Charles L. Duncan

Isolation and Characterization of Multiply Antibiotic-Resistant Clostridium perfringens Strains from Porcine Feces

Multiply antibiotic-resistant strains of Clostridium perfringens were isolated from porcine feces. Strains that were resistant to tetracycline, erythromycin, clindamycin, and lincomycin were isolated, but no penicillin- or chloramphenicol-resistant strains were obtained. Typical minimal inhibitory concentrations for resistant strains were 16 to 64 μg of tetracycline per ml, 64 to >128 μg of erythromycin per ml, ≥128 μg of lincomycin per ml, and 16 to 128 μg of clindamycin per ml. Resistance to erythromycin was always associated with resistance to lincomycin and clindamycin. Minimal inhibitory concentrations were determined for 258 strains from six farms that used antibiotics in their feeds and 240 strains from five farms that did not use antibiotics. The results show that 77.9 and 22.7% of the strains from the former farms were resistant to tetracycline and erythromycin-clindamycin-lincomycin, respectively. The comparable data from the latter farms were 25.0 and 0.8%, respectively. Agarose gel electrophoresis failed to reveal a plasmid band that was common to the resistant strains but absent in the susceptible strains. Attempts to transfer tetracycline, erythromycin, and clindamycin resistance from one strain, CW459, were not successful. Antibiotic-susceptible mutants were not isolated from this strain, despite the use of a variety of curing agents. Images

Identification of a transferable tetracycline resistance plasmid (pCW3) from Clostridium perfringens

Abstract A tetracycline- and chloramphenicol-resistant strain of Clostridium perfringens, CW92, was shown to carry two plasmids, pCW2 and pCW3. Twenty-four independently derived tetracycline-sensitive mutants were isolated using a variety of curing agents. All were missing pCW3 but still carried pCW2. Tetracycline resistance could be transferred to a sensitive recipient strain by what appears to be a conjugation-like process. The efficiency of transfer was 2.8 × 10−5 transcipients per viable donor cell after a 20-h mating. The transcipients transferred tetracycline resistance at a similar frequency. Ten independently derived tetracycline-resistant transcipients all carried pCW3 as shown by agarose gel electrophoresis. The identity of this plasmid in one of these strains was confirmed by electron microscopy and restriction endonuclease analysis. Therefore, pCW3 (30.6 megadaltons) is a transferable tetracycline-resistance plasmid. No chloramphenicol-sensitive mutants or chloramphenicol-resistant transcipients were isolated. Therefore, pCW2 (36.4 megadaltons) remains cryptic.

Effect of Carbohydrates and Control of Culture pH on Beta Toxin Production by Clostridium perfringens Type C

Clostridium perfringens type C strain CN 5384 produced a higher level of beta toxin in a controlled pH medium containing 1% glucose, starch, or sucrose than in media with dextrin, fructose, or raffinose. Toxin synthesis was not related to the growth yield.

Characterization of a Bacteriocinogenic Plasmid in Clostridium perfringens CW55

A bacteriocinogenic strain of Clostridium perfringens was exposed to various curing agents known to accelerate the elimination of extrachromosomal DNA, and 20 independently derived mutants that had lost both the ability to produce bacteriocin and their immunity to it were isolated and characterized. All of the mutants were missing at least two specific plasmid bands seen in the agarose gel plasmid profile of the parent strain. Evidence that the two missing bands represented the open circular and closed circular forms of the same plasmid was obtained by X-ray nicking and restriction endonuclease digestion. The data indicated that bacteriocin production and immunity are controlled by a single plasmid, pCW4, with a molecular weight of 5.6 × 106 in this strain. Attempts to transfer the bacteriocinogenic plasmid were unsuccessful. Images

Sporangila autolysis in Clostridium perfringens type A

Abstract The autolytic system functioning in the release of mature spores and enterotoxin from sporangia of Clostridium prefringens was partially characterized. After sporangial autolysis in buffer, the supernatant fluid of the suspension contained autolysin active against purified sporangial walls. The autolysin was most active at pH 8 and 37°C, in the presence of Co2+ (0.3 · 10−3 M CoCl2) and trypsin (48 μg/ml). Sodium dodecyl sulfate-treated sporangial walls further extracted with trichloroacetic acid to remove teichoic acid were a better enzyme substrate than walls treated only with sodium dodecyl sulfate. N- Acetylmuramyl- l -alanine amidase activity which released N-terminal alanine, and endopeptidase activity which hydrolysed the d -alanyl-glycine linkage liberating N-terminal glycine and C-terminal alanine, were both functional at pH 8. It is not known if one or two enzyme are involved. Autolysin appeared in cells as early as 2 h after inoculation into sporulation medium. Two asporogenic Stage 0 mutants grown in sporulation medium also produced autolysin identical in mode of action to that of the sporogenic wild type. Although the active cellular autolysin concentration subsequently decreased as cells sporilated, the walls of 8-h-old sporangia containing refractile heat-resistant spores were more susceptible to digestion by autolysin, than those of 2-, 4-, or 6-h-old cells grown in sporulation medium or of 4- or 14-h vegetative cells from growth medium. The results suggest that a progressive change may occur in the structure of the sporangial wall during spore morphogenesis, thus increasing its susceptibility to autolysis.