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Effects of Protein Synthesis Inhibitors on the Lethal Action of Kanamycin and Streptomycin

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Effects of Protein Synthesis Inhibitors on the Lethal Action of Kanamycin and Streptomycin 222 THE JOURNAL OF ANTIBIOTICS, SER. A Nov. 1963 EFFECTS OF PROTEIN SYNTHESIS INHIBITORS ON THE LETHAL ACTION OF KANAMYCIN AND STREPTOMYCIN Hrnosm YA MAKI & NoBuo TAN AKA Institute of Applied Microbiology, University of Tokyo (Received for publication June 11, 1963) It was reported by ANAND and DAv1s 1l that chloramphenicol prevents killing of E.coli by streptomycin when the two antibiotics are added to the bacterial culture concomitantly. ANAND, DAvrs and ARMITAGE 2l demonstrated that chloramphenicol blocks accumulation of BC-streptomycin in bacteria. HuRwITz and RosANo 8l assumed the prerequisite of a strepto­ mycin-induced chloramphenicol-sensitive protein synthesis for the lethal action of strepto­ mycin. They•l also observed that chloramphenicol inhibits intrabacterial accumulation of BC-streptomycin and suggested that streptomycin-induced protein may be a specific transport system (permease). However, if the above assumption 1s correct, the induced protein synthesis appears not to be inhibited by streptomycin itself, although the antibiotic is known to interfere with protein synthesis under a certain conditions. W e5l are interested in the mode of action of kanamycin and like to know whether a similar phenomenon occurs with the lethal action of kanamycin. For the purpose of elucidating these problems, the effects of antibiotics, which inhibit bacterial protein synthesis, on the lethal action of kanamycin and streptomycin were studied and the results are presented in this publication. The antibiotics used include chloramphenicol, erythromycin, mikamycin A, puromycin, blasticidin S and tetracycline. The bactericidal action of both streptomycin and kanamycin was blocked by chloramphenicol, erythromycin, mikamycin A, blasticidin S and tetra­ cycline, but not by puromycin. Methods The 18-hour culture of E. coli B was suspended in nutrient broth (turbidity : E680 =0.05~0.07) and incubated at 37°C with gentle agitation. The antibiotics were added to the bacterial culture during the logarithmic phase of growth (approximately 3 hours after the inoculation). The number of viable cells was determined 4 hours after addition of antibiotics, using 0.1 % glucose nutrient agar. The concentrations of antibiotics employed were 20 mcg/ml for streptomycin, kanamycin and chloram­ phenicol; 5 or 20 mcg/ml for tetracycline; 10 or 50 mcg/ml for puromycin; 100 mcg/ml for mikamycin A and blasticidin S; and 1,000 mcg/ml for erythromycin. In most experiments, protein synthesis inhibitors were added to the cell suspensions simultaneously with the addition of streptomycin or kanamycin. However, the effect of puromycin was also investigated, when it was introduced to the bacterial culture an hour before the addition of streptomycin or kanamycin. Mikamycin A was dis­ solved in acetone and was added to the bacterial culture. The final concentration of acetone in the culture medium was approximately 1 % . Vol. XVI No. 6 THE JOURNAL OF ANTIBIOTICS, SER. A 223 Results Effect of Chloramphenicol on Killing of E. coli by Streptomycin or Kanamycin The number of viable cells did not change before the Fig. 1. Effect of chloram­ phenicol on killing of E. addition of chloramphenicol and after 4-hour contact with coli B by streptomycin or chloramphenicol, whereas in antibiotic-free medium the num­ kanamycin. togN ber of viable cells increased 16 times. The number of viable 10 cells markedly decreased in streptomycin- or kanamycin­ containing medium. The addition of chloramphenicol to K streptomycin- or kanamycin-containing culture significantly prevented killing of bacteria by the glycoside antibiotics. The results are illustrated in Fig. 1. 4- SM Effect of Puromycin on Killing of E. coli by 2 '---~--::-----;:; Streptomycin or Kanamycin o .1 S 7 hrs. N : The number of viable cells. As shown in Fig. 2a and 2b, the activity of puromycin Control: No antibiotics. CP : Chloramphenicol 20 mcg/ m! was primarily bacteriostatic and that of streptomycin or KM : Kanamycin 20 mcg/ ml SM : Streptomycin 20 mcg/ml kanamycin was primarily bactericidal. The lethal action of The arrow shows the time, when antibiotics are added to the bac· streptomycin or kanamycin was not significantly affected by terial culture. the presence of puromycin when it was added to the bacterial culture simultaneously with or 1 hour before the addition of kanamycin or streptomycin. Effect of Blasticidin S on Killing of E. coli by Streptomycin or Kanamycin The activity of blasticidin S appeared to be primarily bacteriostatic and it markedly prevented killing of the bacteria by streptomycin or kanamycin in the same conditions as Fig. 2a. Effect of puromycin Fig. 2b. Effect of puromycin on Fig. 3. Effect of blasticidin on killing of E. coli B killing of E. coli B by strepto­ S on killing of E. coli B by streptomycin or kanamycin. mycin or kanamycin. streptomycin or kanamycin. iogN logN ·t /0 10 8 8 ,f PM 6 . t ,.'.f KM 41 4 f SN SM 4 -::::, ~-~/M,~ffl•SM<U XM (JI) 6l~"~, f ~ XM•P/.1 'l .. ll"1--4 2 SM•PM 2 0 2 7 hrs. J s o I .1 .\' 7 hrs O I J s 7/lrs. N: The number of viable cells N: The number of viable cells of N: The number of viable cells of E. coli B E . coli B of E. coli B PM : Puromycin 10 mcg/ml Control : No antibiotics Control : No antibiotics SM : Streptomycin 20 mcg/ml PM : Puromycin 50 mcg/ml BL : Blasticidin S 100 mcg/ml KM : Kanamycin 20 mcg/ml SM : Streptomycin 20 mcg/ml KM : Kanamycin 20 mcg/ ml The arrow shows the time, when KM : Kanamycin 20 mcg/ml SM : Streptomycin 20 mcg/ ml antibiotics are introduced to the PM+SM (I) and PM+KM (I): The arrow shows the time, when culture. Puromycin was added to the culture antibiotics are introduced to the simultaneously with streptomycin or culture. kanamycin at the time of arrow I. PM+SM(II) and PM+KM (II): Puromycin was added at the time of arrow I and streptomycin or kana~ mycin was added at the time of arrow II. 224 THE JOURNAL OF ANTIBIOTICS, SER. A Nov. 1963 Fig. 4. Effect of erythro­ Fig. 5. Effect of mikamycin A the above experiments. The mycin on killing of E.coli on killing of E. coli B by B by streptomycin or streptomycin or kanamycin. results are presented in Fig. kanamycin. 3. It seems interesting that lcq/1 1/N JO the activity of blasticidin S is different from that of puro­ 8 3 mycin in this respect. If/,,/ Effect of Erythromycin SM on Killing of E. coli 4 4 by Streptomycin or KM Kanamycin 2 As illustrated in Fig. 4, J .) 7hrs. 0 J s 7 hrs. N: The number of viable cells N : The number of viable cells of E. erythromycin markedly re­ of E. coli B coli B Control : No antibiotics Control : No antibiotics versed the lethal action of EM : Erythromycin I. 000 mcg/ ml Control (acetone I%): No antibiotics streptomycin or kanamycin. SM : Streptomycin 20 mcg/ ml and 1 % of acetone KM : Kanamycin 20 mcg/ ml MK : Mikamycin A 100 mcg/ml The arrow shows the time, when SM : Streptomycin 20 mcg/ ml Effect of Mikamycin antibiotics are added to the bac­ KM : Kanamycin 20 mcg/m! A on Killing of E. coli terial culture. The arrow shows the time, when anti• biotics are introduced to the bacterial by Streptomycin or culture. Kanamycin The antibacterial activity of mikamycin A was primarily bacteriostatic. By the method employed, mikamycin A conspicuously antagonized the killing effect of streptomycin or kanamycin. The results are illustrated in Fig. 5. Effect of Tetracycline on Killing of E. coli by Streptomycin or Kanamycin Tetracycline slightly reversed the lethal action of sterptomycin or kanamycin at the concentration of 5 mcg/ml and markedly did at the concentration of 20 mcg/ml. The results are illustrated in Figs. 6a and 6b. Combination of Kanamycin and Streptomycin The concomitant addition of streptomycin and kanamycin appeared not significantly to Fig. 6a. Effect of tetracycline Fig. 6b. Effect of tetracycline Fig. 7. Combination of on killing of E. coli B by on killing of E. coli B by streptomycin and kana­ streptomycin or kanamycin. streptomycin or kanamycin. mycin. log/I loqN i11gt1 JO 10 10 3 g KM SM ~ TC+S/.1 SM•/fM SM \"\ TUKf,1 4 4 lfM 4 ' \SM K/1 2 2 o~~~-J~~~s~--=7 hrs. 0 J s 7 hrs N: The number of viable cells of N: The number of viable cells of N : The number of viable cells E. coli B E. coli B of E . coli B Control : No antibiotics Control : No antibiotics SM : Streptomycin 20 mcglml TC : Tetracycline 5 mcg/ml TC: Tetracycline 20 mcg/ml KM: Kanamycin 17 mcg/ ml SM : Streptomycin 20 mcg/ml SM : Streptomycin 20 mcg/ml The arrow shows the time, when KM : Kanamycin 20 mcg/ml KM : Kanamycin 20 mcg/ml antibiotics are added to the The arrow shows the time, when The arrow shows the time, when culture. antibiotics are added to the bac­ antibiotics are introduced t::> the terial culture. bacterial culture. Vol. XVI No. 6 THE JOURNAL OF ANTIBIOTICS, SER. A 225 influence the activity of both antibiotics. The results are presented m Fig. 7. Discussion It was observed in this study that chloramphenicol, erythromycin, mikamycin A, blasticidin S or tetracycline markedly blocks the lethal action of kanamycin or streptomycin, but puromycin does not affect it. Reversal of the lethal effect of streptomycin by chloramphenicol suggested that the strepto­ mycin-sensitive metabolic process may not work in the cells affected by chloramphenicol or an agent, necessary for the action of streptomycin, may be inhibited by chloramphenicol. In the former case, the site of protein synthesis, sensitive to streptomycin might follow the process, inhibited by chloram­ phenicol.
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