Detection of the etx gene (ε-toxin inducer) in plasmids of high molecular weight in Clostridium perfringens type D

The purpose of this work is to correlate the production of epsilon-toxin in a set of strains of Clostridium perfringens type D with the presence of the etx gene, either genomic or in plasmids. Total DNA obtained from strains with a different level of toxin production was explored by PCR and all the strains showed the amplification signal. Different methods were used to obtain plasmid profiles and all of the bands were assayed by PCR. The detection of the etx gene was only shown in several high molecular plasmids. These results were confirmed by a Southern blot. We suggest that the localization of the etx gene in different plasmids could be associated with the epsilon-toxin production level.     

Sequencing and diversity analyses reveal extensive similarities between some epsilon-toxin-encoding plasmids and the pCPF5603 Clostridium perfringens enterotoxin plasmid

Clostridium perfringens type B and D isolates produce epsilon-toxin, the third most potent clostridial toxin. The epsilon-toxin gene (etx) is plasmid borne in type D isolates, but etx genetics have been poorly studied in type B isolates. This study reports the first sequencing of any etx plasmid, i.e., pCP8533etx, from type B strain NCTC8533. This etx plasmid is 64.7 kb, carries tcp conjugative transfer genes, and encodes additional potential virulence factors including beta2-toxin, sortase, and collagen adhesin but not beta-toxin. Interestingly, nearly 80% of pCP8533etx open reading frames (ORFs) are also present on pCPF5603, an enterotoxin-encoding plasmid from type A isolate F5603. Pulsed-field gel electrophoresis and overlapping PCR indicated that a pCP8533etx-like etx plasmid is also present in most, if not all, other type B isolates and some beta2-toxin-positive, cpe-negative type D isolates, while other type D isolates carry different etx plasmids. Sequences upstream of the etx gene vary between type B isolates and some type D isolates that do not carry a pCP8533etx-like etx plasmid. However, nearly all type B and D isolates have an etx locus with an upstream IS1151, and those etx loci typically reside near a dcm ORF. These results suggest that pCPF5603 and pCP8533etx evolved from insertion of mobile genetic elements carrying enterotoxin or etx genes, respectively, onto a common progenitor plasmid.     

Functional identification of conjugation and replication regions of the tetracycline resistance plasmid pCW3 from Clostridium perfringens

Clostridium perfringens causes fatal human infections, such as gas gangrene, as well as gastrointestinal diseases in both humans and animals. Detailed molecular analysis of the tetracycline resistance plasmid pCW3 from C. perfringens has shown that it represents the prototype of a unique family of conjugative antibiotic resistance and virulence plasmids. We have identified the pCW3 replication region by deletion and transposon mutagenesis and showed that the essential rep gene encoded a basic protein with no similarity to any known plasmid replication proteins. An 11-gene conjugation locus containing 5 genes that encoded putative proteins with similarity to proteins from the conjugative transposon Tn916 was identified, although the genes' genetic arrangements were different. Functional genetic studies demonstrated that two of the genes in this transfer clostridial plasmid (tcp) locus, tcpF and tcpH, were essential for the conjugative transfer of pCW3, and comparative analysis confirmed that the tcp locus was not confined to pCW3. The conjugation region was present on all known conjugative plasmids from C. perfringens, including an enterotoxin plasmid and other toxin plasmids. These results have significant implications for plasmid evolution, as they provide evidence that a nonreplicating Tn916-like element can evolve to become the conjugation locus of replicating plasmids that carry major virulence genes or antibiotic resistance determinants.     

TcpA, an FtsK/SpoIIIE homolog, is essential for transfer of the conjugative plasmid pCW3 in Clostridium perfringens

The conjugative tetracycline resistance plasmid pCW3 is the paradigm conjugative plasmid in the anaerobic gram-positive pathogen Clostridium perfringens. Two closely related FtsK/SpoIIIE homologs, TcpA and TcpB, are encoded on pCW3, which is significant since FtsK domains are found in coupling proteins of gram-negative conjugation systems. To develop an understanding of the mechanism of conjugative transfer in C. perfringens, we determined the role of these proteins in the conjugation process. Mutation and complementation analysis was used to show that the tcpA gene was essential for the conjugative transfer of pCW3 and that the tcpB gene was not required for transfer. Furthermore, complementation of a pCW3DeltatcpA mutant with divergent tcpA homologs provided experimental evidence that all of the known conjugative plasmids from C. perfringens use a similar transfer mechanism. Functional genetic analysis of the TcpA protein established the essential role in conjugative transfer of its Walker A and Walker B ATP-binding motifs and its FtsK-like RAAG motif. It is postulated that TcpA is the essential DNA translocase or coupling protein encoded by pCW3 and as such represents a key component of the unique conjugation process in C. perfringens.     

Multiplex PCR assay for toxinotyping Clostridium perfringens isolates obtained from Finnish broiler chickens

AIMS: The aim of the study was to determine the presence of genes coding for alpha (cpa), beta (cpb), epsilon (etx), iota (iA) and enterotoxin (cpe) from Clostridium perfringens broiler chicken isolates, using multiplex PCR assay established in the study. METHODS AND RESULTS: The multiplex PCR assay was shown to be specific when tested with 10 C. perfringens strains representing different toxin types, and 15 strains of other bacterial species. All 118 broiler chicken C. perfringens isolates were shown to carry the cpa gene but not cpb, etx, iap or cpe genes, signifying that all isolates represented type A and were cpe-negative. CONCLUSIONS: The assay established in the study enables the simultaneous detection of the major toxin genes and the cpe gene from C. perfringens isolates. SIGNIFICANCE AND IMPACT OF THE STUDY: The present study offers a new primer pair for detecting cpa, combined with a multiplex PCR assay. In addition, the study provides data of the presence of different toxin genes in C. perfringens isolates obtained from broiler chickens.     

Complete sequencing and diversity analysis of the enterotoxin-encoding plasmids in Clostridium perfringens type A non-food-borne human gastrointestinal disease isolates

Enterotoxin-producing Clostridium perfringens type A isolates are an important cause of food poisoning and non-food-borne human gastrointestinal diseases, e.g., sporadic diarrhea (SPOR) and antibiotic-associated diarrhea (AAD). The enterotoxin gene (cpe) is usually chromosomal in food poisoning isolates but plasmid-borne in AAD/SPOR isolates. Previous studies determined that type A SPOR isolate F5603 has a plasmid (pCPF5603) carrying cpe, IS1151, and the beta2 toxin gene (cpb2), while type A SPOR isolate F4969 has a plasmid (pCPF4969) lacking cpb2 and IS1151 but carrying cpe and IS1470-like sequences. By completely sequencing these two cpe plasmids, the current study identified pCPF5603 as a 75.3-kb plasmid carrying 73 open reading frames (ORFs) and pCPF4969 as a 70.5-kb plasmid carrying 62 ORFs. These plasmids share an approximately 35-kb conserved region that potentially encodes virulence factors and carries ORFs found on the conjugative transposon Tn916. The 34.5-kb pCPF4969 variable region contains ORFs that putatively encode two bacteriocins and a two-component regulator similar to VirR/VirS, while the approximately 43.6-kb pCPF5603 variable region contains a functional cpb2 gene and several metabolic genes. Diversity studies indicated that other type A plasmid cpe+/IS1151 SPOR/AAD isolates carry a pCPF5603-like plasmid, while other type A plasmid cpe+/IS1470-like SPOR/AAD isolates carry a pCPF4969-like plasmid. Tn916-related ORFs similar to those in pCPF4969 (known to transfer conjugatively) were detected in the cpe plasmids of other type A SPOR/AAD isolates, as well as in representative C. perfringens type B to D isolates carrying other virulence plasmids, possibly suggesting that most or all C. perfringens virulence plasmids transfer conjugatively.     

The peptidoglycan hydrolase TcpG is required for efficient conjugative transfer of pCW3 in Clostridium perfringens

Peptidoglycan hydrolases that are specifically associated with bacterial conjugation systems are postulated to facilitate the assembly of the transfer apparatus by creating a temporally and spatially controlled local opening in the peptidoglycan layer. To date little is known about the role of such enzymes in conjugation systems from Gram-positive bacteria. Conjugative plasmids from the Gram-positive pathogen Clostridium perfringens all encode two putative peptidoglycan hydrolases, TcpG and TcpI, within the conserved tcp transfer locus. Mutation and complementation analysis was used to demonstrate that a functional tcpG gene, but not the tcpI gene, was required for efficient conjugative transfer of pCW3. Furthermore, it was also shown that each of the two predicted catalytic domains of TcpG was functional in C. perfringens and that the predicted catalytic site residues, E-111, D-136, and C-238, present within these functional domains were required for optimal TcpG function. Escherichia coli cells producing TcpG demonstrated a distinctive autoagglutination phenotype and partially purified recombinant TcpG protein was shown to have peptidoglycan hydrolase-like activity on cognate peptidoglycan from C. perfringens. Based on these results it is suggested that TcpG is a functional peptidoglycan hydrolase that is required for efficient conjugative transfer of pCW3, presumably by facilitating the penetration of the pCW3 translocation complex through the cell wall.     

Conjugative Plasmids of Neisseria gonorrhoeae

Many clinical isolates of the human pathogen Neisseria gonorrhoeae contain conjugative plasmids. The host range of these plasmids is limited to Neisseria species, but presence of a tetracycline (tetM) determinant inserted in several of these plasmids is an important cause of the rapid spread of tetracycline resistance. Previously plasmids with different backbones (Dutch and American type backbones) and with and without different tetM determinants (Dutch and American type tetM determinants) have been identified. Within the isolates tested, all plasmids with American or Dutch type tetM determinants contained a Dutch type plasmid backbone. This demonstrated that tetM determinants should not be used to differentiate between conjugal plasmid backbones. The nucleotide sequences of conjugative plasmids with Dutch type plasmid backbones either not containing the tetM determinant (pEP5233) or containing Dutch (pEP5289) or American (pEP5050) type tetM determinants were determined. Analysis of the backbone sequences showed that they belong to a novel IncP1 subfamily divergent from the IncP1α, β, γ, δ and ε subfamilies. The tetM determinants were inserted in a genetic load region found in all these plasmids. Insertion was accompanied by the insertion of a gene with an unknown function, and rearrangement of a toxin/antitoxin gene cluster. The genetic load region contains two toxin/antitoxins of the Zeta/Epsilon toxin/antitoxin family previously only found in Gram positive organisms and the virulence associated protein D of the VapD/VapX toxin/antitoxin family. Remarkably, presence of VapX of pJD1, a small cryptic neisserial plasmid, in the acceptor strain strongly increased the conjugation efficiency, suggesting that it functions as an antitoxin for the conjugative plasmid. The presence of the toxin and antitoxin on different plasmids might explain why the host range of this IncP1 plasmid is limited to Neisseria species. The isolated plasmids conjugated efficiently between N. gonorrhoeae strains, but did not enhance transfer of a genetic marker.     

Molecular typing of Clostridium perfringens isolated from turkey meat by multiplex PCR

Aims:  To determine the presence of toxin genes in 22 Clostridium perfringens isolated from turkey meat samples by molecular typing.
Methods and Results:  For this purpose, alpha ( cpa ), beta ( cpb ), beta 2 ( cpb2 ), epsilon ( etx ), iota ( iA ) and enterotoxin ( cpe ) toxin genes were analysed by multiplex PCR. All 22 turkey meat Cl. perfringens isolates were found to carry the cpa , gene but in none of the isolates cpb , etx, iap or cpe genes were detected. Results showed that all isolates represented type A and were cpe negative.
Conclusions:  Our results indicate that Cl. perfringens type A is the most common type in turkey meat. Also multiplex PCR is effective and rapid method for typing of Cl. perfringens .
Significance and Impact of the Study:  It is the first study about molecular typing of Cl. perfringens using multiplex PCR in turkey meat samples in Turkey.     

Effect of epsilon toxin-GFP on MDCK cells and renal tubules in vivo.

Epsilon toxin (epsilon-toxin), produced by Clostridium perfringens types B and D, causes fatal enterotoxemia, also known as pulpy kidney disease, in livestock. Recombinant epsilon-toxin-green fluorescence protein (epsilon-toxin-GFP) and epsilon-prototoxin-GFP were successfully expressed in Escherichia coli. MTT assays on MDCK cells confirmed that recombinant epsilon-toxin-GFP retained the cytotoxicity of the native toxin. Direct fluorescence analysis of MDCK cells revealed a homogeneous peripheral pattern that was temperature sensitive and susceptible to detergent. epsilon-Toxin-GFP and epsilon-prototoxin-GFP bound to endothelia in various organs of injected mice, especially the brain. However, fluorescence mainly accumulated in kidneys. Mice injected with epsilon-toxin-GFP showed severe kidney alterations, including hemorrhagic medullae and selective degeneration of distal tubules. Moreover, experiments on kidney cryoslices demonstrated specific binding to distal tubule cells of a range of species. We demonstrate with new recombinant fluorescence tools that epsilon-toxin binds in vivo to endothelial cells and renal tubules, where it has a strong cytotoxic effect. Our binding experiments indicate that an epsilon-toxin receptor is expressed on renal distal tubules of mammalian species, including human.     

Effect of Genomic Location on Horizontal Transfer of a Recombinant Gene Cassette Between Pseudomonas Strains in the Rhizosphere and Spermosphere of Barley Seedlings

The use of genetically engineered bacteria in natural environments constitutes a risk of transfer of recombinant DNA to the indigenous bacteria. However, chromosomal genes are believed to be less likely to transfer than genes on mobilizable and conjugative plasmids. To study this assumption, horizontal transfer of a recombinant gene cassette inserted into the chromosome of a Pseudomonas stutzeri strain, into a mobilizable plasmid (pAGM42), and into a conjugative plasmid (pKJK5) isolated from barley rhizosphere was investigated. Horizontal transfer efficiencies of the gene cassette inserted into a conjugative plasmid was 8.20 × 10⁻³ transconjugants/(donors × recipients)^1/2 in the rhizosphere and 4.57 × 10⁻² transconjugants/(donors × recipients)^1/2 in the spermosphere. Mobilization of the plasmid pAGM42 by the plasmids RP4 and pKJK5 was also detected at high levels in the microcosms, transfer efficiencies were up to 4.36 × 10⁻³ transconjugants/(donors × recipients)^1/2. Transfer of chromosomal encoded genes could not be detected in the microcosms by conjugation or transformation. However, transformation did occur by using the same bacterial strains under laboratory conditions. The rhizosphere and especially the spermosphere thus proved to be hot spot environments providing favorable conditions for gene transfer by mobilization and conjugation, but these environments did not support transformation at a detectable level. Received: 21 July 2000 / Accepted: 21 August 2000     

Transfer of resistance plasmids from Staphylococcus epidermidis to Staphylococcus aureus: evidence for conjugative exchange of resistance.

The ability of Staphylococcus epidermidis to transfer antimicrobial resistance to Staphylococcus aureus was tested by mixed culture on filter membranes. Two of six clinical isolates examined were able to transfer resistance to S. aureus strains 879R4RF, RN450RF, and UM1385RF. Subsequent S.aureus transconjugants resulting from matings with S. epidermidis donors were able to serve as donors to other S. aureus strains at similar frequencies. Cell-free and mitomycin C-induced filtrates of donors and transconjugants showed no plaque-forming ability. Addition of DNase I, citrate, EDTA, calcium chloride, and human sera to mating mixes and agar showed no effect on transfer. Nonviable donor cells were unable to transfer resistance and transfer did not occur at 4 degrees C. Cell-to-cell contact was required since transfer did not occur in broth or when filters of donor and recipient, respectively, were placed back-to-back so cells were not in direct contact. Analysis of DNA from S. epidermidis isolate UM899, its subsequent S. aureus transconjugants, and cured derivatives demonstrated that all resistance markers which transferred resided on plasmids. Mating experiments suggested a central role for the gentamicin plasmid pAM899-1 in the transfer process. It is concluded that our results are consistent with a conjugative transfer of resistance from S. epidermidis to S. aureus analogous to plasmid transfer demonstrated in streptococcal species for plasmids such as pAM beta 1. This represents a novel mechanism for gene exchange among staphylococci.     

Transferable tetracycline resistance in Clostridium perfringens strains of porcine origin

These studies represent the first systematic survey of the incidence of conjugative antibiotic resistance in Clostridium perfringens. Ninety-two antibiotic-resistant porcine strains were examined to see if they could donate their antibiotic-resistance determinants to sensitive recipient strains. Fifteen of the 89 tetracycline-resistant strains transferred their tetracycline resistance in mixed-plate mating experiments but no transfer of macrolide-lincosamide resistance was detected. The efficiencies of transfer of tetracycline resistance varied from 1.3 X 10(-3) to 1.9 X 10(-6) transconjugants per donor cell. Significantly higher transfer efficiencies were observed when both the donor and recipient strains were derivatives of strain CW 362. These values ranged from 3.7 X 10(-1) to 4.6 X 10(-2) transconjugants per donor cell. This high frequency transfer system should prove invaluable for further genetic studies on this microorganism.     

In situ monitoring of IncF plasmid transfer on semi-solid agar surfaces reveals a limited invasion of plasmids in recipient colonies

Most natural conjugative IncF plasmids encode a fertility inhibition system that represses transfer gene expression in the majority of plasmid-carrying cells. The successful spread of these plasmids in clinically relevant bacteria has been suggested to be supported by a transitory derepression of transfer gene expression in newly formed transconjugants. In this study, we aimed to monitor the extent of transitory derepression during agar surface matings in situ by comparing plasmid spread of the IncF plasmid R1 and its derepressed mutant R1drd19 at low initial cell densities. A zygotic induction strategy was used to visualize the spatial distribution of fluorescent transconjugants within the heterogeneous environment. Epifluorescence and confocal microscopy revealed different transfer patterns for both plasmids, however, spread beyond the first five recipient cell layers adjacent to the donor cells was not observed. Similar results were observed for other prototypical conjugative plasmids. These results cannot rule out that transitory derepression contributes to the limited R1 plasmid invasion, but other factors like nutrient availability or spatial structure seem to limit plasmid spread.     

Exogenous isolation of conjugative plasmids from pesticide contaminated soil

Exogenous plasmid isolation method was used to assess conjugative plasmids conferring pesticide tolerance/multiple metal and antibiotic resistance from contaminated soil using bacteria detached from soil samples as a donor and rifampicin resistant E. coli HMS as a recipient strain on mineral salt agar medium supplemented with γ-HCH, and antibiotics ampicillin, tetracycline, chloramphenicol and kanamycin. Transconjugants were obtained on ampicillin (10?μg/ml) and tetracycline (20?μg/ml) amended MSA plates and frequency of ampicillin and tetracycline resistance gene transfer was 7.2?×?10(-6) and 9.2?×?10(-4) transconjugants/recipient, respectively. PCR typing methods were used to assess the presence of plasmids of the incompatibility groups IncP, IncN, IncW, IncQ and rolling circle plasmids of pMV158 type in DNA derived from transconjugants. All transconjugants were PCR amplified for the detection of Inc group plasmids and rolling circle plasmids of pMV158 family in which TM2, 3, 4, 11 and 12 (tet) transconjugants gave PCR products with the IncP-specific primers for both replication and transfer functions (trfA2 (IncP) and oriT (IncP)), while TM 14 (amp) gave an IncP specific PCR product for the replication gene trfA2 (IncP) only. TM15, 16, 18 and 21 (amp) gave a PCR product for the IncW-specific oriT (IncW). Out of 24 transconjugants, only TM 5 (tet) gave a PCR product with the pMV158 specific primer pair for oriT (RC). Our findings indicate that Inc group plasmids and rolling circle plasmids of pMV158 type may be responsible for transferring multiple antibiotic resistance genes among the bacterial soil community.     

Monitoring the spread of broad host and narrow host range plasmids in soil microcosms

Abstract: Escherichia coli recipient and E. coli donor strains carrying streptothricin-resistance genes were inoculated together into different soil microcosms. These genes were localized on the narrow host range plasmids of incompatibility (Inc) groups FII, Il, and on the broad host range plasmids of IncP1, IncN, IncW3, and IncQ. The experiments were intended to study the transfer of these plasmids in sterile and non-sterile soil with and without antibiotic selective pressure and in planted soil microcosms. Transfer of all broad host range plasmids from the introduced E. coli donor into the recipient was observed in all microcosm experiments. These results indicate that broad host range plasmids encoding short and rigid pili might spread in soil environments by conjugative transfer. In contrast, transfer of the narrow host range plasmids of IncFII and IncI1, into E. coli recipients was not found in sterile or non-sterile soil. These plasmids encoded flexible pili or flexible and rigid pili, respectively. In all experiments highest numbers of transconjugants were detected for the IncP1-plasmid (pTH16). There was evidence with plasmids belonging to IncP group transferred by conjugation into a variety of indigenous soil bacteria at detectable frequencies. Significantly higher numbers of indigenous transconjugants were obtained for the IncP-plasmid under antibiotic selection pressure, and a greater diversity of transconjugants was detected. Availability of nutrients and rhizosphere exudates stimulated transfer in soil. Furthermore, transfer of the IncN-plasmid (pIE1037) into indigenous bacteria of the rhizosphere community could be detected. The transconjugants were determined by BIOLOG as Serratia liquefaciens . Despite the known broad host range of IncW3 and IncQ-plasmids, transfer into indigenous soil bacteria could not be detected.     

Detection and characterization of conjugative degradative plasmids in xenobiotic-degrading Sphingomonas strains

A systematic survey for the presence of plasmids in 17 different xenobiotic-degrading Sphingomonas strains was performed. In almost all analyzed strains, two to five plasmids with sizes of about 50 to 500 kb were detected by using pulsed-field gel electrophoresis. A comparison of plasmid preparations untreated or treated with S1 nuclease suggested that, in general, Sphingomonas plasmids are circular. Hybridization experiments with labeled gene probes suggested that large plasmids are involved in the degradation of dibenzo-p-dioxin, dibenzofuran, and naphthalenesulfonates in S. wittichii RW1, Sphingomonas sp. HH69, and S. xenophaga BN6, respectively. The plasmids which are responsible for the degradation of naphthalene, biphenyl, and toluene by S. aromaticivorans F199 (pNL1) and of naphthalenesulfonates by S. xenophaga BN6 (pBN6) were site-specifically labeled with a kanamycin resistance cassette. The conjugative transfer of these labeled plasmids was attempted with various bacterial strains as putative recipient strains. Thus, a conjugative transfer of plasmid pBN6 from S. xenophaga BN6 to a cured mutant of strain BN6 and to Sphingomonas sp. SS3 was observed. The conjugation experiments with plasmid pNL1 suggested a broader host range of this plasmid, because it was transferred without any obvious structural changes to S. yanoikuyae B1, Sphingomonas sp. SS3, and S. herbicidovorans. In contrast, major plasmid rearrangements were observed in the transconjugants after the transfer of plasmid pNL1 to Sphingomonas sp. HH69 and of pBN6 to Sphingomonas sp. SS3. No indications for the transfer of a Sphingomonas plasmid to bacteria outside of the Sphingomonadaceae were obtained.     

Expression and purification of functional Clostridium perfringens alpha and epsilon toxins in Escherichia coli

The alpha and epsilon toxins are 2 of the 4 major lethal toxins of the pathogen Clostridium perfringens. In this study, the expression of the epsilon toxin (etx) gene of C. perfringens was optimized by replacing rare codons with high-frequency codons, and the optimized gene was synthesized using overlapping PCR. Then, the etx gene or the alpha-toxin gene (cpa) was individually inserted into the pTIG-Trx expression vector with a hexahistidine tag and a thioredoxin (Trx) to facilitate their purification and induce the expression of soluble proteins. The recombinant alpha toxin (rCPA) and epsilon toxin (rETX) were highly expressed as soluble forms in the recipient Escherichia coli BL21 strain, respectively. The rCPA and rETX were purified using Ni(2+)-chelating chromatography and size-exclusion chromatography. And the entire purification process recovered about 40% of each target protein from the starting materials. The purified target toxins formed single band at about 42kDa (rCPA) or 31kDa (rETX) in sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and their functional activity was confirmed by bioactivity assays. We have shown that the production of large amounts of soluble and functional proteins by using the pTIG-Trx vector in E. coli is a good alternative for the production of native alpha and epsilon toxins and could also be useful for the production of other toxic proteins with soluble forms.     

Worldwide distribution of the conjugative Clostridium perfringens tetracycline resistance plasmid, pCW3

The aim of this study was to test the hypothesis that all conjugative R-plasmids of Clostridium perfringens are closely related to the previously characterized tetracycline resistance plasmid, pCW3. Fourteen conjugative R-plasmids derived from 11 C. perfringens strains isolated in Australia, the United States, France, Belgium, and Japan were analyzed. Eleven of the plasmids encoded tetracycline resistance while three carried both tetracycline and chloramphenicol resistance. Each of these plasmids was compared, by restriction analysis, to the reference plasmid, pCW3. Seven of the tetracycline resistance plasmids had EcoRI, XbaI, and ClaI restriction profiles that were identical to those of the corresponding pCW3 digests. The seven remaining R-plasmids were different from pCW3. Comparison of partial restriction maps of these plasmids with a complete map of pCW3 indicated that they contained at least 17 kb of DNA that also was present in pCW3. Hybridization analysis confirmed that these plasmids shared substantial homology with pCW3. The three tetracycline and chloramphenicol resistance plasmids frequently lost a 6-kb chloramphenicol resistance segment during conjugation. Cloning experiments showed that the chloramphenicol resistance determinant was expressed in Escherichia coli and that the chloramphenicol resistance gene of one of these plasmids, pIP401, was contained within a 1.5-kb region of the 6-kb deletion segment. Hybridization analysis indicated that the deletion segment of pIP401 was related to those of the other two chloramphenicol resistance plasmids. During the course of this study, conjugative R-plasmids which appear to be identical to pCW3 or closely related to pCW3 were identified from C. perfringens strains from human, animal and environmental sources in five countries. It is concluded that C. perfringens strains in humans and animals throughout the world have overlapping gene pools and that all the conjugative C. perfringens R-plasmids examined probably evolved from a pCW3-like element.