Molecular analysis of transferable tetracycline resistance plasmids from Clostridium perfringens

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

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

Cloning and analysis of the Clostridium perfringens tetracycline resistance plasmid, pCW3

Clostridium perfringens strain CW92 carries pCW3, a conjugative 47-kb plasmid that confers inducible resistance to tetracycline. The plasmid was examined by restriction endonuclease analysis and by cloning each of the five ClaI fragments of pCW3 in Escherichia coli, using pBR322. Analysis of the recombinant plasmids allowed the deduction of a detailed restriction map of pCW3. The tetracycline resistance determinant of pCW3 was mapped by examining the phenotype of recombinant E. coli clones derived from the cloning, into pUC vector plasmids, of EcoRI fragments from pCW3. The C. perfringens tetracycline resistance determinant was expressed in E. coli and was shown to be located on two juxtaposed EcoRI fragments which together encompass a 4-kb region of pCW3. Deletion experiments showed that the tetracycline resistance gene, and/or its control regions, contained internal EcoRI and SphI sites. E. coli strains that carried recombinant plasmids with only the 4-kb region were found to express tetracycline resistance constitutively. In contrast, recombinant plasmids harboring a 10.5-kb ClaI fragment of pCW3, that included the 4-kb region, coded for an inducible tetracycline resistance phenotype. The existence of a negatively regulated resistance gene, similar to that proposed for several other bacteria is postulated

Molecular cloning of the fimbrial subunit gene from a benign type B isolate of Bacteroides nodosus

The fimbrial subunit gene from the benign type BBacteroides nodosus isolate AC/6 was cloned into theSphI site of the multicopy vector plasmid pUC19. FiveEscherichia coli recombinants that were positive in a colony immunoassay were shown, by Western transfer analysis, to produce an immunologically cross-reacting protein of identical molecular size to fimbrial subunits prepared fromB. nodosus AC/6. Restriction endonuclease analysis showed that 4 of the recombinant plasmids carried a 6.7 kbSphI fragment. Recloning experiments showed that the fimbrial subunit gene was located within a 2.5 kbEcoRI-SphI fragment and that there was aPstI site located within the structural gene or its regulatory region. These recombinant clones will prove useful for the construction of a multivalent recombinant vaccine for the control of ovine footrot

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

Lysogeny and other characteristics of Staphylococcus hyicus isolated from chickens

Lysogeny was readily demonstrated among strains of Staphylococcus hyicus that were isolated from chickens. Susceptibility to phage lysis was affected by prophage immunity, but lipase activity and erythromycin resistance were not affected by the presence of temperate phage. In contrast to previously published results, lipase-negative strains of S. hyicus were relatively common and the use of selective media based on lipase activity would have been unsuitable for detection of the S. hyicus strains examined

Taenia hydatigena: Isolation of mitochondrial DNA, molecular cloning, and physical mitochondrial genome mapping

Mitochondrial DNA was isolated from Taenia hydatigena, T. crassiceps, and Echinococcus granulosus using a cetyltrimethylammonium bromide precipitation technique. The technique is simple, rapid, reproducible, and does not require extensive high speed ultracentrifugation. The advantage of using mitochondrial DNA from taeniid cestodes for comparative restriction analysis was demonstrated. Mitochondrial DNA of T. hydatigena was isolated as covalently closed circular molecules. These were linearized by single digestion with BamHI and the molecular weight was estimated from the linear form of 17.6 kb. The mitochondrial DNA of T. hydatigena is therefore similar in size and structure to that of many other animal species. The entire mitochondrial genome was cloned into pBR322 in Escherichia coli and a restriction map of the recombinant molecule was constructed. The potential of using the cloned mitochondrial genome as a probe in speciation studies as well as for providing functional information on the role of the cestode mitochondrion is discussed

Molecular characterization and antimicrobial susceptibilities of Clostridium difficile clinical isolates from Victoria, Australia

Some Australian strain types of Clostridium difficile appear unique, highlighting the global diversity of this bacterium. We examined recent and historic local isolates, finding predominantly toxinotype 0 strains, but also toxinotypes V and VIII. All isolates tested were susceptible to vancomycin and metronidazole, while moxifloxacin resistance was only detected in recent strains