International Journal of Applied Radiation and Isotopes, 1975, Vol. 26, pp. 387-391. Pergamon Press. Printed in Northern Ireland
The Effect of Ionizing Radiation on the Antibiotic Sensitivity of Micrococcus radiodurans Strains and 5 N A Z A R A G H A , F. M. S A L I H , H. A U D A and S. A L - B A R W A R I Atomic Energy Commission, Nuclear Research Institute, Biology Department, Tuwaitha, Baghdad, Iraq (Received 21 June 1974; in revisedform 18 October 1974) Micrococcus radiodurans strains RI and RII5 were tested for susceptibility to several anti-
biotics by the standardized Bauer-Kirby disc diffusion and tube dilution techniques, using different size of inoculum irradiated at doses of 150, 300, 500 and 750 krad; a control without irradiation. The size of inoculum had a variable effect on susceptibility, depending on the strain of M radiodurans, the antibiotics, and the dose of irradiation. In general, strain /7i was more sensitive to the antibiotics and the radiation than RII5. The minimal inhibitory concentration (MIC) and the minimal bactericidal concentration (MBC) of the antibiotics against .~¢ radiodurans were determined. INTRODUCTION
D I F C O . The strains were maintained on T G Y agar slants of the following composition: resistant coccus originally isolated by ANDERSON 0"5 per cent tryptone, 0"l per cent glucose, and co-workers/a'6) and its resistance to 0"3 per cent yeast extract and 1"5 per cent ionizing and ultraviolet radiation has been agar (pH 7"0). studied.C 4"5"9'at't2) The cell wall of M radiodurans R I contains comparatively large amounts Strains of lipids and a lipoprotein layer, as in gramMicrococcus radiodurans R I and RII 5 were negative bacteriaJ 14) Antibiotic sensitivity of M radiodurans R I and its nonpigmented m u t a n t kindly given by C. EMSORO, of the Research WI were tested for sensitivity to 10 antibiotics. ~1°) Establishment, Riso, Denmark. Loops of Recently a m u t a n t of M radiodurans RII 5 was 48-hr fresh cultures of R I and RII5 maintained isolated which is three times more radiation on T G Y agar plates were transferred into resistant than strain RI .~7) The purpose of our 100ml T G Y broth in Erlenmayer flasks of work was, therefore, to determine the suscepti- 250 ml capacity and grown in a shaking bath bility of R I and RII 5 irradiated by g a m m a (Precision Scientific Co., 115 vibrations/rain) rays at different doses using different size of at 30°C for 18-24 hr. At the end of the incuinoculum (cell/ml) to antibiotics. The antibation period, 10-ml samples were placed in biotic susceptibility of R I and RII5 with respect to irradiation has not been studied yet. I n centrifuge tubes. The samples centrifuged at this paper an attempt was made to determine 5000 rev/min for 15 rain and the pellets were the sensitivity of both strains of M radiodurans irradiated with the following doses: 150, 300, to different antibiotics of different potencies 500 and 750 krad of cobalt-60 g a m m a rays in a "Gammacell 200" unit (Atomic Energy of combined with the effect of radiation. Canada Ltd., Ottawa). The irradiated and MATERIALS AND METHODS non-irradiated pellets were resuspended into sterile physiological saline at room temperature. Medium Tryptone Glucose Yeast extract (TGY) The number of organisms in the inoculum was broth and T G Y agar were purchased from determined by dilution counting method. 5 387 M radiodurans R I is a gram-positive radiation-
388
N. Agha, F. M. Salih, H. Auda and S. Al-Barwari
Irradiation
of all tested antibiotics were prepared, 8 ml Irradiations were carried out aerobically containing 8000 #g was diluted to 20 ml yield at room temperature with the g a m m a rays of 400/~g/ml, passed through 0"45 /~m membrane filter (Millipore Filter Corp., Bedford, Mass., 161 rad/see. U.S.A.), dispensed as 10-ml samples into sterile Antibiotics vials, and frozen and kept stored at --15 to The sources of sensitivity discs were as --20°G. The vials were never refrozen after follows: rifamycin sv (R), and rifampicin (RA) they had been thawed. from Lepetit, spa-Milan, Italy; cloxacillin (CX), ampicillin (AP), earbencillin (CC), Antibiotic susceptibility tests aminosidine sulphate (AS) and gentamiein Disc diffusion susceptibility tests were persulphate (GM), from Mast Laboratories Ltd., formed by the technique of BAUER et al., ~3) Liverpool, England; cefalotin (GF). from Eli with the exception that the large plates (100 Lilly & Co., Indianapolis, U.S.A.; tetracyclin by 15 mm) contained 6 0 m l of T G Y agar, (TG), chloramphenicol (GM) and penicillin resulting in an agar depth of roughly 4 mm, (s) (P), from Oxoid Limited, London, England. and the size of inoculum for each strain was The sources of antibiotics were as follows: determined ( R I = 6 × 105, 6 X 106 cells/ml) rifamycin sv and rifampicin, from Lepetit, and RII5 = 4'2 × 105, 4'2 × 108 cells/ml). spa-Milan, Italy; cloxacillin and carbencillin, Duplicate discs of eleven antibiotics (R, Cx, from Beecham Research Laboratories, Brent- RA, AP, AS, CC, GM, CF, TC, P and CM) ford, England; aminosidine sulphate, from were used with each strain. The plates were Soc. Farmaceutici Italia, Milano, Italy; chlor- incubated for 24 hr and the inhibition zones amphenicol, from Carlo Erba, Milano, Italy; were then measured. Simultaneous broth cefalotin, from Eli Lilly & Co., Indianapolis, dilution tests were performed with the bacterial U.S.A. ; ampicillin, from Ankerfarm spa, inocula adjusted approximately in the range Cinisello B., Milan, Italy; penicillin G sodium 1 to 6 × 104 cells/ml. The strains were exposed salt and tetraeyclin, Laboratory standards, to serial twofold dilutions of ten antibiotics from State C o m p a n y for Drugs Industries, (Tables 1 and 2); 200, 100, 50, 25, 12"5, Samrra, Iraq. The above-mentioned anti- 6"3, 3"2, 1"6, 0"8 and 0"4 #g/ml (final concentrabiotics except penicillin and tetracyclin were tions). The (MIC) of each of the antibiotics obtained commercially. Dilution of 1000/~g/ml was read after overnight incubation at 30°C TABLE 1. Antibiotic susceptibility of M radiodurans R1 and RII5 irradiated at doses of 500 and 750 krad MIC (/~g/ml) M radiodurans Ru 5
M radiodurans R1
irradiated at doses Antibiotic Rifamycin sv Cloxacillin Rifampicin Ampicillin Aminosidine sulphate Carbencillin Cefalotin Tetracyclin HC1 Penicillin G Ghloramphenicol
0.0 krad
500 krad
750 krad
size of inoculum cells/ml
6-3 1-6 3.2 0-4* 6.3 25.0 1.6 0.4* 0-4* 25.0
1.6 0.8 1.6 0.4* 1.6 3.2 0-8 0.4* 0.4* 12.5
0.8 0.8 0.8 0.4* 0-8 3.2 0'8 0.4* 0-4* 6.3
1-0 1.6 6.0 1.0 1.0 4-0 1.6 2.1 2.0 1.0
* The MIG is less than 0.4 pg/ml.
× × × x × × × × × ×
104 104 104 104 104 104 104 104 10~ 104
irradiated at doses 0.0 krad
500 krad
750 krad
size of inoculum cells/ml
6.3 1.6 3.2 0-4* 3.2 6.3 0-8 0.4* 0-4* 25.0
1.6 0-8 1.6 0.4* 1.6 3.2 0.4 0.4* 0.4* 25.0
0.8 0.8 0-8 0.4* 1.6 3.2 0.4 0.4* 0.4* 12.5
1.2 1.2 4.0 3.8 1.0 1.0 1.8 5.0 2-1 1.4
× × x × × × × × x ×
104 104 104 104 104 104 104 104 104 104
Effect of radiation on antibiotic sensitivity of Micrococcus radiodurans
389
TABLE 2. T h e m i n i m a l bactericidal c o n c e n t r a t i o n (MBC) of antibiotics against M radiodurans Rt a n d RII5 i r r a d i a t e d at doses o f 500 a n d 750 k r a d /vIBC (/~g/ml)
Antibiotic R i f a m y c i n sv Cloxacillin Rifampiein Ampicillin A m i n o s i d i n e sulphate Carbencillin Cefalotin T e t r a c y c l i n HC1 Penicillin G Chlorarnphenicol
M radiodurans RI
M radiodurans RII5
i r r a d i a t e d at doses
i r r a d i a t e d at doses
0-0 krad
500 krad
750 krad
size of inoculum cells]ml
0.0 krad
500 krad
750 krad
size of inoculum eells]ml
200* 50 200 50 25 200* 200 200 50 200*
200* 25 50 50 25 200* 100 100 200 200*
50 25 50 50 12.5 200* 100 100 50 200*
1.1 1.6 6.0 1.0 I-0 4,0 1.6 2.1 2-0 1-I
200* 25 200* 100 25 200* 200 200 50 200*
203 12.5 50 100 12.5 200* 50 200 200 200*
100 12.5 25 100 12.5 200* 50 200 100 200*
1.2 1.2 4.0 3.8 1.0 1.0 1.8 5.1 2-1 1.4
x
x × × × x × x x ×
10a 104 104 104 104 104 104 104 104 10 *
x
× x × × x × × x ×
104 104 104 104 104 104 10 ~ 104 104 104
* T h e M B C is m o r e t h a n 200/*g]ml. as t h e l o w e s t c o n c e n t r a t i o n o f a n t i b i o t i c t h a t inhibited visible growth. The (MBC) was d e f i n e d as t h e l o w e s t c o n c e n t r a t i o n o f a n t i b i o t i c w h e r e 0' 1 m l s u b c u l t u r e o n t o a g a r p l a t e s showed no growth at 48 hr. The growth rates of irradiated and nonirradiated of both R I and Rn5 were determined.
RESULTS
AND DISCUSSION
The effect of radiation and size of inoculum on susceptibility of M radiodurans R I and RII5 to eleven antibiotics (disc diffusion techniques) is shown in Figs. 1 and 2. The potencies of antibiotics (#g/disc) were as follows: R, 30; (b)
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FIe. 1. Effect of r a d i a t i o n of i n o c u l u m o n susceptibility of M radiodurans strain R1 to eleven a n t i b i o t i c (a = 6.0 / 105, b = 6-0 × 106 cells]ml).
390
N. Agha, F. M. Salih, H. Auda and S. AI-Barwari
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The Effect of Ionizing Radiation on the Antibiotic Sensitivity of Micrococcus radiodurans Strains and 5 N A Z A R A G H A , F. M. S A L I H , H. A U D A and S. A L - B A R W A R I Atomic Energy Commission, Nuclear Research Institute, Biology Department, Tuwaitha, Baghdad, Iraq (Received 21 June 1974; in revisedform 18 October 1974) Micrococcus radiodurans strains RI and RII5 were tested for susceptibility to several anti-
biotics by the standardized Bauer-Kirby disc diffusion and tube dilution techniques, using different size of inoculum irradiated at doses of 150, 300, 500 and 750 krad; a control without irradiation. The size of inoculum had a variable effect on susceptibility, depending on the strain of M radiodurans, the antibiotics, and the dose of irradiation. In general, strain /7i was more sensitive to the antibiotics and the radiation than RII5. The minimal inhibitory concentration (MIC) and the minimal bactericidal concentration (MBC) of the antibiotics against .~¢ radiodurans were determined. INTRODUCTION
D I F C O . The strains were maintained on T G Y agar slants of the following composition: resistant coccus originally isolated by ANDERSON 0"5 per cent tryptone, 0"l per cent glucose, and co-workers/a'6) and its resistance to 0"3 per cent yeast extract and 1"5 per cent ionizing and ultraviolet radiation has been agar (pH 7"0). studied.C 4"5"9'at't2) The cell wall of M radiodurans R I contains comparatively large amounts Strains of lipids and a lipoprotein layer, as in gramMicrococcus radiodurans R I and RII 5 were negative bacteriaJ 14) Antibiotic sensitivity of M radiodurans R I and its nonpigmented m u t a n t kindly given by C. EMSORO, of the Research WI were tested for sensitivity to 10 antibiotics. ~1°) Establishment, Riso, Denmark. Loops of Recently a m u t a n t of M radiodurans RII 5 was 48-hr fresh cultures of R I and RII5 maintained isolated which is three times more radiation on T G Y agar plates were transferred into resistant than strain RI .~7) The purpose of our 100ml T G Y broth in Erlenmayer flasks of work was, therefore, to determine the suscepti- 250 ml capacity and grown in a shaking bath bility of R I and RII 5 irradiated by g a m m a (Precision Scientific Co., 115 vibrations/rain) rays at different doses using different size of at 30°C for 18-24 hr. At the end of the incuinoculum (cell/ml) to antibiotics. The antibation period, 10-ml samples were placed in biotic susceptibility of R I and RII5 with respect to irradiation has not been studied yet. I n centrifuge tubes. The samples centrifuged at this paper an attempt was made to determine 5000 rev/min for 15 rain and the pellets were the sensitivity of both strains of M radiodurans irradiated with the following doses: 150, 300, to different antibiotics of different potencies 500 and 750 krad of cobalt-60 g a m m a rays in a "Gammacell 200" unit (Atomic Energy of combined with the effect of radiation. Canada Ltd., Ottawa). The irradiated and MATERIALS AND METHODS non-irradiated pellets were resuspended into sterile physiological saline at room temperature. Medium Tryptone Glucose Yeast extract (TGY) The number of organisms in the inoculum was broth and T G Y agar were purchased from determined by dilution counting method. 5 387 M radiodurans R I is a gram-positive radiation-
388
N. Agha, F. M. Salih, H. Auda and S. Al-Barwari
Irradiation
of all tested antibiotics were prepared, 8 ml Irradiations were carried out aerobically containing 8000 #g was diluted to 20 ml yield at room temperature with the g a m m a rays of 400/~g/ml, passed through 0"45 /~m membrane filter (Millipore Filter Corp., Bedford, Mass., 161 rad/see. U.S.A.), dispensed as 10-ml samples into sterile Antibiotics vials, and frozen and kept stored at --15 to The sources of sensitivity discs were as --20°G. The vials were never refrozen after follows: rifamycin sv (R), and rifampicin (RA) they had been thawed. from Lepetit, spa-Milan, Italy; cloxacillin (CX), ampicillin (AP), earbencillin (CC), Antibiotic susceptibility tests aminosidine sulphate (AS) and gentamiein Disc diffusion susceptibility tests were persulphate (GM), from Mast Laboratories Ltd., formed by the technique of BAUER et al., ~3) Liverpool, England; cefalotin (GF). from Eli with the exception that the large plates (100 Lilly & Co., Indianapolis, U.S.A.; tetracyclin by 15 mm) contained 6 0 m l of T G Y agar, (TG), chloramphenicol (GM) and penicillin resulting in an agar depth of roughly 4 mm, (s) (P), from Oxoid Limited, London, England. and the size of inoculum for each strain was The sources of antibiotics were as follows: determined ( R I = 6 × 105, 6 X 106 cells/ml) rifamycin sv and rifampicin, from Lepetit, and RII5 = 4'2 × 105, 4'2 × 108 cells/ml). spa-Milan, Italy; cloxacillin and carbencillin, Duplicate discs of eleven antibiotics (R, Cx, from Beecham Research Laboratories, Brent- RA, AP, AS, CC, GM, CF, TC, P and CM) ford, England; aminosidine sulphate, from were used with each strain. The plates were Soc. Farmaceutici Italia, Milano, Italy; chlor- incubated for 24 hr and the inhibition zones amphenicol, from Carlo Erba, Milano, Italy; were then measured. Simultaneous broth cefalotin, from Eli Lilly & Co., Indianapolis, dilution tests were performed with the bacterial U.S.A. ; ampicillin, from Ankerfarm spa, inocula adjusted approximately in the range Cinisello B., Milan, Italy; penicillin G sodium 1 to 6 × 104 cells/ml. The strains were exposed salt and tetraeyclin, Laboratory standards, to serial twofold dilutions of ten antibiotics from State C o m p a n y for Drugs Industries, (Tables 1 and 2); 200, 100, 50, 25, 12"5, Samrra, Iraq. The above-mentioned anti- 6"3, 3"2, 1"6, 0"8 and 0"4 #g/ml (final concentrabiotics except penicillin and tetracyclin were tions). The (MIC) of each of the antibiotics obtained commercially. Dilution of 1000/~g/ml was read after overnight incubation at 30°C TABLE 1. Antibiotic susceptibility of M radiodurans R1 and RII5 irradiated at doses of 500 and 750 krad MIC (/~g/ml) M radiodurans Ru 5
M radiodurans R1
irradiated at doses Antibiotic Rifamycin sv Cloxacillin Rifampicin Ampicillin Aminosidine sulphate Carbencillin Cefalotin Tetracyclin HC1 Penicillin G Ghloramphenicol
0.0 krad
500 krad
750 krad
size of inoculum cells/ml
6-3 1-6 3.2 0-4* 6.3 25.0 1.6 0.4* 0-4* 25.0
1.6 0.8 1.6 0.4* 1.6 3.2 0-8 0.4* 0.4* 12.5
0.8 0.8 0.8 0.4* 0-8 3.2 0'8 0.4* 0-4* 6.3
1-0 1.6 6.0 1.0 1.0 4-0 1.6 2.1 2.0 1.0
* The MIG is less than 0.4 pg/ml.
× × × x × × × × × ×
104 104 104 104 104 104 104 104 10~ 104
irradiated at doses 0.0 krad
500 krad
750 krad
size of inoculum cells/ml
6.3 1.6 3.2 0-4* 3.2 6.3 0-8 0.4* 0-4* 25.0
1.6 0-8 1.6 0.4* 1.6 3.2 0.4 0.4* 0.4* 25.0
0.8 0.8 0-8 0.4* 1.6 3.2 0.4 0.4* 0.4* 12.5
1.2 1.2 4.0 3.8 1.0 1.0 1.8 5.0 2-1 1.4
× × x × × × × × x ×
104 104 104 104 104 104 104 104 104 104
Effect of radiation on antibiotic sensitivity of Micrococcus radiodurans
389
TABLE 2. T h e m i n i m a l bactericidal c o n c e n t r a t i o n (MBC) of antibiotics against M radiodurans Rt a n d RII5 i r r a d i a t e d at doses o f 500 a n d 750 k r a d /vIBC (/~g/ml)
Antibiotic R i f a m y c i n sv Cloxacillin Rifampiein Ampicillin A m i n o s i d i n e sulphate Carbencillin Cefalotin T e t r a c y c l i n HC1 Penicillin G Chlorarnphenicol
M radiodurans RI
M radiodurans RII5
i r r a d i a t e d at doses
i r r a d i a t e d at doses
0-0 krad
500 krad
750 krad
size of inoculum cells]ml
0.0 krad
500 krad
750 krad
size of inoculum eells]ml
200* 50 200 50 25 200* 200 200 50 200*
200* 25 50 50 25 200* 100 100 200 200*
50 25 50 50 12.5 200* 100 100 50 200*
1.1 1.6 6.0 1.0 I-0 4,0 1.6 2.1 2-0 1-I
200* 25 200* 100 25 200* 200 200 50 200*
203 12.5 50 100 12.5 200* 50 200 200 200*
100 12.5 25 100 12.5 200* 50 200 100 200*
1.2 1.2 4.0 3.8 1.0 1.0 1.8 5.1 2-1 1.4
x
x × × × x × x x ×
10a 104 104 104 104 104 104 104 104 10 *
x
× x × × x × × x ×
104 104 104 104 104 104 10 ~ 104 104 104
* T h e M B C is m o r e t h a n 200/*g]ml. as t h e l o w e s t c o n c e n t r a t i o n o f a n t i b i o t i c t h a t inhibited visible growth. The (MBC) was d e f i n e d as t h e l o w e s t c o n c e n t r a t i o n o f a n t i b i o t i c w h e r e 0' 1 m l s u b c u l t u r e o n t o a g a r p l a t e s showed no growth at 48 hr. The growth rates of irradiated and nonirradiated of both R I and Rn5 were determined.
RESULTS
AND DISCUSSION
The effect of radiation and size of inoculum on susceptibility of M radiodurans R I and RII5 to eleven antibiotics (disc diffusion techniques) is shown in Figs. 1 and 2. The potencies of antibiotics (#g/disc) were as follows: R, 30; (b)
(0) 7O
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CK RA A P AS CC GM CF TC CM P
Dose (o = O.0, A = 150, V = 3 0 0 , × = . 5 0 0 , [] = 7 5 0 kred )
FIe. 1. Effect of r a d i a t i o n of i n o c u l u m o n susceptibility of M radiodurans strain R1 to eleven a n t i b i o t i c (a = 6.0 / 105, b = 6-0 × 106 cells]ml).
390
N. Agha, F. M. Salih, H. Auda and S. AI-Barwari
(b)
(a)
voj 6O [3 "O [] []
)
