Journal of Antimicrobial Chemotherapy (1992) 30, Suppl. A, 19-23
In-vitro antibacterial activity of RP 59500, a semisyntfaetic streptogramin, against Streptococcus pneumoniae A. Fremanx, G. Sissia, R. Cohen and P. Geslin Centre
The in-vitro activity of the new streptogramin RP 59500 was determined against 100 strains of Streptococcus pneumoniae. In all, 48 erythromycin-sensitive strains and 52 erythromycin-resistant strains were tested by an agar dilution method (MIC determination), 20 strains (eight erythromycin-sensitive and 12 erythromycin-resistant) were tested by a broth microdilution technique (MIC and MBC determination) and ten strains (two erythromycin-sensitive and eight erythromycin-resistant) were tested for bactericidal kinetics. The study showed that RP 59500 had good bacteristatic and bactericidal activity against both erythromycin-sensitive and erythromycinresistant strains. On the basis of these results, and because the number of erythromycin-resistant strains of S. pneumoniae isolated in France has increased steadily since 1985, RP 59500 might be useful for the treatment of pneumococcal infections and warrants further investigation. Introduction Until recently the macrolides, a group of safe and effective antibiotics, have been useful as first-line treatment for respiratory tract infections (Societe Francaise de la Tuberculose et des Maladies Respiratoires, 1981). However, since 1985 the increase in erythromycin-resistant strains of 5. pneumoniae isolated from different hospitals in France has been a problem limiting the effectiveness of these antibiotics (Duval, 1985; Geslin, 1986). In contrast, streptogramins are still effective against S. pneumoniae (Emond et al., 1989). RP 59500 is a new injectable streptogramin which is reported to have activity against Gram-positive bacteria. The purpose of the present work was to study the in-vitro antibacterial activity of RP 59500 against erythromycin-sensitive (ES) and -resistant strains (ER) of S. pneumoniae by determining MICs and MBCs, together with the kinetics of bacterial killing by RP 59500. Materials and methods Bacterial strains and
antibiotics
One hundred strains of S. pneumoniae, serotyped by latex agglutination (Table I), were isolated from blood culture (57), CSF (17), middle ear aspirate (17), pleural fluid (6), gastric fluid (1), peritoneal fluid (1) and vaginal swab (1). These strains, isolated between 1986 and 1989, were sent by hospitals involved in a national co-operative survey of pneumococcal infections. When tested by the disc diffusion method, 48 strains were susceptible to erythromycin and 52 strains were resistant. MICs and MBCs 0305-7453/92/30A019+05 $03.00/0
19 © 1992 The British Society for Antimicrobial Chemotherapy
Downloaded from http://jac.oxfordjournals.org/ at Carleton University on June 23, 2015
Centre National de Reference des Pneumocoques, Laboratoire de Microbiologie, Hospitalier Intercommunal, Creteil, France
20
A. Frananx et aL Table L Serotype and group distribution of 100 strains of S. pneumonias Serotype
Serotype
No. of strains
5
14 1SB 17 A 18 C 19 A 19 F 20 22F 23 A 23F 24 F 33 F 34 38
18 1 1 2 4 13
4 3 1 6 9 4 i
I
2 1
were determined for 20 strains selected from these 100 isolates, of which eight were erythromycin-sensitive strains and 12 were erythromycin-resistant strains. Ten strains were selected (two erythromycin-sensitive and eight erythromycin-resistant strains) to investigate the kinetics of bactericidal activity. All strains were maintained frozen (—80°Q in Brain Heart Infusion broth containing glycerol 15% v/v. The antimicrobial agents used (and their respective manufacturers) were RP 59500 (Rhone-Poulenc Rorcr) and erythromycin (Abbott). MIC determinations by an agar dilution technique Strains were grown shaken in Brain Heart infusion broth supplemented with peritoneal fluid (10%) for 3 h at 37°C. A final inoculum of lOMO* cfu was delivered with a Steers replicator on to horse blood enriched Mueller-Hinton agar plates containing erythromycin or RP 59500 at concentrations in the range of 0-004 to 128 mg/L. The plates were incubated overnight at 37CC in an atmosphere with 5% CO2. Staphylococcus aureus strain ATCC 25293 was used as a control. The MIC was defined as the lowest concentration of antimicrobial agent which completely inhibited bacterial growth. MIC and MBC determinations by a broth microdilution technique Dilutions of antibiotic were prepared in Mueller-Hinton broth supplemented with 5% horse blood; 100 //L portions of the antibiotic dilutions were delivered to the wells of a microtitre plate, after which an inoculum of 10* cfu (10 uL) was added to each well. MICs were read after incubation for 18 h at 37°C in the presence of 5% COj. MBCs were determined after subculturing samples from each well by means of an inoculator pin (Dynatech) which delivered 10 uL portions on to blood agar plates. Plates were incubated overnight at 37°C in the presence of 5% CO3. The MBC was defined as the lowest concentration of antibiotic that yielded no growth.
Downloaded from http://jac.oxfordjournals.org/ at Carleton University on June 23, 2015
1 3 4 5 6A 6B 7F 8 9N 9V 10 A 10 F 11 A 12 F 13
No. of strains
Activity of RP 59500 tgainst S. pmemamdae
21
Tatde II. MICs of erythromycin and RP 59500 for 100 strains of S. pneumonku. as determined by the agar dilution technique Antibiotic (no. strains tested)
0O64 0-125
Erythromycin (100) RP 59500 (48 ES)
— —
17 —
RP 59500 (52 ER)
—
—
MIC (mg/L) 0-5 1
0-25 28 2
3 33
2 2 8
2
4
>128
— 12
— 1
— —
52 —
19
3
—
—
MICJO-O-5/MIQO-I-O
ES, erythromycin-tcnsitive strains; ER, erythromycin-resistant strains.
In-vitro bactericidal activity was determined by exposing a mid-log phase culture (103 cfu/mL) in Mueller-Hinton broth to 2 x MIC of RP 59500. After incubation for 4, 6 and 24 h at 37°C in an atmosphere with 5% CO2, appropriate dilutions of the culture were subcultured on blood agar plates to determine viable counts. Bactericidal activity was defined as a > 4 x logl0 reduction in the viable count of the culture when compared with the original inoculum. Results
The in-vitro inhibitory activity of RP 59500 and erythromycin, as determined by the agar dilution method, against 100 strains of 5. pnewnoniae is presented in Table II. The 48 erythromycin-sensitive strains had erythromycin MICs of < 0-5 mg/L, while the 52 erythromycin-resistant strains had erythromycin MICs of > 128 mg/L. In contrast, the RP 59500 MICs were between 0-25 and 2 mg/L for all tested strains. The MICj, and MIQo values for RP 59500 were 0-5 mg/L and 1 mg/L for erythromycin-sensitive and -resistant strains, respectively. MICs and MBCs of RP 59500 for 20 strains (eight erythromycin-sensitive and 12 erythromycin-resistant), as determined by a microdilution technique, were similar for 18 strains. For 15 strains, the MIC and MBC were both 1 mg/L, and a further three strains had MICs and MBCs of 2 mg/L. Of the remaining two strains, one had an MIC of 1 mg/L and an MBC of 2 mg/L, while the other had an MIC of 2 mg/L and an MBC of 4 mg/L (Table III). Table ID. MICs and MBCs of RP 59500 for 20 strains of S. pnewnoniae, as determined by the micro-dilution technique No. of strains MIC (mg/L)
MBC (mg/L) 0-5 1-0 2-0 4-0
0-5
1-0
20
15
1
3 1
40
Downloaded from http://jac.oxfordjournals.org/ at Carleton University on June 23, 2015
Bactericidal kinetics
22
A. Frananx et aL Table IV. Kinetics of bacterial killing by RP 59500 at 2 x MIC for ten isolates of S. pneumoniae
Strains
Type
Erythromycin-sensitive
18C
Erythromycin-resistant
6B 1 14
10 F
A F F A F
Bactericidal activity* 4h 6h 24 h
2 4 2 1 1 2 2 1 0-5
-4 -5 -5 -5 -5 -5 -3 -5 -5
-4 -5 -5 -5 -5 -5 -5 -5 -5
-4 -5 -5 -1 -5 -5 -5 -5 -5
4
-5
-5
-5
*Log,0 decrease in viable count after exposure for the indicated period.
The kinetics of bacterial killing by RP 59500, studied with ten strains (two erythromycin-sensitive and eight erythromycin-resistant) at 2 x MIC, are presented in Table IV. For the two erythromycin-sensitive strains, viable counts were reduced by approximately four to five logs after exposure for 4, 6 or 24 h. For six of the eight erythromycin-resistant strains, viable counts were reduced by > 4 logs after 4, 6 and 24 h. For all ten strains studied, RP 59500 at 2 x MIC was bactericidal after exposure for 6 h. Discussion
Pneumococcal resistance to macrolides, lincosamides and streptogramin B (MLSj resistance phenotype) was first detected in France in 1976 (Buu-Hoi, Goldstein & Acar, 1988), but by 1989 was found in 25% of clinical isolates, and 43-8% of strains isolated from patients with otitis media (Geslin, 1991). These data, and the more recent emergence of multiply-resistant strains, demonstrate the need for alternative antimicrobial therapy. The results of the present study with RP 59500 are in agreement with those reported previously by N'Guyen (1988). While the erythromycin MICs for the strains of S. pneumoniae studied allowed two separate populations (erythromycin-sensitive and erythromycin-resistant strains) to be distinguished, the MICs of RP 59500 were fairly homogeneous, ranging from 0-25-2 mg/L for both erythromycin-sensitive and -resistant strains. RP 59500 exhibited good bactericidal activity following exposure for 6 h at 2 x MIC for both erythromycin-sensitive and -resistant strains. We conclude that RP 59500 may be of value in the treatment of respiratory tract infections caused by resistant pneumococci. References Buu-Hoi, A. Y., Goldstein, F. W. & Acar, J. F. (1988). A seventeen-year epidemiologjcal survey of antimicrobial resistance in pneumococci in two hospitals. Journal of Antimicrobial Chemotherapy 22, Suppt. B, 41-52.
Downloaded from http://jac.oxfordjournals.org/ at Carleton University on June 23, 2015
19 19 19 6 23
RP 59500 MIC x 2 (mg/L)
Acdrtty of RP 59500 against 5. pneumonia*
23
Duval, J. (1985). Evolution and epidemiology of MLS resistance. Journal of Antimicrobial Chemotherapy 16, Suppl. A. 137-9. Emond, J. Ph., Frcmaux, A., Dublanchet, A., Sissia, G., Geslin, P. et al. (1989). Resistance of two strains of Streptococcus pneumoniae to pristinamycin associated with 16-membercd macrolides. Pathologie Biologie 37, 791-2. Geslin, P. (1986). Rapport du Centre National de Reference du Pneumocoque (Annce 1985). Bulletin Epidimiologique Hebdomadaire, number 50, 197-9. Geslin, P. (1991). Rapport du Centre National de Reference du Pneumocoque (Annee 1989). Bulletin Epidimiologique Hebdomadaire, number 2, 6-7. N'Guyen, J. (1988). Activite in vitro antipneumococcique des streptogramines. In Macrolides et Synergistines, pp. 115-8. Journies de I'Hdpital Claude-Bernard (Paris). Arnette. Societe Francaise de la Tuberculose et des Maladies Respiratoires (1981). Recommandations. Revue Franfaise des Maladies Respiratoires 9, 523-4. Downloaded from http://jac.oxfordjournals.org/ at Carleton University on June 23, 2015
In-vitro antibacterial activity of RP 59500, a semisyntfaetic streptogramin, against Streptococcus pneumoniae A. Fremanx, G. Sissia, R. Cohen and P. Geslin Centre
The in-vitro activity of the new streptogramin RP 59500 was determined against 100 strains of Streptococcus pneumoniae. In all, 48 erythromycin-sensitive strains and 52 erythromycin-resistant strains were tested by an agar dilution method (MIC determination), 20 strains (eight erythromycin-sensitive and 12 erythromycin-resistant) were tested by a broth microdilution technique (MIC and MBC determination) and ten strains (two erythromycin-sensitive and eight erythromycin-resistant) were tested for bactericidal kinetics. The study showed that RP 59500 had good bacteristatic and bactericidal activity against both erythromycin-sensitive and erythromycinresistant strains. On the basis of these results, and because the number of erythromycin-resistant strains of S. pneumoniae isolated in France has increased steadily since 1985, RP 59500 might be useful for the treatment of pneumococcal infections and warrants further investigation. Introduction Until recently the macrolides, a group of safe and effective antibiotics, have been useful as first-line treatment for respiratory tract infections (Societe Francaise de la Tuberculose et des Maladies Respiratoires, 1981). However, since 1985 the increase in erythromycin-resistant strains of 5. pneumoniae isolated from different hospitals in France has been a problem limiting the effectiveness of these antibiotics (Duval, 1985; Geslin, 1986). In contrast, streptogramins are still effective against S. pneumoniae (Emond et al., 1989). RP 59500 is a new injectable streptogramin which is reported to have activity against Gram-positive bacteria. The purpose of the present work was to study the in-vitro antibacterial activity of RP 59500 against erythromycin-sensitive (ES) and -resistant strains (ER) of S. pneumoniae by determining MICs and MBCs, together with the kinetics of bacterial killing by RP 59500. Materials and methods Bacterial strains and
antibiotics
One hundred strains of S. pneumoniae, serotyped by latex agglutination (Table I), were isolated from blood culture (57), CSF (17), middle ear aspirate (17), pleural fluid (6), gastric fluid (1), peritoneal fluid (1) and vaginal swab (1). These strains, isolated between 1986 and 1989, were sent by hospitals involved in a national co-operative survey of pneumococcal infections. When tested by the disc diffusion method, 48 strains were susceptible to erythromycin and 52 strains were resistant. MICs and MBCs 0305-7453/92/30A019+05 $03.00/0
19 © 1992 The British Society for Antimicrobial Chemotherapy
Downloaded from http://jac.oxfordjournals.org/ at Carleton University on June 23, 2015
Centre National de Reference des Pneumocoques, Laboratoire de Microbiologie, Hospitalier Intercommunal, Creteil, France
20
A. Frananx et aL Table L Serotype and group distribution of 100 strains of S. pneumonias Serotype
Serotype
No. of strains
5
14 1SB 17 A 18 C 19 A 19 F 20 22F 23 A 23F 24 F 33 F 34 38
18 1 1 2 4 13
4 3 1 6 9 4 i
I
2 1
were determined for 20 strains selected from these 100 isolates, of which eight were erythromycin-sensitive strains and 12 were erythromycin-resistant strains. Ten strains were selected (two erythromycin-sensitive and eight erythromycin-resistant strains) to investigate the kinetics of bactericidal activity. All strains were maintained frozen (—80°Q in Brain Heart Infusion broth containing glycerol 15% v/v. The antimicrobial agents used (and their respective manufacturers) were RP 59500 (Rhone-Poulenc Rorcr) and erythromycin (Abbott). MIC determinations by an agar dilution technique Strains were grown shaken in Brain Heart infusion broth supplemented with peritoneal fluid (10%) for 3 h at 37°C. A final inoculum of lOMO* cfu was delivered with a Steers replicator on to horse blood enriched Mueller-Hinton agar plates containing erythromycin or RP 59500 at concentrations in the range of 0-004 to 128 mg/L. The plates were incubated overnight at 37CC in an atmosphere with 5% CO2. Staphylococcus aureus strain ATCC 25293 was used as a control. The MIC was defined as the lowest concentration of antimicrobial agent which completely inhibited bacterial growth. MIC and MBC determinations by a broth microdilution technique Dilutions of antibiotic were prepared in Mueller-Hinton broth supplemented with 5% horse blood; 100 //L portions of the antibiotic dilutions were delivered to the wells of a microtitre plate, after which an inoculum of 10* cfu (10 uL) was added to each well. MICs were read after incubation for 18 h at 37°C in the presence of 5% COj. MBCs were determined after subculturing samples from each well by means of an inoculator pin (Dynatech) which delivered 10 uL portions on to blood agar plates. Plates were incubated overnight at 37°C in the presence of 5% CO3. The MBC was defined as the lowest concentration of antibiotic that yielded no growth.
Downloaded from http://jac.oxfordjournals.org/ at Carleton University on June 23, 2015
1 3 4 5 6A 6B 7F 8 9N 9V 10 A 10 F 11 A 12 F 13
No. of strains
Activity of RP 59500 tgainst S. pmemamdae
21
Tatde II. MICs of erythromycin and RP 59500 for 100 strains of S. pneumonku. as determined by the agar dilution technique Antibiotic (no. strains tested)
0O64 0-125
Erythromycin (100) RP 59500 (48 ES)
— —
17 —
RP 59500 (52 ER)
—
—
MIC (mg/L) 0-5 1
0-25 28 2
3 33
2 2 8
2
4
>128
— 12
— 1
— —
52 —
19
3
—
—
MICJO-O-5/MIQO-I-O
ES, erythromycin-tcnsitive strains; ER, erythromycin-resistant strains.
In-vitro bactericidal activity was determined by exposing a mid-log phase culture (103 cfu/mL) in Mueller-Hinton broth to 2 x MIC of RP 59500. After incubation for 4, 6 and 24 h at 37°C in an atmosphere with 5% CO2, appropriate dilutions of the culture were subcultured on blood agar plates to determine viable counts. Bactericidal activity was defined as a > 4 x logl0 reduction in the viable count of the culture when compared with the original inoculum. Results
The in-vitro inhibitory activity of RP 59500 and erythromycin, as determined by the agar dilution method, against 100 strains of 5. pnewnoniae is presented in Table II. The 48 erythromycin-sensitive strains had erythromycin MICs of < 0-5 mg/L, while the 52 erythromycin-resistant strains had erythromycin MICs of > 128 mg/L. In contrast, the RP 59500 MICs were between 0-25 and 2 mg/L for all tested strains. The MICj, and MIQo values for RP 59500 were 0-5 mg/L and 1 mg/L for erythromycin-sensitive and -resistant strains, respectively. MICs and MBCs of RP 59500 for 20 strains (eight erythromycin-sensitive and 12 erythromycin-resistant), as determined by a microdilution technique, were similar for 18 strains. For 15 strains, the MIC and MBC were both 1 mg/L, and a further three strains had MICs and MBCs of 2 mg/L. Of the remaining two strains, one had an MIC of 1 mg/L and an MBC of 2 mg/L, while the other had an MIC of 2 mg/L and an MBC of 4 mg/L (Table III). Table ID. MICs and MBCs of RP 59500 for 20 strains of S. pnewnoniae, as determined by the micro-dilution technique No. of strains MIC (mg/L)
MBC (mg/L) 0-5 1-0 2-0 4-0
0-5
1-0
20
15
1
3 1
40
Downloaded from http://jac.oxfordjournals.org/ at Carleton University on June 23, 2015
Bactericidal kinetics
22
A. Frananx et aL Table IV. Kinetics of bacterial killing by RP 59500 at 2 x MIC for ten isolates of S. pneumoniae
Strains
Type
Erythromycin-sensitive
18C
Erythromycin-resistant
6B 1 14
10 F
A F F A F
Bactericidal activity* 4h 6h 24 h
2 4 2 1 1 2 2 1 0-5
-4 -5 -5 -5 -5 -5 -3 -5 -5
-4 -5 -5 -5 -5 -5 -5 -5 -5
-4 -5 -5 -1 -5 -5 -5 -5 -5
4
-5
-5
-5
*Log,0 decrease in viable count after exposure for the indicated period.
The kinetics of bacterial killing by RP 59500, studied with ten strains (two erythromycin-sensitive and eight erythromycin-resistant) at 2 x MIC, are presented in Table IV. For the two erythromycin-sensitive strains, viable counts were reduced by approximately four to five logs after exposure for 4, 6 or 24 h. For six of the eight erythromycin-resistant strains, viable counts were reduced by > 4 logs after 4, 6 and 24 h. For all ten strains studied, RP 59500 at 2 x MIC was bactericidal after exposure for 6 h. Discussion
Pneumococcal resistance to macrolides, lincosamides and streptogramin B (MLSj resistance phenotype) was first detected in France in 1976 (Buu-Hoi, Goldstein & Acar, 1988), but by 1989 was found in 25% of clinical isolates, and 43-8% of strains isolated from patients with otitis media (Geslin, 1991). These data, and the more recent emergence of multiply-resistant strains, demonstrate the need for alternative antimicrobial therapy. The results of the present study with RP 59500 are in agreement with those reported previously by N'Guyen (1988). While the erythromycin MICs for the strains of S. pneumoniae studied allowed two separate populations (erythromycin-sensitive and erythromycin-resistant strains) to be distinguished, the MICs of RP 59500 were fairly homogeneous, ranging from 0-25-2 mg/L for both erythromycin-sensitive and -resistant strains. RP 59500 exhibited good bactericidal activity following exposure for 6 h at 2 x MIC for both erythromycin-sensitive and -resistant strains. We conclude that RP 59500 may be of value in the treatment of respiratory tract infections caused by resistant pneumococci. References Buu-Hoi, A. Y., Goldstein, F. W. & Acar, J. F. (1988). A seventeen-year epidemiologjcal survey of antimicrobial resistance in pneumococci in two hospitals. Journal of Antimicrobial Chemotherapy 22, Suppt. B, 41-52.
Downloaded from http://jac.oxfordjournals.org/ at Carleton University on June 23, 2015
19 19 19 6 23
RP 59500 MIC x 2 (mg/L)
Acdrtty of RP 59500 against 5. pneumonia*
23
Duval, J. (1985). Evolution and epidemiology of MLS resistance. Journal of Antimicrobial Chemotherapy 16, Suppl. A. 137-9. Emond, J. Ph., Frcmaux, A., Dublanchet, A., Sissia, G., Geslin, P. et al. (1989). Resistance of two strains of Streptococcus pneumoniae to pristinamycin associated with 16-membercd macrolides. Pathologie Biologie 37, 791-2. Geslin, P. (1986). Rapport du Centre National de Reference du Pneumocoque (Annce 1985). Bulletin Epidimiologique Hebdomadaire, number 50, 197-9. Geslin, P. (1991). Rapport du Centre National de Reference du Pneumocoque (Annee 1989). Bulletin Epidimiologique Hebdomadaire, number 2, 6-7. N'Guyen, J. (1988). Activite in vitro antipneumococcique des streptogramines. In Macrolides et Synergistines, pp. 115-8. Journies de I'Hdpital Claude-Bernard (Paris). Arnette. Societe Francaise de la Tuberculose et des Maladies Respiratoires (1981). Recommandations. Revue Franfaise des Maladies Respiratoires 9, 523-4. Downloaded from http://jac.oxfordjournals.org/ at Carleton University on June 23, 2015
