Identification and Characterization of a Shuttle Plasmid with Antibiotic Resistance Gene from <Emphasis Type="Italic">Staphylococcus aureus</Emphasis>

While studying antibiotic-resistant plasmids from multi-drug-resistant nosocomial Staphylococcus aureus strains, we isolated a small (2.889 kb) chloramphenicol-resistant (Cm^r) plasmid, which was designated as pMC524/MBM. The molecular size of pMC524/MBM was close to that of pC194 (2.910 kb), a well-known Cm^r staphylococcal plasmid. Unlike pC194, this plasmid can replicate and express itself efficiently and stably in Escherichia coli. However, Cm is needed for stable maintenance of pMC524/MBM in different hosts. In this study, the nucleotide sequences of these two plasmids were compared after sequencing of pMC524/MBM [EMBL Accession No. AJ312056 SAU312056]. Although these two plasmids have striking nucleotide sequence homology, the Plus Origin, Minus Origin, the replication protein (Rep), and the chloramphenicol acetyl transferase (Cat) have considerable variations. Possibly, these changes have modulated pMC524/MBM into an efficient shuttle-plasmid. Received: 8 April 2002 / Accepted: 27 July 2002     

Differentiation of ruminal and human Streptococcus bovis strains by DNA homology and 16s rRNA probes

Streptococcus bovis is commonly present in the rumen, but strains of S. bovis have also occasionally been isolated from human blood or fecal samples. Studies were undertaken with 16s rRNA gene sequences and DNA hybridizations to define the genetic relationships between these two groups of strains. Ruminal strains were found to yield genomic DNA restriction endonuclease digest patterns different from human strains when either the 16s rRNA gene amplified from ruminal S. bovis strain JB1 or a conserved universal 23s rRNA fragment was used as probes. A DNA probe based on the V1 region of the 16s rRNA of S. bovis JB1 was found to hybridize to DNAs of other ruminal S. bovis strains K27FF4, 21-09-6C, five new ruminal isolates, and weak hybridization was found with DNAs from S. bovis 33317 (type strain), S. equinus 9812, and six other ruminal isolates. No hybridization occurred with strains representing different major human biotypes/homology groups (43143, 43144, 27960, V1387). All ruminal S. bovis strains had a guanosine plus cytosine DNA content of 37.4–38.8 mol% and, based on DNA-DNA genomic hybridizations, could be separated into two homology groups, one of which included S. equinus 9812 and S. bovis 33317. Both ruminal groups had less than 38% DNA homology to the human strains, indicating ruminal strains are clearly two separate species distinct from the human strains.     

Plaque forming specialized transducing phage P1: Isolation of P1CmSmSu, a precursor of P1Cm

Summary E. coli strains lysogenic for various types of P1-R hybrids were isolated. These carry all the essential genes for vegetative phage production and lysogenization including P1 immunity and P1 incompatibility, together with drug resistance genes derived from the R plasmid NR1. In particular, P1Cm and P1CmSmSu derivatives were studied. When strains lysogenic for these phages were induced in the absence of helper phage, yields of phage particles as high as with wild type P1 were obtained. All P1Cm phages isolated were of plaque forming type and usually every plaque contained Cm^r lysogens. Lysates of P1CmSmSu lysogens transduced Cm^rSm^rSu^r at high frequency and they formed plaques with an efficiency of 10^-4 to 10^-2 per phage particle. Only a minority of these plaques contained drug resistant bacteria. Cm^rSm^rSu^r transductants isolated from bacteria infected at a high multiplicity with phage P1CmSmSu were lysogens for the original P1CmSmSu. In contrast, Cm^rSm^rSu^r transductants isolated after infection at low multiplicity appeared to carry the Cm^rSm^rSu^r markers integrated into the host chromosome. The results described suggest that P1CmSmSu prophages carry the resistance genes transposed into the P1 genome without in principle causing a loss of essential gene functions. However, since these prophages are longer than the wild type P1 genome, the DNA packaged into phage particles has a reduced redundancy which seriously affects the reproduction and lysogenization abilities.Plaque forming P1Cm can be obtained from P1CmSmSu. Thus, P1CmSmSu is a precursor of P1Cm. P1Cm is also obtainable from P1 and NR1 under the recA ^- condition. The mechanism of formation of plaque forming P1Cm is discussed.     

Integration of vector-containing Bacillus subtilis chromosomal DNA by a Campbell-like mechanism

Summary Using plasmid pHV60, which contains a chloramphenicol resistance (Cm^r) gene that is expressed in Bacillus subtilis, a set of transformation-deficient strains of B. subtilis was isolated by insertional mutagenesis. When chromosomal DNA from these mutants was used to transform a transformation-proficient B. subtilis strain, almost all of the Cm^r transformants had the mutant phenotype as expected. However, with a frequency of approximately 3×10^-4 atypical transformants with the wild-type phenotype were produced. Data concerning amplification of the DNA containing the Cm^r marker and duplication of DNA sequences are presented that suggest that these atypical transformants are the result of a Campbell-like integration of the chromosomal DNA containing the integrated plasmid. Transductional mapping showed that in the atypical transformants the vector-containing DNA had a strong tendency to integrate at sites adjacent to the original site of integration, although integration at sites elsewhere on the chromosome was also observed. The production of atypical transformants is explained on the basis of integration of chromosomal DNA by a Campbell-like mechanism. Circularization of vector-containing chromosomal DNA is thought to occur through joining of the extremities of single-stranded DNA molecules by fortuitous base pairing with an independently entered single-stranded DNA molecule.     

Regions of DNA sequence homology between an octopine and a nopaline Ti plasmid of Agrobacterium tumefaciens

Summary Despite the fact that pTiC58 and pTiB6S3 functionally, have been shown to date to have only tumorigenicity and phage AP1 exclusion in common, many restriction fragments of the plasmids contain DNA sequences common to both. The bulk of this homologous DNA is concentrated in a few restriction endonuclease fragments and the remainder is organized in short discontinuous regions spread over many fragments. In pTiB6S3 the bulk of the homology is distributed throughout a 29x10⁶ dalton segment comprising 8 Sma I fragments. This region includes those sequences which are transferred to and transcribed in tumorigenic plant cells induced by B6-806 or closely related strains. The pattern of homology within this portion of the plasmid shows a region of low sequence homology (Sma I Fragment 3 b) apparently corresponding to the gene or genes coding for octopine synthesis in the plant tumor cells, surrounded by regions of high sequence homology. The extent of inter-plasmid homology then decreases with increasing distance from fragment 3b. The remainder of the homology is distributed throughout a segment of maximum size 21.5x10⁶ daltons comprising two Sma I fragments and cannot yet be definitely linked with any specific plasmid function.     

A novel method for the rapid cloning in Escherichia coli of Bacillus subtilis chromosomal DNA adjacent to Tn917 insertions

Summary A rapid and general procedure has been devised for the pBR322-mediated cloning in Escherichia coli of Bacillus subtilis chromosomal DNA extending in a specified direction from any Tn917 insertion. Derivatives of Tn917 have been constructed that contain a pBR322-derived replicon, together with a chloramphenicol-resistance (Cm^r) gene of Gram-positive origin (selectable in B. subtilis), inserted by ligation in two orientations into a SalI restriction site located near the center of the transposon. When linearized plasmid DNA carrying such derivatives was used to transform to Cm^r B. subtilis bacteria already containing a chromosomal insertion of Tn917, the pBR322 sequences efficiently became integrated into the chromosomal copy of the transposon by homologous recombination. It was then possible to clone chromosomal sequences adjacent to either transposon insertion junction into E. coli, using a selection for ampicillin-resistance, by transforming CaCl₂-treated cells with small amounts of insert-containing DNA that had been digested with various restriction enzymes and then ligated at a dilute concentration. Because pBR322 sequences may be inserted by recombination in either orientation with respect to the transposon arms, a single restriction enzyme (such as EcoRi or SphI) that has a unique recognition site in pBR322 DNA may be used to separately clone chromosomal DNA extending in either direction from the site of any transposon insertion. A family of clones generated from the region of an insertional spo mutation (spoIIH::Tn917) was used in Southern hybridization experiments to verify that cloned material isolated with this procedure accurately reflected the arrangement of sequences present in the chromosome. Strategies are discussed for taking advantage of certain properties inherent in the structure of clones generated in this way to facilitate the identification and study of promoters of insertionally mutated genes.     

Epidemiological and structural studies of Staphylococcus aureus R plasmids mediating resistance to tobramycin and streptogramin

We have studied the genetic properties of five S. aureus strains (serotypes 18 and III) isolated in France during a recent outbreak and carrying two new resistance characters (tobramycin and streptogramin). After transfer by transduction of these resistances, recipient cells were found in each case to harbor a single multicopy plasmid carrying the two resistance characters plus resistance to cadmium salts and/or resistance to tetracycline. The five Sg^r-Tm^r plasmids recovered by transduction are related epidemiologically; all five belong to the same incompatibility group and they share from 50 to 100% base sequence homology. Three of the five have similar restriction endonuclease patterns.     

Plasmid RFLP profiling and DNA homology inThermus isolated from hot springs of different geographical areas

Thirty-four strains belonging to various species of the genus Thermus (T. aquaticus, "T. thermophilus," "T. brockianus," T. scotoductus, and genomic species 2) isolated from hot springs of different geographical areas were examined for plasmid content and restriction fragment length polymorphism (RFLP) of plasmid DNAs. The four strains of the numerical taxonomy cluster E of genomic species 2 did not harbor plasmid DNA. Overall examination of the HindIII-RFLP profiling of plasmid DNA showed considerable variability between and within genomic species, with the exception of presumed clonal isolates. In spite of this heterogeneity, HindIII plasmid digests within a numerical taxonomic cluster gave a subset of restriction fragments of similar or identical length. Strains belonging to genomic species 2 or unclassified isolates from S. Pedro do Sul that harbored plasmid DNA (7 of the 14 strains studied) exhibited strong DNA homology between plasmid regions. No homologous sequences to these plasmid regions were found in chromosomal DNA from strains isolated from S. Pedro do Sul in which no plasmids were detected. The strains belonging to T. scotoductus formed two plasmid DNA homology groups, as estimated by probing with a plasmid fragment that coincided with the two numerical taxonomy clusters proposed previously. Among the other species, homology of plasmid regions was also found between some strains. Strong homology was also found between plasmid regions from some strains of different taxonomic groups, isolated from the same and from different sources, suggesting that these sequences are highly conserved in plasmids present in Thermus. For plasmid-containing strains, results of plasmid RFLP profiling/DNA homology appear promising for the typing of Thermus at the level of biotypes or of individual strains, namely, for monitoring the diversity and frequency of isolates from a particular hot spring. Received: 24 October 1994 / Accepted: 6 March 1995     

Restriction and modification in Bacillus subtilis Marburg 168: target sites and effects on plasmid transformation

Summary The effects of the restriction system of Bacillus subtilis strain M on plasmid transformation were studied. Plasmid pHV1401 DNA prepared from B. subtilis transformed the restriction-proficient M strain 100 times more efficiently than the DNA prepared from Escherichia coli, while the two DNA preparations transformed restriction-deficient derivatives of that strain with similar efficiencies. This indicates that transformation with pHV1401 is sensitive to the M restriction system. pHV1401 contains three CTCGAG (XhoI sites). Successive removal of these abolished the effect of restriction. This indicates that the XhoI sites are the targets for the M restriction system.Abbreviations used Ap^r resistance to ampicillin - Cm^r resistance to chloramphenicol - R/M restriction and modification - Tc^r resistance to tetracycline     

Cloning and DNA sequence analysis of the mercury resistance genes of Streptomyces lividans

Summary A broad-spectrum mercury resistance locus (mer) from a spontaneous chloramphenicol-sensitive (Cm^s), arginine auxotrophic (Arg^–) mutant of Streptomyces lividan 1326 was isolated on a 6 kb DNA fragment by shotgun cloning into the mercury-sensitive derivative S. lividans TK64 using the vector pIJ702. The mer genes form part of a very large amplifiable DNA sequence present in S. lividans 1326. This element was amplified to about 20 copies per chromosome in the Cm^s Arg^– mutant and was missing from strains like S. lividans TK64, cured for the plasmid SLP3. DNA sequence analysis of a 5 kb region encompassing the whole region required for broad-spectrum mercury resistance revealed six open reading frames (ORFs) transcribed in opposite directions from a common intercistronic region. The protein sequences predicted from the two ORFs transcribed in one direction showed a high degree of similarity to mercuric reductase and organomercurial lyase from other gram-negative and gram-positive sources. Few, if any, similarities were found between the predicted polypeptide sequences of the other four ORFs and other known proteins.     

Vibrio ordalii sp. nov.: A causative agent of vibriosis in fish

Vibrio ordalii sp. nov. is the name proposed for the bacterium previously designated asV. anguillarum biotype 2. The change in the classification of this fish pathogen is based on differences between the classicalV. anguillarum andV. ordalii in cultural and biochemical characteristics, and in deoxyribonucleic acid (DNA) sequence relatedness. Phenotypically,V. ordalii was distinguishable fromV. anguillarum based on: negative Voges-Proskauer reaction; negative reaction with arginine in Moeller's medium; negative Simmons' and Christensen's citrate test; negative ONPG test; failure to hydrolyze starch; failure to show lipase activity; inability to grow at 37°C; and failure to ferment cellobiose, glycerol, sorbitol, and trehalose. Genotypically, strain ofV. ordalii formed a highly conserved DNA homology group which showed 83 to 100% within-group homology and only 58 to 69% relatedness toV. anguillarum. In contrast, theV. anguillarum strains tested showed greater than 70% withingroup homology and 53 to 67% relatedness toV. ordalii. NeitherV. ordalii norV. anguillarum were related toV. parahaemolyticus orV. alginolyticus. The proposed type strain (holotype) ofV. ordalii is ATCC 33509 (=DF₃K=Dom F₃ kid).     

Antibacterial Activity of Marine Culturable Bacteria Collected from a Global Sampling of Ocean Surface Waters and Surface Swabs of Marine Organisms

The purpose of the present study was to isolate marine culturable bacteria with antibacterial activity and hence a potential biotechnological use. Seawater samples (244) and 309 swab samples from biotic or abiotic surfaces were collected on a global Danish marine research expedition (Galathea 3). Total cell counts at the seawater surface were 5 × 10⁵ to 10⁶ cells/ml, of which 0.1–0.2% were culturable on dilute marine agar (20°C). Three percent of the colonies cultured from seawater inhibited Vibrio anguillarum, whereas a significantly higher proportion (13%) of colonies from inert or biotic surfaces was inhibitory. It was not possible to relate a specific kind of eukaryotic surface or a specific geographic location to a general high occurrence of antagonistic bacteria. Five hundred and nineteen strains representing all samples and geographic locations were identified on the basis of partial 16S rRNA gene sequence homology and belonged to three major groups: Vibrionaceae (309 strains), Pseudoalteromonas spp. (128 strains), and the Roseobacter clade (29 strains). Of the latter, 25 strains were identified as Ruegeria mobilis or pelagia. When re-testing against V. anguillarum, only 409 (79%) retained some level of inhibitory activity. Many strains, especially Pseudoalteromonas spp. and Ruegeria spp., also inhibited Staphylococcus aureus. The most pronounced antibacterial strains were pigmented Pseudoalteromonas strains and Ruegeria spp. The inhibitory, pigmented Pseudoalteromonas were predominantly isolated in warmer waters from swabs of live or inert surfaces. Ruegeria strains were isolated from all ocean areas except for Arctic and Antarctic waters and inhibitory activity caused by production of tropodithietic acid.     

Phenotypic Characteristics and Virulence of Vibrio anguillarum-Related Organisms

The phenotypic, molecular, and virulence properties of 46 Vibrio anguillarum-related (VAR) strains isolated from diseased fish and shellfish and from the environment were investigated. Twelve reference strains belonging to the 10 serotypes of V. anguillarum and the Vibrio splendidus type strain were included for comparison. Numerical taxonomy studies allowed us to group the isolates into four phena. The main phenotypic traits to differentiate VAR strains from V. anguillarum were fermentation of arabinose and mannitol, indole and Voges-Proskauer reactions, gelatin and casein hydrolysis, hemolytic activity, growth at 37 and 4°C, and resistance to ampicillin. Serological analysis confirmed that phena I and II were composed mainly of strains of V. anguillarum, while phena III and IV included VAR strains. Excluding the reference strains, the typeable isolates belonged to serotypes O3 (15 strains), O4 (3 strains), and O5 (2 strains) of V. anguillarum. The infectivity trials showed that only 9 of a total of 24 strains tested displayed virulence for rainbow trout. Virulent strains (50% lethal dose ranging from 10² to 10⁶ cells) included V. anguillarum strains belonging to serotypes O1 (one strain), O2 (one strain), O3 (three isolates), and O4 (one isolate) and only three strains of the VAR group. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of lipopolysaccharide and outer membrane proteins showed heterogeneity not only among the 10 V. anguillarum serotypes but also within the VAR group. Immunoblot assays demonstrated a close relationship among V. anguillarum strains from the same serotype, while strains from different serotypes were not antigenically related. The VAR strains did not share antigenic components with the serotypes of V. anguillarum tested (serotypes O1 to O5). Plasmids were detected in only 19 of the total of 59 strains. The majority of the strains carrying plasmids were grouped within phenon IV, in which plasmid bands of 27 and 36 MDa were found in all the isolates. No correlation between the plasmid content of VAR microorganisms and their phenotypic or virulence characteristics was observed. From these results it can be concluded that VAR strains associated with disease should be included together with V. anguillarum in the formulation of vaccines against vibriosis.     

DNase-resistant transfer of chromosomal cat and tet insertions by filter mating in Pneumococcus

Genes for chloramphenicol resistance (Cm^r) and tetracycline resistance (Tc^r), which are present as heterologous insertions in the chromosomes of some clinical isolates of Streptococcus pneumoniae (pneumococcus) and derivative strains, were transferred at a low frequency to other pneumococci by a DNase-resistant filter mating process that resembles conjugation. Cotransfer of Cm^r and Tc^r was the most common event. Tetracycline resistance was transferred alone from one Tc^r strain or rarely from Cm^rTc^r donors, whereas Cm^r was never transferred alone. Neither the donor strains nor the transconjugants contained detectable plasmids. Transconjugants acted as donors for transformation and for filter mating and had properties similar to those of the parent strain. The presence of the conjugative plasmid pIP501 in the donor did not appear to influence the transfer properties of the Cm^r or Tc^r determinants. No transfer of Cm^r or Tc^r toStreptococcus faecalis JH2-2 was observed.     

Genetic relationships among clinical and environmental isolates ofVibrio cholerae from Australia

DNA-DNA homology among twenty-nine isolates having the phenotypic properties ofVibrio cholerae was studied using the S1 endonuclease method. Ten strains ofV. cholerae O1 isolated from patients and from the environment in Australia showed greater than 88% homology with the neotype strain ofV. cholerae NCTC 8021. Strains of the non-O1 serotype isolated from a variety of clinical and environmental sources also showed a high level of relatedness, including four luminescent strains and a reference strain of the biotypealbensis. A group of sucrose-negative strains showed a low level of homology (40 to 43%) withV. cholerae, but 75% and 82% homology within the group.     

Genetic and physical characterization of the ColIb plasmid using ColIb-R222 hybrids

Summary The presence of the ColIb plasmid in Escherichia coli cells inhibits the growth of bacteriophages BF23 and T5 (Ibf phenotype; inhibition of BF23 and T5 growth). To understand this abortive infection, we devised a method of isolating mutants that were defective in some ColIb phenotypes including Ibf. This method consisted of transduction of the tet (Tc^r; tetracycline resistance) or cml (Cm^r; chloramphenicol resistance) gene of plasmid R222 with phage P22 into ColIb, construction of Tc^rCm^rIbf⁺ Imm⁺ (immunity to colicin Ib) Cib^- (no production of colicin Ib) recombinants by crossing between the transductants, and isolation of deletion mutants from the recombinants by phage P1 transduction. By this procedure, pKM25-2 (Tc^rCm^sIbf^-Imm^-Cib^-) and pKM25-1 (Tc^rCm^sIbf⁺Imm⁺Cib^-) were isolated. Construction of the cleavage map of the ColIb plasmid by restriction endonucleases and comparative analyses of the DNA fragments produced from the mutant plasmids revealed that the genes determining Ibf and Imm mapped on a 4.60 Mdal HindIII fragment (H-3) and the gene determining Cib on a 1.71 Mdal EcoRI fragment (E-12).These results together with other observations (Wilkins et al. 1981; Hama personal communication) also show the approximate positions of the genes for Rep (replication), Inc (incompatibility), and Sog (suppression of dnaG) as well as Ibf, Imm, and Cib phenotypes on the cleavage map of the ColIb plasmid.Preliminary data were reported in the 1979 Annual Meeting of the Japan Molecular Biology Society (Uemura and Mizobuchi, Abst Ann Mol Biol Meet 1979, p 36)     

Incompatibility and molecular relationships between small staphylococcal plasmids carrying the same resistance marker

Incompatibility relationships between staphylococcal plasmids carrying the same, single resistance marker were studied by means of appropriate recombinant plasmids. Naturally occurring plasmids encoding streptomycin, tetracycline, or chloramphenicol resistance, respectively, were used in this study, four of each phenotype. The plasmids responsible for tetracycline resistance proved to belong to a single incompatibility set. Similarly, the four streptomycin resistance plasmids fall in the same incompatibility set. On the other hand, plasmids encoding chloramphenicol resistance were divided in four distinct incompatibility sets, three of them being newly defined. Study of the molecular relationships between these plasmids by DNA-DNA hybridization and restriction endonuclease cleavage supported the conclusions from genetic tests that the four Tc^r and the four Sm^r plasmids are essentially identical, whereas the four Cm^r plasmids are diverse.     

Brachyspira (Serpulina) hyodysenteriae gyrB Mutants and Interstrain Transfer of Coumermycin A1 Resistance

To further develop genetic techniques for the enteropathogen Brachyspira hyodysenteriae, the gyrB gene of this spirochete was isolated from a λZAPII library of strain B204 genomic DNA and sequenced. The putative protein encoded by this gene exhibited up to 55% amino acid sequence identity with GyrB proteins of various bacterial species, including other spirochetes. B. hyodysenteriae coumermycin A₁-resistant (Cn^r) mutant strains, both spontaneous and UV induced, were isolated by plating B204 cells onto Trypticase soy blood agar plates containing 0.5 μg of coumermycin A₁/ml. The coumermycin A₁ MICs were 25 to 100 μg/ml for the resistant strains and 0.1 to 0.25 μg/ml for strain B204. Four Cn^r strains had single nucleotide changes in their gyrB genes, corresponding to GyrB amino acid changes of Gly₇₈ to Ser (two strains), Gly₇₈ to Cys, and Thr₁₆₆ to Ala. When Cn^r strain 435A (Gly₇₈ to Ser) and Cm^r Km^r strain SH (ΔflaA1::cat Δnox::kan) were cultured together in brain heart infusion broth containing 10% (vol/vol) heat-treated (56°C, 30 min) calf serum, cells resistant to chloramphenicol, coumermycin A₁, and kanamycin could be isolated from the cocultures after overnight incubation, but such cells could not be isolated from monocultures of either strain. Seven Cn^r Km^r Cm^r strains were tested and were determined to have resistance genotypes of both strain 435A and strain SH. Cn^r Km^r Cm^r cells could not be isolated when antiserum to the bacteriophage-like agent VSH-1 was added to cocultures, and the numbers of resistant cells increased fivefold when mitomycin C, an inducer of VSH-1 production, was added. These results indicate that coumermycin resistance associated with a gyrB mutation is a useful selection marker for monitoring gene exchange between B. hyodysenteriae cells. Gene transfer readily occurs between B. hyodysenteriae cells in broth culture, a finding with practical importance. VSH-1 is the likely mechanism for gene transfer.     

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.