Journal of Antimicrobial Chemotherapy (1992) 30, Suppl. A. 95-99
In-vitro and in-vivo synergic activity and fractional inhibitory concentration (FIQ of the components of a semisynthetic streptogramin, RP 59500 D. H. Boaaocbaod Biology Department, Rhdne-Poulenc Rorer S. A., 94403 Vitry-sw-Seine, France
Introduction RP 59500, a new semisynthetic streptogramin, is a combination of two semisynthetic compounds: RP 57669 (referred to as RPI) and RP 54476 (referred to as RPII) combined in a 30:70 ratio. The two constituent compounds of RP 59500 are not metabolized at the same rate; therefore, their proportions in the body do not remain the same over time, which may affect the antibacterial properties of the combination. The goal of the present study was to determine, in vitro and in vivo, the proportions at which RPI and RPII exhibit synergy in their action against Staphylococcus aureus and Streptococcus pneumoniae. For this purpose we found it useful to calculate the fractional inhibitory concentration ( F I Q index, defined by Elion, Singer & Hitchings (1954) and applied by Krogstad & Moellering (1986) to the study of antimicrobial combinations. Materials and methods Bacterial strains Fourteen strains of S. aureus were clinical isolates obtained from several French hospitals; strain Smith was obtained from the collection of Institut Pasteur (Paris). Five 0305-7453/92/30A095+05 $03.00/0
95 © 1992 The British Society for Antimicrobial Chemotherapy
Downloaded from http://jac.oxfordjournals.org/ by guest on December 3, 2014
RP 59500 is a new semisynthetic injectable streptogramin antibiotic composed of two compounds which interact synergically, RP 57669 and RP 54476, derived from pristinamycin IA and pristinamycin IIB, respectively. The bacteristatic and bactericidal activities of RP 57669 and RP 54476 alone or combined in various proportions were tested by the chequerboard dilution technique. The fractional inhibitory concentration (FIQ index was determined for 14 Staphylococcus aureus isolates (including methicdllin- and macrolide-resistant strains) and one culture collection strain. The F1C index was found to be much lower than 0-5, indicating the presence of synergy for all strains tested, whatever their resistance pattern. The EDW of RP 57669 and RP 54476 in various combinations were also determined in three experimental murine models of septicaemia, caused by either S. aureus or Streptococcus pneumoniae, and a thigh abscess model caused by S. aureus. The combinations which performed best in the model of septicaemia were those in which the RP 57669: RP 54476 ratio ranged from 16:84 to 92:8, while those active against the thigh abscess model had ratios ranging from 8:92 to 84:16. That the drugs were active over a wide range of ratios suggests that synergy will be maintained even if one drug is cleared more rapidly than the other. The combination of 30:70, referred to as RP 59500, was selected for further studies, both in vitro and in various experimental models of infections.
96
D. H. Boaandumd
strains were methicillin-susceptiblc S. aureus (MSSA) that were susceptible to macrolides, lincosamides and streptogramin B (MLSB); five were methicillin-resistant S. aureus (MRSA) with induciblc resistance to MLS^ and five were MRSA with constitutive resistance to MLS^ S. pneumoniae strain 4552/03 was obtained from the French Pneumococcal Reference Centre, Creteil, France (courtesy of Dr. P. Geslin). Media and growth conditions S. aureus strains were grown in Muellcr-Hinton broth or agar and were incubated aerobically for 18 h. S. pneumoniae was grown in Mucller-Hinton broth and agar, supplemented with 10% human ascitic fluid, and incubated in a 5% CO2-enriched atmosphere.
RPI, RPII and RP 59500 methane sulphonate (Rhone-Poulenc Rorer) were diluted in sterile distilled water, and aliquots of 1 mL (50 mg/L) were stored at — 80°C before use. MICs and FICs MICs were determined by an agar dilution method (Ericsson & Sherris, 1971), in which a Denley Multipoint Inoculator was used to apply approximately 10* cfu of each bacterial strain per spot. Plates were incubated at 35 to 37°C, with added CO2 for the S. pneumoniae strain. The FIC index was determined by the chequerboard dilution technique (Krogstad & Moellering, 1986), with concentrations of RPI and RPII ranging from 0-06 to 64 mg/L. The respective concentrations of RPI and RPII were: 0:100, 1:99, 8:92, 16:84, 32:68, 68:32, 84:16, 92:8, 99:1, and 100:0; i.e. ten combinations. An inoculum in logarithmic phase and appropriate dilutions of antimicrobials were added to Mueller-Hinton broth in the wells of microtitre plates, so as to obtain 2x 10s cfu/mL. Plates were incubated for 24 h at 37°C. The FIC for each component was derived by dividing the concentration of the component necessary to inhibit growth when the two components were combined by the MIC of that component when it was tested alone; the FIC index was calculated by summing the separate FICs for each component, according to the equation (Krogstad & Moellering, 1986): (A) in the presence of B + (B) in the presence of A = (MICJ (MICB)
n c
nc^
= n c
indcx
Animal experiments Swiss Mice (average weight: 20 g) were used in animal models of septicaemia and intramuscular abscess. In the case of the model of septicaemia, mice were injected intraperitoneally either with MLSB-susceptible MSSA (strain Smith) in a 5% w/v mucin suspension, or with S. pneumoniae suspended in normal saline. Mice were treated subcutaneously twice a day for two days, and ED M were calculated seven days after infection as described by Miraglia (1976). Thigh abscesses were induced by injecting an inoculum of either MLSB-susceptible MSSA or MLSB-constitutively resistant MRSA into the right thigh muscle (Rolin,
Downloaded from http://jac.oxfordjournals.org/ by guest on December 3, 2014
Antibiotics
RP 59500 synergic activity
97
Huet & Bouanchaud, 1986). Mice used in this model were treated by subcutaneous administration of the antibiotic 1 and 6 h after infection. Ratios of RPI: RPII used were: 0:100, 8:92, 16:84, 32:68, 68:32, 84:16, 92:8 and 100:0. Ten days after the last treatment, the mice were killed and examined for the presence of septic abscess. The number of viable bacteria in muscle tissue was counted and the EDM was calculated, as described previously. Experiments have been carried out to conform with directive EEC/86/606 concerning the protection of animals used for experimental scientific purposes. Results In-vitro testing
Table I. Inhibitory activity of RPI, RPII and RP 595O0, together with the FIC index for the combination, against 15 strains of 5. aureus representing three groups of susceptibility to methitillin and MLS, antibiotics Phenotype"
Strains
RPI
M(S) MLSB(S)
Smi 133 CN491 ROG WEI
05
M(R)
MLSBO)
M(R) MLSB(C)
1 1
1 1
MICS (mg/L) RPII
RP 59500
FIC index*
2 2 2 4 4
012 025 012 025 025
012 012 006 006 006
006 012 006 012 012
009 0045 018 006 004
025 O50 025 050 050
009 004 004 004 003
PRI FAU DEA MAI VDG261
2 2 1 2 8
4 2 2 4
DUC LEF VDG262 SCH BUY
30 30 30 30 60
2 2 4 2
4
4
*M(S) MLS^S) = MethkilliD-scnsilive, MLS, sensitive: M(R) MLS^I) « methkalUn-resistaiit, MLS, inducibly resistant; M(R) MLS^C) - methicillin-renstant, MLS, constitutively resistant. *Synergy it defined as an FIC index of < 05.
Downloaded from http://jac.oxfordjournals.org/ by guest on December 3, 2014
The MICs of RPI, RPII and RP 59500 for the 15 5. aureus strains tested are presented in Table I. The MICs of RPI ranged from 0-5 to 60 mg/L. For the MSSA MLSB-susceptible and the MRSA MLSB-inducibly resistant strains, the RPI MIC range was 0-5-1 and 1-8 mg/L, respectively, whereas the MIC range for MRSA strains constitutively resistant to MLSB antibiotics was 30-60 mg/L. The range of MICs of RPII was much more restricted; this compound inhibited the growth of all S. aureus strains examined at concentrations ranging from 2 to 4 mg/L. The MICs of RP 59500 ranged from 0-06 mg/L (for two MRSA, MLS,,-indurible strains) to 0-5 mg/L (for three MRSA, MLSo-constitutive strains). Five strains had MICs of RP 59500 of 0-12 and five had MICs of 0-25 mg/L. All the strains examined had FIC indices of < 0-5, indicating that the combination of RPI and RPII was, in each case, synergic. The
98
D. H. Bonancfaand
Table H. Geometric mean MIC (mg/L) of various proportions of RPI and RPII forfivestrains of sensitive S. aureus (S), five strains of MRSA with inducible MLSB resistance (I) and five strains of MRSA with constitutive resistance ( Q to MLS, antibiotics Phenotype
0:100
1:99
8:92
Ratio of RPI:RPII 16:84 32:68 68:32 84:16 92:8
99:1
100:0
2 4 4
1 2 2
0-25 0-5 1
0-12 0-5 1
0-25 1 1
0-5 1 125
MSSA, MLSB(S) MRSA, MLS,(I) MRSA, MLSB(C)
0-12 0-5 0-5
0-12 0-25 0-5
0-12 0-25 0-5
0-12 1 1
In-vivo testing The results obtained with the septicaemia and the thigh abscess models, caused by S. aureus or S. pneumoniae, are presented in Table III. The lowest ED,,, that conferred protection against both 5. aureus-induced and S. pneumoniae-induccd septicaemia were seen with RPI: RPII ratios of between 16:84 and 84:16, inclusive. For S. aureus-induced septicaemia, ED^s less than or equal to 30 mg/kg were obtained with all the combinations tested, except the 92:8 ratio, where the ED,, was 44 mg/kg. For S. pneumoniae-induced septicaemia. ED^s lower than 30 mg/kg were obtained with RPI: RPII ratios ranging from 16/84 to 92/8. For thigh abscesses caused by MLSB-sensitive MSSA, the EDjoS obtained with RPI and RPII in ratios of 16:84 to 84:16, inclusive, were lower than 15 mg/kg. The lowest EDJO for thigh abscesses caused by MRSA constitutively-resistant to the MLSB antibiotics was 22 mg/kg, obtained with the RPI: RPII ratio of 68:32. ED^s lower than 40 mg/kg were obtained with ratios ranging from 8/92 to 84/16 for infections caused by this bacterial strain.
Table IIL Results of protection studies in the mouse septicaemia and abscess models with various ratios of RPI and RPII EDJO (mg/kg)
Strains and phenotypes
0:100
8:92
ratios of RPI: RPII 16:84 32:68 68:32 84:16
S. aureus and S. pneumoniae septicaemia model MSSA, MLSB(S) > 300 16 20 S. pneumoniae > 150 95 10
20 9
28 11
30 18
92:8
100:0
14 25
— 150
22
—
S. aureus thigh abscess model MSSA,MLSB(S) MRSA, MLSB(C)
150
29
11
8-5
10
14
95
34
30
24
22
38
S, Sensitive; C, ConjtitutiveJy resistant.
> 125 > 125
Downloaded from http://jac.oxfordjournals.org/ by guest on December 3, 2014
bacteristatic effect of RPI and RPII, combined in proportions pf 1:99, 8:92, 16:84, 32:68, 68:32, 84:16, 92:8 and 99:1 was evaluated by determining their MICs for the 15 S. aureus strains. The MICs of each component alone were also determined. The results are shown in Table II. The MICs ranged from 012 to 8-0 mg/L. The lowest MICs (0-12-0.5 mg/L) were observed with RPI/RPII ratios of 32/64, 68/32 and 84/16.
RP 59500 synergic activity
99
Discussion
Acknowledgements The author thanks K. Pepper for her help with preparation of the manuscript, and J. J. Garaud for helpful discussions. I acknowledge the expert technical help of F. Duvoux, M. Godreau, Y. Huet, J. J. Rousseau-Blanchery, M. Selingue and J. Thomas-Martin. References Elion, G. B., Singer, S. & Hitchings, G. H. (1954). Antagonists of nucleic acid derivatives. VIII. Synergism in combination of biochemically related antimetabolites. Journal of Biological Chemistry 208, 477-38. Ericsson, H. M. & Sherris, J. C. (1971). Antibiotic sensitivity testing. Acta Pathologica el Microbiologica Scandinavica, Suppl. 217, 1-90. Krogstad, D. J. & Moellering, R. C. (1986). Antimicrobial combinations. In Antibiotics in Laboratory Medicine (Lorian, V., Ed.), pp. 537-95. Williams & Wilkins, Baltimore, MD. Miraglia, G. J. (1976). Systemic models of bacterial infections. In Chemotherapy of Infectious Diseases (Gadebush, H. H., Ed.), pp. 1-19. CRC Press, London. Rolin, O., Huet, Y. & Bouanchaud, D. H. (1986). Comparative efficacy of pefloxacin and six other antimicrobial agents on Staphylococcus aureus experimental abscesses. Journal of Antimicrobial Chemotherapy 17, Suppl. B, 49-52.
Downloaded from http://jac.oxfordjournals.org/ by guest on December 3, 2014
. This study was conducted to determine whether the two components of RP 59500, the first injectable streptogramin, act synergically in vitro and in vivo, and within what range of ratios their synergy is maintained. Separately, the two components, RPI and RPII, were effective bacteristatic agents against the MRSA strains tested. Not surprisingly, MRSA strains with constitutive resistance to the MLSj, antibiotics displayed resistance to RPI (MICs ^ 30 mg/L). Synergy between antibiotics in vitro is a phenomenon mainly found at concentrations close to the MIC. This was true with RPI and RPII for strains sensitive to MLSg, or strains with inducible MLS,, resistance, but not for strains with constitutive resistance. High level RPI resistance did not reduce significantly the activity of RPI:RPII in combination, and in fact, for each strain examined, the FIC index was < 0-5. In vivo, the components of RP 59500 may display distinct pharmacokinetic characteristics; in independent studies (data not shown), it has been shown that RPII is cleared more rapidly than RPI from mouse serum. That the combination of RPI and RPII remained synergic in vitro and in vivo, even as their ratio varied over a wide range (i.e. from 16:84 to 84:16), is significant for the eventual clinical application of RP 59500.
In-vitro and in-vivo synergic activity and fractional inhibitory concentration (FIQ of the components of a semisynthetic streptogramin, RP 59500 D. H. Boaaocbaod Biology Department, Rhdne-Poulenc Rorer S. A., 94403 Vitry-sw-Seine, France
Introduction RP 59500, a new semisynthetic streptogramin, is a combination of two semisynthetic compounds: RP 57669 (referred to as RPI) and RP 54476 (referred to as RPII) combined in a 30:70 ratio. The two constituent compounds of RP 59500 are not metabolized at the same rate; therefore, their proportions in the body do not remain the same over time, which may affect the antibacterial properties of the combination. The goal of the present study was to determine, in vitro and in vivo, the proportions at which RPI and RPII exhibit synergy in their action against Staphylococcus aureus and Streptococcus pneumoniae. For this purpose we found it useful to calculate the fractional inhibitory concentration ( F I Q index, defined by Elion, Singer & Hitchings (1954) and applied by Krogstad & Moellering (1986) to the study of antimicrobial combinations. Materials and methods Bacterial strains Fourteen strains of S. aureus were clinical isolates obtained from several French hospitals; strain Smith was obtained from the collection of Institut Pasteur (Paris). Five 0305-7453/92/30A095+05 $03.00/0
95 © 1992 The British Society for Antimicrobial Chemotherapy
Downloaded from http://jac.oxfordjournals.org/ by guest on December 3, 2014
RP 59500 is a new semisynthetic injectable streptogramin antibiotic composed of two compounds which interact synergically, RP 57669 and RP 54476, derived from pristinamycin IA and pristinamycin IIB, respectively. The bacteristatic and bactericidal activities of RP 57669 and RP 54476 alone or combined in various proportions were tested by the chequerboard dilution technique. The fractional inhibitory concentration (FIQ index was determined for 14 Staphylococcus aureus isolates (including methicdllin- and macrolide-resistant strains) and one culture collection strain. The F1C index was found to be much lower than 0-5, indicating the presence of synergy for all strains tested, whatever their resistance pattern. The EDW of RP 57669 and RP 54476 in various combinations were also determined in three experimental murine models of septicaemia, caused by either S. aureus or Streptococcus pneumoniae, and a thigh abscess model caused by S. aureus. The combinations which performed best in the model of septicaemia were those in which the RP 57669: RP 54476 ratio ranged from 16:84 to 92:8, while those active against the thigh abscess model had ratios ranging from 8:92 to 84:16. That the drugs were active over a wide range of ratios suggests that synergy will be maintained even if one drug is cleared more rapidly than the other. The combination of 30:70, referred to as RP 59500, was selected for further studies, both in vitro and in various experimental models of infections.
96
D. H. Boaandumd
strains were methicillin-susceptiblc S. aureus (MSSA) that were susceptible to macrolides, lincosamides and streptogramin B (MLSB); five were methicillin-resistant S. aureus (MRSA) with induciblc resistance to MLS^ and five were MRSA with constitutive resistance to MLS^ S. pneumoniae strain 4552/03 was obtained from the French Pneumococcal Reference Centre, Creteil, France (courtesy of Dr. P. Geslin). Media and growth conditions S. aureus strains were grown in Muellcr-Hinton broth or agar and were incubated aerobically for 18 h. S. pneumoniae was grown in Mucller-Hinton broth and agar, supplemented with 10% human ascitic fluid, and incubated in a 5% CO2-enriched atmosphere.
RPI, RPII and RP 59500 methane sulphonate (Rhone-Poulenc Rorer) were diluted in sterile distilled water, and aliquots of 1 mL (50 mg/L) were stored at — 80°C before use. MICs and FICs MICs were determined by an agar dilution method (Ericsson & Sherris, 1971), in which a Denley Multipoint Inoculator was used to apply approximately 10* cfu of each bacterial strain per spot. Plates were incubated at 35 to 37°C, with added CO2 for the S. pneumoniae strain. The FIC index was determined by the chequerboard dilution technique (Krogstad & Moellering, 1986), with concentrations of RPI and RPII ranging from 0-06 to 64 mg/L. The respective concentrations of RPI and RPII were: 0:100, 1:99, 8:92, 16:84, 32:68, 68:32, 84:16, 92:8, 99:1, and 100:0; i.e. ten combinations. An inoculum in logarithmic phase and appropriate dilutions of antimicrobials were added to Mueller-Hinton broth in the wells of microtitre plates, so as to obtain 2x 10s cfu/mL. Plates were incubated for 24 h at 37°C. The FIC for each component was derived by dividing the concentration of the component necessary to inhibit growth when the two components were combined by the MIC of that component when it was tested alone; the FIC index was calculated by summing the separate FICs for each component, according to the equation (Krogstad & Moellering, 1986): (A) in the presence of B + (B) in the presence of A = (MICJ (MICB)
n c
nc^
= n c
indcx
Animal experiments Swiss Mice (average weight: 20 g) were used in animal models of septicaemia and intramuscular abscess. In the case of the model of septicaemia, mice were injected intraperitoneally either with MLSB-susceptible MSSA (strain Smith) in a 5% w/v mucin suspension, or with S. pneumoniae suspended in normal saline. Mice were treated subcutaneously twice a day for two days, and ED M were calculated seven days after infection as described by Miraglia (1976). Thigh abscesses were induced by injecting an inoculum of either MLSB-susceptible MSSA or MLSB-constitutively resistant MRSA into the right thigh muscle (Rolin,
Downloaded from http://jac.oxfordjournals.org/ by guest on December 3, 2014
Antibiotics
RP 59500 synergic activity
97
Huet & Bouanchaud, 1986). Mice used in this model were treated by subcutaneous administration of the antibiotic 1 and 6 h after infection. Ratios of RPI: RPII used were: 0:100, 8:92, 16:84, 32:68, 68:32, 84:16, 92:8 and 100:0. Ten days after the last treatment, the mice were killed and examined for the presence of septic abscess. The number of viable bacteria in muscle tissue was counted and the EDM was calculated, as described previously. Experiments have been carried out to conform with directive EEC/86/606 concerning the protection of animals used for experimental scientific purposes. Results In-vitro testing
Table I. Inhibitory activity of RPI, RPII and RP 595O0, together with the FIC index for the combination, against 15 strains of 5. aureus representing three groups of susceptibility to methitillin and MLS, antibiotics Phenotype"
Strains
RPI
M(S) MLSB(S)
Smi 133 CN491 ROG WEI
05
M(R)
MLSBO)
M(R) MLSB(C)
1 1
1 1
MICS (mg/L) RPII
RP 59500
FIC index*
2 2 2 4 4
012 025 012 025 025
012 012 006 006 006
006 012 006 012 012
009 0045 018 006 004
025 O50 025 050 050
009 004 004 004 003
PRI FAU DEA MAI VDG261
2 2 1 2 8
4 2 2 4
DUC LEF VDG262 SCH BUY
30 30 30 30 60
2 2 4 2
4
4
*M(S) MLS^S) = MethkilliD-scnsilive, MLS, sensitive: M(R) MLS^I) « methkalUn-resistaiit, MLS, inducibly resistant; M(R) MLS^C) - methicillin-renstant, MLS, constitutively resistant. *Synergy it defined as an FIC index of < 05.
Downloaded from http://jac.oxfordjournals.org/ by guest on December 3, 2014
The MICs of RPI, RPII and RP 59500 for the 15 5. aureus strains tested are presented in Table I. The MICs of RPI ranged from 0-5 to 60 mg/L. For the MSSA MLSB-susceptible and the MRSA MLSB-inducibly resistant strains, the RPI MIC range was 0-5-1 and 1-8 mg/L, respectively, whereas the MIC range for MRSA strains constitutively resistant to MLSB antibiotics was 30-60 mg/L. The range of MICs of RPII was much more restricted; this compound inhibited the growth of all S. aureus strains examined at concentrations ranging from 2 to 4 mg/L. The MICs of RP 59500 ranged from 0-06 mg/L (for two MRSA, MLS,,-indurible strains) to 0-5 mg/L (for three MRSA, MLSo-constitutive strains). Five strains had MICs of RP 59500 of 0-12 and five had MICs of 0-25 mg/L. All the strains examined had FIC indices of < 0-5, indicating that the combination of RPI and RPII was, in each case, synergic. The
98
D. H. Bonancfaand
Table H. Geometric mean MIC (mg/L) of various proportions of RPI and RPII forfivestrains of sensitive S. aureus (S), five strains of MRSA with inducible MLSB resistance (I) and five strains of MRSA with constitutive resistance ( Q to MLS, antibiotics Phenotype
0:100
1:99
8:92
Ratio of RPI:RPII 16:84 32:68 68:32 84:16 92:8
99:1
100:0
2 4 4
1 2 2
0-25 0-5 1
0-12 0-5 1
0-25 1 1
0-5 1 125
MSSA, MLSB(S) MRSA, MLS,(I) MRSA, MLSB(C)
0-12 0-5 0-5
0-12 0-25 0-5
0-12 0-25 0-5
0-12 1 1
In-vivo testing The results obtained with the septicaemia and the thigh abscess models, caused by S. aureus or S. pneumoniae, are presented in Table III. The lowest ED,,, that conferred protection against both 5. aureus-induced and S. pneumoniae-induccd septicaemia were seen with RPI: RPII ratios of between 16:84 and 84:16, inclusive. For S. aureus-induced septicaemia, ED^s less than or equal to 30 mg/kg were obtained with all the combinations tested, except the 92:8 ratio, where the ED,, was 44 mg/kg. For S. pneumoniae-induced septicaemia. ED^s lower than 30 mg/kg were obtained with RPI: RPII ratios ranging from 16/84 to 92/8. For thigh abscesses caused by MLSB-sensitive MSSA, the EDjoS obtained with RPI and RPII in ratios of 16:84 to 84:16, inclusive, were lower than 15 mg/kg. The lowest EDJO for thigh abscesses caused by MRSA constitutively-resistant to the MLSB antibiotics was 22 mg/kg, obtained with the RPI: RPII ratio of 68:32. ED^s lower than 40 mg/kg were obtained with ratios ranging from 8/92 to 84/16 for infections caused by this bacterial strain.
Table IIL Results of protection studies in the mouse septicaemia and abscess models with various ratios of RPI and RPII EDJO (mg/kg)
Strains and phenotypes
0:100
8:92
ratios of RPI: RPII 16:84 32:68 68:32 84:16
S. aureus and S. pneumoniae septicaemia model MSSA, MLSB(S) > 300 16 20 S. pneumoniae > 150 95 10
20 9
28 11
30 18
92:8
100:0
14 25
— 150
22
—
S. aureus thigh abscess model MSSA,MLSB(S) MRSA, MLSB(C)
150
29
11
8-5
10
14
95
34
30
24
22
38
S, Sensitive; C, ConjtitutiveJy resistant.
> 125 > 125
Downloaded from http://jac.oxfordjournals.org/ by guest on December 3, 2014
bacteristatic effect of RPI and RPII, combined in proportions pf 1:99, 8:92, 16:84, 32:68, 68:32, 84:16, 92:8 and 99:1 was evaluated by determining their MICs for the 15 S. aureus strains. The MICs of each component alone were also determined. The results are shown in Table II. The MICs ranged from 012 to 8-0 mg/L. The lowest MICs (0-12-0.5 mg/L) were observed with RPI/RPII ratios of 32/64, 68/32 and 84/16.
RP 59500 synergic activity
99
Discussion
Acknowledgements The author thanks K. Pepper for her help with preparation of the manuscript, and J. J. Garaud for helpful discussions. I acknowledge the expert technical help of F. Duvoux, M. Godreau, Y. Huet, J. J. Rousseau-Blanchery, M. Selingue and J. Thomas-Martin. References Elion, G. B., Singer, S. & Hitchings, G. H. (1954). Antagonists of nucleic acid derivatives. VIII. Synergism in combination of biochemically related antimetabolites. Journal of Biological Chemistry 208, 477-38. Ericsson, H. M. & Sherris, J. C. (1971). Antibiotic sensitivity testing. Acta Pathologica el Microbiologica Scandinavica, Suppl. 217, 1-90. Krogstad, D. J. & Moellering, R. C. (1986). Antimicrobial combinations. In Antibiotics in Laboratory Medicine (Lorian, V., Ed.), pp. 537-95. Williams & Wilkins, Baltimore, MD. Miraglia, G. J. (1976). Systemic models of bacterial infections. In Chemotherapy of Infectious Diseases (Gadebush, H. H., Ed.), pp. 1-19. CRC Press, London. Rolin, O., Huet, Y. & Bouanchaud, D. H. (1986). Comparative efficacy of pefloxacin and six other antimicrobial agents on Staphylococcus aureus experimental abscesses. Journal of Antimicrobial Chemotherapy 17, Suppl. B, 49-52.
Downloaded from http://jac.oxfordjournals.org/ by guest on December 3, 2014
. This study was conducted to determine whether the two components of RP 59500, the first injectable streptogramin, act synergically in vitro and in vivo, and within what range of ratios their synergy is maintained. Separately, the two components, RPI and RPII, were effective bacteristatic agents against the MRSA strains tested. Not surprisingly, MRSA strains with constitutive resistance to the MLSj, antibiotics displayed resistance to RPI (MICs ^ 30 mg/L). Synergy between antibiotics in vitro is a phenomenon mainly found at concentrations close to the MIC. This was true with RPI and RPII for strains sensitive to MLSg, or strains with inducible MLS,, resistance, but not for strains with constitutive resistance. High level RPI resistance did not reduce significantly the activity of RPI:RPII in combination, and in fact, for each strain examined, the FIC index was < 0-5. In vivo, the components of RP 59500 may display distinct pharmacokinetic characteristics; in independent studies (data not shown), it has been shown that RPII is cleared more rapidly than RPI from mouse serum. That the combination of RPI and RPII remained synergic in vitro and in vivo, even as their ratio varied over a wide range (i.e. from 16:84 to 84:16), is significant for the eventual clinical application of RP 59500.
