Sebald. 1977.Characterization and transferability of Clostridium perfringensplasmids. Plasmid 1:52-66.

9. Chang, A. C. Y., and S. N. Cohen. 1978. Construction andcharacterization of amplifiable multicopy DNA cloning vehiclesderived from the P15A cryptic miniplasmid. J. Bacteriol. 134:1141-1156.

10. Dutta, G. N., and L. A. Devriese. 1980. Susceptibility of Clos-tridium perfringens of animal origin to fifteen antimicrobialagents. J. Vet. Pharmacol. Ther. 3:227-236.

1'. Duvall, E. J., D. M. Williams, S. Mongkolsuk, and P. S. Lovett.1984. Regulatory regions that control expression of two chlo-ramphenicol-inducible cat genes cloned in Bacillius subtilis. J.Bacteriol. 158:784-790.

12. Gil, J. A., H. M. Kaiser, and D. A. Hopwood. 1985. Cloning ofa chloramphenicol acetyltransferase gene of Streptomyces ac-rimycini and its expression in Streptomyces and Escherichiacoli. Gene 38:1-8.

13. lonesco, H. 1980. Transfert de la resistance a la tetracycline chezClostridium difficile. Ann. Microbiol. (Paris) 131A:171-179.

14. Iordanescu, S., M. Surdeanu, P. D. Latta, and R. Novick. 1978.Incompatibility and molecular relationships between small sta-phylococcal plasmids carrying the same resistance marker.Plasmid 1:468-479.

15. Lutticken, R. 1985. The genetics of GBS. Antibiot. Chemother.(Basel) 35:71-82.

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17. Maniatis, T., E. F. Fritsch, and J. Sambrook. 1982. Molecularcloning: a laboratory manual. Cold Spring Harbor Laboratory,Cold Spring Harbor, N.Y.

18. Rood, J. I., J. R. Buddle, A. J. Wales, and R. Sidhu. 1985. Theoccurrence of antibiotic resistance in Clostridium perfringensfrom pigs. Aust. Vet. J. 62:276-278.

19. Rood, J. I., A. J. Laird, and J. W. Williams. 1980. Cloning of theEscherichia coli K-12 dihydrofolate reductase gene followingMu-mediated transposition. Gene 8:255-265.

20. 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.

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

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23. Smith, H. W. 1959. The effect of the continuous administrationof diets containing tetracyclines and penicillin on the number ofdrug-resistant and drug-sensitive Clostridium welchii in thefaeces of pigs and chickens. J. Pathol. Bacteriol. 77:79-92.

24. Steffen, C., and H. Matzura. 1989. Nucleotide sequence analysisand expression studies of a chloramphenicol-acetyltransferase-coding gene from Clostridium perfringens. Gene 75:349-354.

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