A fully virulent classical type A strain of Clostridium perfringens was treated during its logarithmic growth phase with 100 μg/ml of N-méthyl-N′-nitro-N-nitrosoguanidine, the bacteria being exposed to the mutagen for 30 min at 37 °C in a phosphate buffer adjusted to pH 6.2; after treatment the suspension was streaked on sheep blood agar plates, and colonies that showed an alteration in the theta-hemolysis pattern were selected for isolation. The virulence of two mutants, thus altered in their thêta-hemolysis, was studied. One, designated LNG 5, was still capable of killing most of the inoculated guinea pigs in less than 24 h with all the clinical, macroscopic, and bacteriological signs of gas gangrene; however, histological sections showed that tissue damage was not as marked as with the wild strain. On the contrary, the second mutant, labelled LNG 11, was completely avirulent as far as gas gangrene was concerned; indeed, the injection of fluid cultures containing 1 × 108-109/ml viable bacteria, was not followed by any clinical, bacteriological, or histological signs of gas gangrene. However, strain LNG 11 did give rise to a firm swelling of the inoculated thigh with a corresponding acute inflammatory response of the connective tissue, although the muscle fiber was unaltered. Eventually, this local reaction was followed by necrosis of the skin accompanied by an acute or subacute inflammation with fibroblastic proliferation. These superficial lesions healed spontaneously. They could not be reproduced with crude filtrate alone or with washed bacilli. Strain LNG 11 was therefore considered to be solely an attenuated strain since, although avirulent as far as gas gangrene is concerned, it is still capable of producing low levels of toxic material. This appears to be the first time that such a strain of C. perfringens type A has been obtained by nitrosoguanidine treatment.
Use of Genetically Manipulated Strains of Clostridium perfringens Reveals that Both Alpha-Toxin and Theta-Toxin Are Required for Vascular Leukostasis To Occur in Experimental Gas Gangrene