S. G. Bradley

MECHANISMS OF ACTION OF ANTIBIOTICS

’l’he discovery and development of antibiotics have brought about a new era in medical and biological sciences. Tuberculosis, which was responsible for 45.2 deaths/100,000 citizens of the United States in 1930, caused only 7.5 deaths/100,000 in 1057. Mortality resulting from acute rheumatic fever, appendicitis, and syphilis has also decreased sharply during this period (3.5 to 0.5, 9.9 to 1.2, and 11.2 to 2.2 per 100,000 citizens of the United states, respectively).l The widespread use of penicillin, streptomycin, the tetracyclines, and other antibacterial antibiotics has been responsible, to a great extent, for these dramatic reductions. Effective antifungal antibiotics have been developed only recently, part ially because severe mycotic infections are less frequent and less contagious than grave bacterial infect.ions. Moreover, the prevalence of fungus diseases has been underestimated; it is seldom realized that North American blastomycosis, candidiasis, cryptococcosis, and aspergillosis cause as many tleat.hs as tetanus, salmonellosis, chicken pox, and bacillary dysentery. I n 1055, aspergillosis, blastomycosis, bacillary dysenl ery, salmonellosis, and tetanus each was responsible for the death of 2 Minnesotans; candidiasis was responsible for 3 deaths. In 1956, cryptococcosis and chicken pox each was the cause of 2 deaths in MinnesotaP I n Iiddition, the incidence of candidiasis has been increasing, especially following prolonged treatment with broad-spectrum antibacterial antibiotics3 Cancer patients, particularly those receiving X-ray therapy, and diabetics seem to be more susceptible to mycoses than the population as a whole.4. Nystatin, the first clinically accepted antifungal antibiotic, alters favorably the course of candidiasis,6 but not of dermatophytoses or deep-seated mycoses.7 Clinical results reported in this publication indicate that griseofulvin and amphotericin €3 are becoming firmly established as effective drugs-of-choice for dermatophytosesand deep mycoses, respectively (J. H. Seabury, H. E. Dascomb, and F. J, Roth). Unfortunately, our current understanding of the mode of action of these antibiotics is limited. This discussion, therefore, reviews briefly known and postulated mechanisms of action, and outlines our approach to this problem, using Candida stellatoidea and nystatin as the test system. In order to elucidate the mode of action of an antibiotic, the cellular function(s) affected by the drug must be determined. Possible physiological loci include: (1) protein synthcsis; (2) nucleic acid synthesis; (3) synthesis or activity of coenzymes; (4) energy generation or traiisfer; ( 5 ) permeation; (6) cellular and subcellular organization; and (7) the metabolic self-regulating “feedback” system. The forenamed categories can not always be delineated sharply; for example, anabolism and catabolism are dependent on protein (enzyme) synthesis. A similar series of interactions involves active transport of precursors, fabrication of complex molecules, and integration of these into cellular structure and function; all of these steps require abundant energy.

Suppression by Endotoxin of the Immune Response to Actinophage in the Mouse.

Summary Repeated injections of 4 mg endotoxin/kg, subsequent to antigenic stimulation with actinophage MSP8, retarded the early and late immune response in the mouse and resulted in an accelerated loss of preformed antibody. Cessation of endotoxin treatment permitted phage neutralizing activity in stimulated animals to increase.

Antibody production in neonatal chickens following injection of adult cells mixed with antigen in vitro.

Summary Antibody production to Brucella abortus antigen was initiated in neonatal chicks by transfer of adult homologous hen spleen cells mixed with antigen in vitro. The studies indicate a wide variation in response and that a pathologic picture consisting primarily of splenomegaly is regularly associated with good agglutinin titers in the recipient.

Synergistic Toxicity of Endotoxin with Pactamycin or Sparsomycin

Summary Sublethal doses of the antitumor antibiotics, sparsomycin and pactamycin, act synergistically with bacterial endotoxin to produce ocular hemorrhage and even death of the treated BALB/sy mice. Another antitumor antibiotic, psicofuranine, in combination with endotoxin, does not result in enhanced toxicity for mice. Mice made immune to endotoxin are protected from the synergistic toxicity of endotoxin with sparsomycin.

Interactions between Phosphate and Nystatin in Candida stellatoidea .

Summary Glucose fermentation by Candida stellatoidea was inhibited by sodium arsenate and by the fungistatic antibiotic, nystatin. Mutants selected for resistance to one inhibitor simultaneously developed increased resistance to the other inhibitor. The inhibitory effects of arsenate and nystatin on glucose fermentation by the sensitive parental strain were reversed by appropriate concentrations of phosphate.

Immune Response to Actinophage in the Mouse.

Summary Actinophage injected intraperitoneally into inbred Balb mice appeared almost instantaneously in blood, brain, liver, kidney, spleen, lung and testis. Viable virus was isolated from the blood for 2 days following challenge, from lung, liver and kidney for 6 days, and from testis and spleen for more than 10 days. Mice produced neutralizing antibody within 48 hours after antigenic stimulation. Antibody production was elicited by as few as 107 actinophage particles.

Compatibility System Controlling Heterokaryon Formation in Streptomyces coelicolor.

Summary Heretofore, complementary pairs of growth-factor dependent mutants of Streptomyces coelicolor have interacted to yield colonies which grow readily in the absence of added supplements. Recently however certain combinations have been found which did not interact successfully. This failure of nutritionally dependent mutants to form growth-factor independent growth was not the result of allelism, but of a compatibility system controlling heterokaryon formation. The compatibility system was apparently determined by two factors, one of which probably arose by mutation.

Preferential Compatibility in Streptomyces violaceoruber

SUMMARY: A compatibility system, or system of relative mating potency, has been found among mutants of Streptomyces violaceoruber (S. coelicolor). Certain combinations of growth factor-dependent mutants failed to form nutritionally independent (prototrophic) recombinants. Incompatibility was not the result of inability of identical mutant genes to interact, failure of hyphae to fuse, or inhibition of prototroph development by incompatible mixtures. No infective or diffusible factors were found which induced or suppressed fertility. The yield of recombinants was affected by the relative growth rates of the two parental types, the time elapsed between the planting of each parent and the relative and absolute numbers of the parents. Sporogenesis was not a prerequisite for recombination.

THE ACTION OF ULTRASONIC VIBRATIONS ON ACTINOPHAGES.

Summary: High-frequency sonic oscillations destroyed the plaque-forming ability of actinophages. Among the 31 actinophage strains studied, the fraction of viral particles which survived an exposure for 1 min. to vibrations of 20 kcyc./sec. varied from 100 to 0·0002%. Coliphage T2hr+ was more susceptible to ultrasonic treatment than were some actinophages and was more resistant than other actinophages. In general, large actinophage particles were more sensitive to sonic treatment than small particles. A 60 W. 20 kcyc./sec. M.S.E. Ultrasonic Disintegrator destroyed actinophages more rapidly than a 50 W., 9 kcyc./sec. Raytheon Sonic Oscillator. Electron micrographs of actinophage MSP8 treated with sonic vibrations showed progressive disruption of the viral particles. Susceptibility to sonic inactivation gave an additional criterion for grouping actinophages.

Immune response by the mouse to orally administered actinophage.

Summary Actinophage added to the drinking water of adult BALB mice appeared in the blood within minutes; continued oral exposure to phage elicited production of phage neutralizing antibody. The initial immune response was 19S globulin; later both 7S and 19S globulins were present. Injection of antigen into mice previously fed phage elicited an enhanced response, but oral administration of phage to mice previously immunized by one injection of antigen did not cause an increase in antibody titer.