Avirulent Clostridium perfringens strains obtained by euflavine treatment.

Clostridium perfringens was incubated in the presence of euflavine (EU); the resistant mutants which were thus isolated had highly reduced capacity to release alpha-toxin. This fact was confirmed by lecithinase determinations and by immunoelectrophoresis. Injected into guinea pigs and into 6- to 7-day-old chicks, these mutants were completely avirulent. A study of their properties indicated biochemical differences between wild types and mutants.

Properties of four temperate bacteriophages active on Clostridium perfringens type A.

Four temperature bacteriophages (designated as PF1, PF2, PF3 and PF4) were isolated from lysogenic strains of Clostridium perfringens type A. On the basis of plaque morphology, pH stability, DNase and RNase resistance, buoyant density, one-step growth parameters and electron microscope phage dimensions, it seems that these phages are different and unrelated. Calcium was required for better phage replication. Bacterial strain S107 appears to be the only UV-inducible strain as compared with the other three lysogenic strains. PF2 has a unique pattern of pH stability showing two optima values: one at pH 5 and the second at pH 8-9. Generally, all four phages have a better resistance in acid than in alkaline pH values. The CsC1 equilibrium centrifugation patterns reveal low figures for phage PF1, PF2 and PF3 and show off the fact that PF4 lysates contain two viral particules different with respect to their densities, a property which other determinations failed to demonstrate. Each phage, except PF4, is well characterized by the parameters of the one-step growth cycle.

Numerical data concerning the sensitivity of anaerobic bacteria to oxygen.

Upon comparison of results obtained from growing eight different species of anaerobic bacteria in deep agar under two and three atm of pure oxygen with results of growing them in air under atomospheric pressure, it appears possible that the old terms of "microaerophobic," "very strict anaerobe," and "microaerophilic" can be replaced by precise values corresponding to the size of the inhibition zone produced by oxygen under various pressures when the bacteria are grown in a solid medium. The simultaneous effect of the dilution of the inoculum and the pressure of oxygen used was determined with the help of an electronic computer according to the standard multiple regression method. Two indices have been worked out which express the sensitivity of the species at different oxygen pressures when varying numbers of anaerobic bacteria are used: X sp, which is the size in millimeters of the inhibition zone when 1:10 dilutions of the culture are grown under pure oxygen at atmospheric pressure (lp=0), and B sp, which is the result of multiplying by 1.43 the difference in size of inhibition zone when 1:10 dilutions of the culture are grown under atmospheric air (lp = -0.7) and under pure oxygen at 1 atm absolute.

[Nitrosoguanidine-induced attenuated Clostridium perfringens type A mutant in gas gangrene]. / Mutant atténué nitrosoguanidine-induit dans la gangrène gazeuse à Clostridium perfringens type A

A fully virulent classical type A strain of Clostridium perfringens was treated during its logarithmic growth phase with 100 mug/ml of N-méthyl-N'-nitro-N-nitrosoguanidine, the bacteria being exposed to the mutagen for 30 min at 37 degrees 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 theta-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 times 10(8) - 10(9)/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 soletly 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.

Correlations between virulence and other characters of Clostridium perfringens type A.

The correlation analysis which has already proved its value in ecology has not yet been applied to the determination of virulence indicators. Its application to a group of Clostridium perfringens type A strains has brought out some characters that may be considered as virulence indicators. This study suggests that the toxicity of the culture supernatant fluids for mice is significantly correlated with the virulence for mice and guinea pigs. A significant correlation was found between the virulence of the fluid cultures for mice or guinea pigs and the coagulation of milk, production of gas (in deep agar), hydrogen sulfide production, and fermentation of glucose, sucrose, maltose, and levulose.