174 ANTIBACTERIAL SUBSTANCES FOR TREATMENT OF INFECTIONS 



of dead cocci. The medium favoured the growth of the soil bacilli most capable 

 of utilizing the coccal protoplasm as a source of food, and, by selection, certain 

 spore-bearing bacilli were finally obtained from which antibacterial substances 

 could be isolated (Dubos 1939) (see also Stokes and Woodward 1942). Similar 

 bacilli were isolated from manure, sewage and cheese. Working mainly with one 

 of them, B. brevis, Dubos and Hotchkiss (1941) obtained a mixture of bactericidal 

 substances, tyrothricin, from which they separated two crystalline substances, 

 gramicidin and tyrocidin. 



Tyrocidin had the following properties. It was a polypeptide with a free basic NHg 

 group, a weakly acidic or phenolic group, and contained tryptophan, tyrosine and dicarb- 

 oxylic ammo-acids ; about one-fifth of the amino-acid produced on hydrolysis was com- 

 posed of cZ-amino-acids. The molecular weight was in the region of 2,500. It was bacteri- 

 cidal in vitro for Gram-positive and Gram-negative organisms, but was also markedly 

 toxic for animal tissues and leucocytes, losing much of its antibacterial activity when 

 in contact with them. It appeared to act as a general protoplasmic poison. (See also 

 Downs 1943.) 



Gramicidin was also a polypeptide, but with no free acid or basic groups, about half 

 the amino-acids being of the d-fonn. The molecular weight was about 1,400. Gramicidin 

 killed only Gram-positive organisms i7i vitro. It protected mice against infections by 

 pneumococci, streptococci and staphylococci ; one /tig. of the substance, for example, 

 was effective when injected intraperitoneally along with 10,000 lethal doses of pneumo- 

 cocci. Though highly toxic when given l)y the parenteral route, the curative dose was 

 well below the dose tolerated by the animal. 



The high content of the " unnatural " d-amino-acids in both substances suggested 

 that their peculiar properties might in part be dependent on these bodies. Tyrocidin 

 on the whole behaved like an antiseptic ; gramicidin acted in a more specific manner, 

 like the sulphonamides (Hotchkiss and Dubos 1940, Dubos and Hotchkiss 1941, Hotchkiss 

 1941, Lipmami, Hotchkiss and Dubos 1941, Dubos, Hotchkiss and Coburn 1942). (See 

 also Tishler et al. 1941.) It is noteworthy that Fox, Fhng and BoUenback (1944) found 

 that d-leucine inhibited the growth of L. arabinosus, an organism for which /-leucine is 

 an essential nutrient, and suggested that the (^-isomer might act by interfering with the 

 uptake of the Msomer (cf. the antagonism of ^-aminobenzoic acid and sulphanilamide.) 



As the result of a study of the products of acid-hydrolysis, Gordon, Martin and Synge 

 (1943) conclude that the structural unit of gramicidin is a cyclopeptide made up of 24 

 ammo-acid residues : — 6 (Z-leucine, 6 Z-tryptophan, 5 dZ-vaUne, 3 /-alanine, 2 glycine and 

 2 unknown hydroxy amino compounds. Tyrocidin they found to contam eight different 

 amino-acids, of which phenylalanine was in the (Z-form, the rest being mainly in the Z-form. 

 (See also Christensen 1943, 1944 and Synge 1944.) 



Gramicidin is hsemolytic, and lowers surface tension, though destruction of its anti- 

 bacterial activity by heat does not alter the surface activity (Heilman and HerreU i941a, b). 

 Tyrocidin lyses both red cells and leucocytes, and both substances are toxic when injected 

 into animal tissues. Administered by mouth to dogs and mice, neither is toxic and neither 

 has any effect on infections (Rammelkamp and Weinstetn 1942, Robinson and MoUtor 

 1942). For successful therapy, gramicidhi and tyrocidin must be brought into immediate 

 contact with the infecting bacteria. Thus oral, subcutaneous or intravenous adminis- 

 tration has no effect on intraperitoneally infected mice, and mice infected systemically 

 are not protected by intraperitoneal administration (Robinson and Graessle 1942). Serial 

 subculture in low, but increasing concentration of gramicidin induced resistance in a 

 susceptible strain of Staph, aureus, and the development of resistance was accompanied 

 by the production of colony variants (Phillips and Barnes 1942). The antibacterial 

 activity of gramicidin is destroyed by gentle hydrolysis in dilute alkali, insuflflcient to 

 destroy its polypeptide structxire. 



