Journal of Antimicrobial Chemotherapy (1992) 30, Suppl. A. 29-37
In-vitro activity of RP 59500, a new semisyntbetk streptogramin antibiotic, against Gram-positive bacteria W. Brnmfttt, J.M.T. HarnDtoo-Mflkr* and S. Shah
A study of the in-vitro activity of RP 59500, a semisynthetic derivative of pristinamycin, against a range of Gram-positive bacteria including erythrotnycin-resistant strains was undertaken. MICs were determined by plate dilution in IsoSensitest agar and MBCs by velvet pad replication. RP 59500 was found to have in-vitro activity almost identical to that of its parent compound, pristinamydn. Sixty methicillinresistant Staphylococcus aureus (MRSA) and 60 methirillin-iensitivc 5. aureus (MSSA) were found to be sensitive to RP 59500, with MICs of 0-13-1 mg/L. All the MSSA and most MRSA showed an MBC < 2 mg/L. Erythromycin-resistant S. aureus (62) were as sensitive to RP 59500 as were erythromycin-senstive strains (58). RP 59500 was more active against MRSA than fusidate, vancomycin, amikacin, ciprofloxacin, imipenem or erythromycin. Forty strains of coagulase-negative staphylococci (11 were erythromycin-resistant) showed MICs of 0-25-1 mg/L, and RP 59500 was more active than methitillin, erythromycin, imipenem, cefotaxime or vancomycin. Sixty strains of streptococci (20 pneumococci and 40 of groups A, B, C, or G) and 20 enterococci were inhibited by 0-13-1 mg/L and 0-25-4 mg/L, respectively. Gram-positive bacilli (five each of diphtheroids, lactobaciUi, Usteria monocytogenes and Bacillus spp., and 19 Clostridium spp.) were also sensitive, with MICs of between 0-06 and 4 mg/L. All 279 strains tested were judged to be sensitive to RP 59500, which was bactericidal and showed a small inoculum effect. The activity against MRSA, and against erythromycin-resistant strains of all species, was particularly interesting.
Introduction
Pristinamydn is a mixture of a macrocyclic lactone (macrolide) and a cyclic depsipeptide antibiotic, belonging to the streptogramin family, which interact synergically. As 'Pyostadne', it has been used for many years in France for treating staphylococcal infections (Janbon et al., 1962). There is no cross-resistance between pristinamydn and other antibiotics because of the unique structures and interactions of the pristinamydn components IA and IIA. Thus, pristinamydn is active against highly-resistant isolates of bacteria such as- methidllin-resistant Staphylococcus aureus (MRSA) (Maple, Hamilton-Miller & Brumfitt, 1989). Unfortunately, pristinamydn is too poorly soluble to be administered by injection, which has severely limited its use. RP 59500 is a mixture of chemically-modified pristinamydn components IA and IIA that is watersoluble (Barriere et al., 1990). We report here an in-vitro assessment of RP 59500 •Corresponding luthor.
29 0305-7453/92/30A029+09 $03.00/0
© 1992 The British Society for Antimicrobial Chemotherapy
Downloaded from http://jac.oxfordjournals.org/ at University of Arizona on June 6, 2015
Department of Medical Microbiology, Royal Free Hospital School of Medicine, London NW3, UK
30
W. Bnnnfltt et aL
against a wide range of Gram-positive pathogens, including typically resistant species such as MRSA, coagulase-negative staphylococci (CNS) and enterococci. Materials and methods Bacterial strains MRSA were taken from our collection of strains obtained from around the world (Maple et al., 1989). All other strains were originally isolated from specimens sent for microbiological investigation from patients at The Royal Free Hospital. Antibiotics
Microbiological methods Erythromycin susceptibility was classified as follows: strains were spread on IsoSensitest agar (ISA, Oxoid) with approximately 104 cfu/plate. Discs of erythromycin (10 ng) and of clindamycin (2 /*g) were placed on each plate, 12 mm apart. Zones of inhibition were read after overnight incubation. Erythromycin-sensitive strains showed symmetrical zones around both discs; those with inducible resistance were indicated by no zone around the erythromycin disc and a flattening of the zone around the clindamycin disc proximal to the erythromycin disc; constitutive resistance resulted in no zone (or very small zones) around each disc. MICs were determined by the agar plate dilution method with ISA. Separate sets of plates were inoculated with 10* and 104 cfu/spot, prepared from overnight cultures in IsoSensitest broth (ISB) of appropriate test organisms. The MIC was read after overnight incubation at 37CC in air for the aerobes and facultative species, and in 80% N 2 +10% H 2 +10% CO2 for the obligate anaerobes. MBCs were determined by replication from the plates which had been inoculated with 10* cfu, with sterile velvet pads on to fresh ISA; the velvet pad method has a sampling efficiency of 1%. Plates were incubated under appropriate conditions at 37°C overnight. The endpoint was taken as growth of ten or fewer colonies (at least 99-9% kill). Bacterial killing kinetics were determined by inoculating sets of six 500 mL conical flasks, each containing 100 mL ISB, with approximately 5x 107cfu (0-1 mL of an overnight broth culture). RP 59500 was added to five of the flasks to give multiples of the MIC from x 1 to x 16; the sixth flask was not supplemented and acted as a control.
Downloaded from http://jac.oxfordjournals.org/ at University of Arizona on June 6, 2015
RP 59500 methane sulphonate and pristinamycin were given by Rhone-Poulenc Rorer. Comparator compounds were as follows: erythromycin base (Lilly Laboratories), ethanolamine salt of fusidic acid (Leo Laboratories), vancomycin hydrochloride (Sigma), amikacin sulphate (Bristol Laboratories), ciprofloxacin hydrochloride (Bayer Pharmaceuticals), imipenem (Merck, Sharp & Dohme), sodium ampicillin and sodium methicillin (Beecham Research Laboratories), sodium cefotaxime and gentamicin sulphate (Roussel Laboratories), sodium benzylpenicillin (Glaxo Laboratories), chloramphenicol (Parke Davis), metronidazole (May & Baker). The following solvents were used: methanol for pristinamycin, ethanol for erythromycin and chloramphenicol, a minimal volume of M-NaOH for metronidazole and water for all other antibiotics.
Io-ritro •ctfrity of RP 59500
31
Flasks were shaken at 100 rpm at 37°C in an orbital incubator (Gallenkamp) and samples were taken for viable counting at 0, 2, 4, 6, 8 and 24 h. Plates were counted after 40 h incubation. The post-antibiotic effect (PAE) was measured by preparing flasks as above, four for each strain. To three were added RP 59500 at x 1, x 3 and x 10 the MIC; the fourth acting as control. After shaking for 1 h at 37°C, 1 mL was removed from each flask and added to flasks containing 99 mL ISB, which were shaken as before. The numbers of viable organisms in each flask were determined at hourly intervals by the method described above. Plots were made of viable count against time, and the PAE was calculated thus: time (h) for cultures exposed to antibiotic to increase by one log minus the time for the control culture to increase by one log.
Comparative activity of RP 59500 and pristinamycin were compared for 11 erythromycin-sensitive, 32 inducibly-rcsistant and 36 constitutively-resistant MRS A strains. The results (Table I) show that pristinamycin and RP 59500 are of very similar activity. All the strains tested were sensitive, irrespective of their erythromycin susceptibility status, but it is clear that the constitutively-resistant strains have slightly higher Inhibitory activity of RP 59500 Results with 279 isolates are shown in Table II. RP 59500 was inhibitory to the majority of these strains at a concentration of 1 mg/L, and all isolates were inhibited by 4 mg/L. The obligate anaerobes tested were as susceptible as the facultative and aerobic species of bacteria. The wide activity of RP 59500 was in contrast to that of many other antibiotics (Table III), particularly against such frequently antibiotic-resistant species as MRSA, CNS and enterococci, and the Gram-positive bacilli. It can be seen that RP 59500 had a wider range of activity against MRSA than fusidic acid, amikacin, ciprofloxacin, erythromycin and imipenem, and it was more active than vancomycin. RP 59500 was active against more strains of CNS than methicillin, erythromycin and vancomycin. The inoculum effect for RP 59500 was small (Table IV). Bactericidal activity of RP 59500 In terms of MBC, RP 59500 was bactericidal against more than half the strains tested (excluding, predictably, the enterococci). Table L Activity of pristinamycin and RP 59500 against MRSA sensitive to erythromycin, with inducible and constitutive resistance
Antibiotic Pristinamycin RP 59500
Sensitive strains (11) MIC50 MIC*, 018 018
0-23 023
MIC (mg/L) Inducibly resistant strains (32) MICJ0 MIC*, 015 018
022 024
Constitutively resistant (36) MIC30 MIC*, 039 048
O9 083
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Results
Microorganism (/i) Methicillin-resistant S. aureus (MRSA) (60) Methicillin-sensitivc S. aureus (MSSA) (60) Coagulase-negative staphylococci (CNS) (40) Enterococcus spp. (20) Lancefield Group A, B, C & G streptococci (40) Streptococcus pnewnonlae (19) Other Gram-positive aerobic bacteria (diphtheroids, Bacillus sp, Usteria and Lactobacilhis sp.) (19) Gram-positive anaerobic bacteria (20)
Erythromycin sensitive resistant (n) (n)
range
MIC (mg/L) MIC J 0
2 59 29 11 37 19
58 1 11 9 3 0
0-125-1-0 0-25-0-5 0-25-1 0-25-4 0-125-1 0-125-1
0-28 0-21 0-35
16 18
3 2
006-4 0125-OS
0-3 0-3
10 0-28 0-15
MIC, 0 0-71
range
0-26
0-25->8O 0-25-2-0 O-5->32 0-25->64 0-25-64 0-125-2
3-2 046
006-64 0 2 5 - > 64
(Ml 067 3-4 045
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Table IL In-vitro activity of RP 59500 against Gram-positive bacteria with an inoculum of 10* cfu
MBC (mg/L) MBC,
,0
065 042 080 >64 029 021 O64 062
>8 08 >32 >64 050 O50 171 >64
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Table IIL In-vitro activity of comparator antibacterial agents against the collection of Gram-positive bacteria reported in Table II. Dashed lines indicate that the species was not tested Comparator antibacterial agent Erythromycin Fusidk acid Vancoraycin Amikacin Ciprofloxacin Inupenem Methictllin Cefotaxime Ampicillin Benzyl penicillin Chloramphenicol Metronidazole Gentamicin
Bacterial species MIC range (mg/L) streptococci Gram-positive Gram-positive enterococci A, B, C & G S. pneumontae aerobes anaerobes
MRSA
MSSA
CNS
O25->128 16 05-80 32 O125->16 O015->32 — — — — — —
025-> 2O 05-1 2O-4O 20-16 O25-4O 0015-003 — — — — — —
128 — 1-16 — — < 003-64 64 O5->32 — — — —
256 — 1-8 — — 1-32 — — 025-4 — — —
0007-128
64 003-8
0007-006 006-8
0007-025 0007-2
64 —
l->64 O06->64
— —
—
16-64
— — O06->32
1-8 0125-2 —
34
W. Brnmfltt et aL
Table IV. Inoculum effect for RP 59500 as defined by the ratio of the MIC,, with an inoculum of 10* and If/cfu for 107MIC,, for 104
Bacterium MRSA MSSA
CNS Enterococci Lancefield groups streptococci of A, B, C & G S. pneumoniae Other Gram-positive aerobes Gram-positive anaerobes
1-3 11 1-3 1-0 11 10 10 16
e Conctntration of RP 5 9 5 0 0 (multiplt a) MIC )
Flgarc Graph ihowing the effect of concentration of RP 59500 on rate of killing over 8 h of MRSA, 5. aureus and enterococci. Values are meanj±s.E.M. for six strains of MRSA (OX four strains of S. aureus ( • ) and four strains of enterococci (D)-
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The usual finding in studies of killing kinetics with S. aureus was a slow progressive killing of bacteria by concentrations in excess of the MIC (maximally 2 to 2-5 log,0 in 8 h). Little or no additional killing occurred during the next 16 h, except at the highest antibiotic concentration. There was a trend for more rapid killing to occur with higher antibiotic concentrations (Figure). On some occasions, regrowth was observed overnight, but the bacteria isolated from these flasks remained sensitive to RP 59500. Enterococci were not killed so readily as other bacteria and, up to 8 h, killing was no more rapid with the higher antibiotic concentrations (Figure). Regrowth usually
In-Titro activity of RP 59500
35
Table V. Post-antibiotic effect of RP 59500 Microorganism S. aureus Enterococci
Number of strains tested 10 3
Duration (h) of PAE x 1 MIC x 3 MIC x10 MIC 2-4±0-67* 2-4±0*
4-4±l >5-5
>5-5 >5-5
•Mean value+S.D.
occurred at all antibiotic concentrations less than 4xMIC, and again the bacteria isolated under these circumstances were found to be sensitive to RP 59500.
A striking property of RP 59500 is that it produces a long PAE. We tested ten strains of S. aureus (four erythromycin-sensitive, four with induciblc resistance and two with constitutive resistance), and three enterococci (two erythromycin-sensitive and one resistant). A mean PAE of 2-4 h was found after 1 h exposure to 1 x MIC of RP 59500 for both species, and increasing the antibiotic concentration lengthened the duration of the PAE (Table V). With antibiotic concentrations of 10 x MIC, the PAE was too long to be measured by our methodology. There was no correlation between erythromycin susceptibility status and the duration of the PAE.
Discussion The uniform activity of RP 59500 against such commonly-resistant bacteria as MRSA, CNS and enterococci is encouraging. New therapeutic agents to counter the increasing resistance in these species are urgently needed, and of the novel parenteral agents currently being assessed, only daptomycin (Benson, Beaudette & Trenholm, 1987) seems to have such a broad spectrum. RP 59500 was found to be active against all the strains tested, irrespective of their erythromycin susceptibility status. This shows that all the erythromycin resistance in this population is due to the MLS,, type of resistance (Duval, 1985), caused by methylation of adenine in the 23S fraction of ribosomal RNA. Constitutively-resistant strains are resistant to type B streptogramins (in this case, the modified pristinamycin LA moiety), but retain sensitivity to RP 59500 because of the activity of the type A component (the modified pristinamycin IIA molecule) and synergy between the components. It has been suggested (Chabbert & Courvalin, 1971; Gibert, Domart & Chastre, 1985) that caution should be exercised in the use of pristinamycin against strains that show erythromycin resistance. The incidence of resistance to pristinamycin in staphylococci in France has remained low, despite this antibiotic being in general use. Figures of 1-5% and 0-5% have been reported (Modai, 1985; Thabaut, Meyran & Huerre, 1985). Resistance appears to be more common in MRSA than in methicillin-sensitive strains (Duval, 1985), but we have found no resistance to RP 59500 in more than 150 strains of MRSA tested. This type of resistance to pristinamycin is plasmid-mediated (Le Goffic et al., 1977; Duval, 1985), and involves acetylation of the antibiotic.
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Post-antibiotic effect
36
W. Bramfitt et *L
Acknowledgement Part of this work was presented as a poster at the 30th Interscience Conference on Antimicrobial Agents and Chemotherapy, Atlanta, 1990. References Barrierc, J. C , Bouanchaud, D. H., Harris, N. V., Paris, J. M., Rolin, O. & Smith, C. (1990). The design, synthesis and properties of RP 59500 and related semi-synthetic strcptogramin antibiotics. Abstracts of 30th Interscience Conference on Antimicrobial Agents and Chemotherapy, Atlanta. Abstract 768. Benson, C. A., Beaudette, F. & Trenhohn, G. (1987). Comparative in-vitro activity of LY146032 a new peptolide with vancomycin and eight other agents against Gram-positive organisms. Antimicrobial Agents and Chemotherapy 20, 191-6. Chabbert, Y. & Courvatin, P. (1971). Synergic des composants des antibiotiques du groupe de la streptogramine. Pathologie Biologie 19, 613-9. Duval, J. (1985). Evolution and epidemiology of MLS resistance. Journal of Antimicrobial Chemotherapy 16, Suppl. A. 137-49. Gibert, C , Domart, Y. & Chastre, J. (1985). Pathologie intra hospitaliere due aux staphylocoques resistants a la meticiline. Presse Mtdicale 14, 2081-3. Janbon, N., Brunei, D., Bertrand, A. & Micbel-Briand, Y. (1962). La pristinamycin (7293 RP), antibiotique antistaphylococcique. Etude comparative in-vitro, premiere experience clinique. Semaine Thirapeutique 38, 25-33. Le Goffic, F., Capmau, M.-L., Bonnet, D., Cerceau, C , Soussy, C , Dublanchet, A. A Duval, J. (1977). Plasmid-mediatcd pristinamycin resistance. PAC HA a new enzyme which modifies pristinamycin IIA. Journal of Antibiotics 30, 665-9. Maple, P. A. C , Hamilton-Miller, J. M. T. & Brumfitt, W. (1989). World-wide antibiotic resistance in methicillin-rcsistant Staphylococcus aureus. Lancet i, 537-40. Maskell, J. P., Sefton, A. M., Yong, Chi, S. J. & Williams, J. D. (1988). Comparative in-vitro activity of erythromycin, vancomycin and pristinamycin. Infection 16, 365-70. Modal, J. (1985). Comparative evaluation of macroh'des, lincosamides and streptogramins in staphylococcal infections. Journal of Antimicrobial Chemotherapy 16, Suppl. A, 195-7.
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Entcrococci are resistant to the A component of streptogramins, and thus one would expect that erythromydn-resistant strains showing the MLSB pattern would be resistant to pristinamycin and to RP 59500. However, we did not find this to be the case for RP 59500, mirroring the experience of Maskell et al. (1988) with pristinamycin. The long PAE found here confirms the findings of Nougayrcde, Berthaud & Bouanchaud (1992), who suggested that this might be explained by the high stability of the complex between RP 59500 and the bacterial ribosome. Agents that inhibit protein synthesis usually show a PAE of not more than 4 h against Gram-positive cocci (Odenholt-Tornqvist, 1989), so our figures of > 6 h for RP 59500 and the values > 7 h reported by Nougayrede et al. (1992) emphasize a remarkable property of this antibiotic. Besides the obvious pharmacodynamic implications, the long PAE has an important practical application: when viable counting of cultures exposed to RP 59500 is being carried out, an incubation period of at least 40 h should be used, otherwise a false impression of killing rates will be obtained. Our results make it clear that the development of a water-soluble derivative of pristinamycin, which can be given by the intravenous route, is important. Seriously ill patients can be started on treatment with intravenous RP 59500, and therapy can be continued with oral pristinamycin once the patient's condition has improved sufficiently for oral antibiotic administration to be possible. Precise dosage regimens await results of pharmacokinctic investigations.
In-vttro activity of RP 59500
37
Nougayrede, A., Berthaud, N. & Bouanchaud, D. H. (1990). Post-antibiotic effects of RP 59500 with Staphylococcus aureus. Journal of Antimicrobial Chemotherapy 30, Suppl. A. 101-106. Odenholt-Tomqvist, I. (1989). Phannacodynamics of beta-lactam antibiotics. Scandinavian Journal of Infectious Diseases Suppl. 58, 1-55. Thabaut, A., Meyran, M. & Huerre, M. (1985). Evolution and present situation of Staphylococcus aureus sensitivity to MLS in hospital (1975-83). Journal of Antimicrobial Chemotherapy, Suppl. A, 16, 205-7.
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In-vitro activity of RP 59500, a new semisyntbetk streptogramin antibiotic, against Gram-positive bacteria W. Brnmfttt, J.M.T. HarnDtoo-Mflkr* and S. Shah
A study of the in-vitro activity of RP 59500, a semisynthetic derivative of pristinamycin, against a range of Gram-positive bacteria including erythrotnycin-resistant strains was undertaken. MICs were determined by plate dilution in IsoSensitest agar and MBCs by velvet pad replication. RP 59500 was found to have in-vitro activity almost identical to that of its parent compound, pristinamydn. Sixty methicillinresistant Staphylococcus aureus (MRSA) and 60 methirillin-iensitivc 5. aureus (MSSA) were found to be sensitive to RP 59500, with MICs of 0-13-1 mg/L. All the MSSA and most MRSA showed an MBC < 2 mg/L. Erythromycin-resistant S. aureus (62) were as sensitive to RP 59500 as were erythromycin-senstive strains (58). RP 59500 was more active against MRSA than fusidate, vancomycin, amikacin, ciprofloxacin, imipenem or erythromycin. Forty strains of coagulase-negative staphylococci (11 were erythromycin-resistant) showed MICs of 0-25-1 mg/L, and RP 59500 was more active than methitillin, erythromycin, imipenem, cefotaxime or vancomycin. Sixty strains of streptococci (20 pneumococci and 40 of groups A, B, C, or G) and 20 enterococci were inhibited by 0-13-1 mg/L and 0-25-4 mg/L, respectively. Gram-positive bacilli (five each of diphtheroids, lactobaciUi, Usteria monocytogenes and Bacillus spp., and 19 Clostridium spp.) were also sensitive, with MICs of between 0-06 and 4 mg/L. All 279 strains tested were judged to be sensitive to RP 59500, which was bactericidal and showed a small inoculum effect. The activity against MRSA, and against erythromycin-resistant strains of all species, was particularly interesting.
Introduction
Pristinamydn is a mixture of a macrocyclic lactone (macrolide) and a cyclic depsipeptide antibiotic, belonging to the streptogramin family, which interact synergically. As 'Pyostadne', it has been used for many years in France for treating staphylococcal infections (Janbon et al., 1962). There is no cross-resistance between pristinamydn and other antibiotics because of the unique structures and interactions of the pristinamydn components IA and IIA. Thus, pristinamydn is active against highly-resistant isolates of bacteria such as- methidllin-resistant Staphylococcus aureus (MRSA) (Maple, Hamilton-Miller & Brumfitt, 1989). Unfortunately, pristinamydn is too poorly soluble to be administered by injection, which has severely limited its use. RP 59500 is a mixture of chemically-modified pristinamydn components IA and IIA that is watersoluble (Barriere et al., 1990). We report here an in-vitro assessment of RP 59500 •Corresponding luthor.
29 0305-7453/92/30A029+09 $03.00/0
© 1992 The British Society for Antimicrobial Chemotherapy
Downloaded from http://jac.oxfordjournals.org/ at University of Arizona on June 6, 2015
Department of Medical Microbiology, Royal Free Hospital School of Medicine, London NW3, UK
30
W. Bnnnfltt et aL
against a wide range of Gram-positive pathogens, including typically resistant species such as MRSA, coagulase-negative staphylococci (CNS) and enterococci. Materials and methods Bacterial strains MRSA were taken from our collection of strains obtained from around the world (Maple et al., 1989). All other strains were originally isolated from specimens sent for microbiological investigation from patients at The Royal Free Hospital. Antibiotics
Microbiological methods Erythromycin susceptibility was classified as follows: strains were spread on IsoSensitest agar (ISA, Oxoid) with approximately 104 cfu/plate. Discs of erythromycin (10 ng) and of clindamycin (2 /*g) were placed on each plate, 12 mm apart. Zones of inhibition were read after overnight incubation. Erythromycin-sensitive strains showed symmetrical zones around both discs; those with inducible resistance were indicated by no zone around the erythromycin disc and a flattening of the zone around the clindamycin disc proximal to the erythromycin disc; constitutive resistance resulted in no zone (or very small zones) around each disc. MICs were determined by the agar plate dilution method with ISA. Separate sets of plates were inoculated with 10* and 104 cfu/spot, prepared from overnight cultures in IsoSensitest broth (ISB) of appropriate test organisms. The MIC was read after overnight incubation at 37CC in air for the aerobes and facultative species, and in 80% N 2 +10% H 2 +10% CO2 for the obligate anaerobes. MBCs were determined by replication from the plates which had been inoculated with 10* cfu, with sterile velvet pads on to fresh ISA; the velvet pad method has a sampling efficiency of 1%. Plates were incubated under appropriate conditions at 37°C overnight. The endpoint was taken as growth of ten or fewer colonies (at least 99-9% kill). Bacterial killing kinetics were determined by inoculating sets of six 500 mL conical flasks, each containing 100 mL ISB, with approximately 5x 107cfu (0-1 mL of an overnight broth culture). RP 59500 was added to five of the flasks to give multiples of the MIC from x 1 to x 16; the sixth flask was not supplemented and acted as a control.
Downloaded from http://jac.oxfordjournals.org/ at University of Arizona on June 6, 2015
RP 59500 methane sulphonate and pristinamycin were given by Rhone-Poulenc Rorer. Comparator compounds were as follows: erythromycin base (Lilly Laboratories), ethanolamine salt of fusidic acid (Leo Laboratories), vancomycin hydrochloride (Sigma), amikacin sulphate (Bristol Laboratories), ciprofloxacin hydrochloride (Bayer Pharmaceuticals), imipenem (Merck, Sharp & Dohme), sodium ampicillin and sodium methicillin (Beecham Research Laboratories), sodium cefotaxime and gentamicin sulphate (Roussel Laboratories), sodium benzylpenicillin (Glaxo Laboratories), chloramphenicol (Parke Davis), metronidazole (May & Baker). The following solvents were used: methanol for pristinamycin, ethanol for erythromycin and chloramphenicol, a minimal volume of M-NaOH for metronidazole and water for all other antibiotics.
Io-ritro •ctfrity of RP 59500
31
Flasks were shaken at 100 rpm at 37°C in an orbital incubator (Gallenkamp) and samples were taken for viable counting at 0, 2, 4, 6, 8 and 24 h. Plates were counted after 40 h incubation. The post-antibiotic effect (PAE) was measured by preparing flasks as above, four for each strain. To three were added RP 59500 at x 1, x 3 and x 10 the MIC; the fourth acting as control. After shaking for 1 h at 37°C, 1 mL was removed from each flask and added to flasks containing 99 mL ISB, which were shaken as before. The numbers of viable organisms in each flask were determined at hourly intervals by the method described above. Plots were made of viable count against time, and the PAE was calculated thus: time (h) for cultures exposed to antibiotic to increase by one log minus the time for the control culture to increase by one log.
Comparative activity of RP 59500 and pristinamycin were compared for 11 erythromycin-sensitive, 32 inducibly-rcsistant and 36 constitutively-resistant MRS A strains. The results (Table I) show that pristinamycin and RP 59500 are of very similar activity. All the strains tested were sensitive, irrespective of their erythromycin susceptibility status, but it is clear that the constitutively-resistant strains have slightly higher Inhibitory activity of RP 59500 Results with 279 isolates are shown in Table II. RP 59500 was inhibitory to the majority of these strains at a concentration of 1 mg/L, and all isolates were inhibited by 4 mg/L. The obligate anaerobes tested were as susceptible as the facultative and aerobic species of bacteria. The wide activity of RP 59500 was in contrast to that of many other antibiotics (Table III), particularly against such frequently antibiotic-resistant species as MRSA, CNS and enterococci, and the Gram-positive bacilli. It can be seen that RP 59500 had a wider range of activity against MRSA than fusidic acid, amikacin, ciprofloxacin, erythromycin and imipenem, and it was more active than vancomycin. RP 59500 was active against more strains of CNS than methicillin, erythromycin and vancomycin. The inoculum effect for RP 59500 was small (Table IV). Bactericidal activity of RP 59500 In terms of MBC, RP 59500 was bactericidal against more than half the strains tested (excluding, predictably, the enterococci). Table L Activity of pristinamycin and RP 59500 against MRSA sensitive to erythromycin, with inducible and constitutive resistance
Antibiotic Pristinamycin RP 59500
Sensitive strains (11) MIC50 MIC*, 018 018
0-23 023
MIC (mg/L) Inducibly resistant strains (32) MICJ0 MIC*, 015 018
022 024
Constitutively resistant (36) MIC30 MIC*, 039 048
O9 083
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Results
Microorganism (/i) Methicillin-resistant S. aureus (MRSA) (60) Methicillin-sensitivc S. aureus (MSSA) (60) Coagulase-negative staphylococci (CNS) (40) Enterococcus spp. (20) Lancefield Group A, B, C & G streptococci (40) Streptococcus pnewnonlae (19) Other Gram-positive aerobic bacteria (diphtheroids, Bacillus sp, Usteria and Lactobacilhis sp.) (19) Gram-positive anaerobic bacteria (20)
Erythromycin sensitive resistant (n) (n)
range
MIC (mg/L) MIC J 0
2 59 29 11 37 19
58 1 11 9 3 0
0-125-1-0 0-25-0-5 0-25-1 0-25-4 0-125-1 0-125-1
0-28 0-21 0-35
16 18
3 2
006-4 0125-OS
0-3 0-3
10 0-28 0-15
MIC, 0 0-71
range
0-26
0-25->8O 0-25-2-0 O-5->32 0-25->64 0-25-64 0-125-2
3-2 046
006-64 0 2 5 - > 64
(Ml 067 3-4 045
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Table IL In-vitro activity of RP 59500 against Gram-positive bacteria with an inoculum of 10* cfu
MBC (mg/L) MBC,
,0
065 042 080 >64 029 021 O64 062
>8 08 >32 >64 050 O50 171 >64
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Table IIL In-vitro activity of comparator antibacterial agents against the collection of Gram-positive bacteria reported in Table II. Dashed lines indicate that the species was not tested Comparator antibacterial agent Erythromycin Fusidk acid Vancoraycin Amikacin Ciprofloxacin Inupenem Methictllin Cefotaxime Ampicillin Benzyl penicillin Chloramphenicol Metronidazole Gentamicin
Bacterial species MIC range (mg/L) streptococci Gram-positive Gram-positive enterococci A, B, C & G S. pneumontae aerobes anaerobes
MRSA
MSSA
CNS
O25->128 16 05-80 32 O125->16 O015->32 — — — — — —
025-> 2O 05-1 2O-4O 20-16 O25-4O 0015-003 — — — — — —
128 — 1-16 — — < 003-64 64 O5->32 — — — —
256 — 1-8 — — 1-32 — — 025-4 — — —
0007-128
64 003-8
0007-006 006-8
0007-025 0007-2
64 —
l->64 O06->64
— —
—
16-64
— — O06->32
1-8 0125-2 —
34
W. Brnmfltt et aL
Table IV. Inoculum effect for RP 59500 as defined by the ratio of the MIC,, with an inoculum of 10* and If/cfu for 107MIC,, for 104
Bacterium MRSA MSSA
CNS Enterococci Lancefield groups streptococci of A, B, C & G S. pneumoniae Other Gram-positive aerobes Gram-positive anaerobes
1-3 11 1-3 1-0 11 10 10 16
e Conctntration of RP 5 9 5 0 0 (multiplt a) MIC )
Flgarc Graph ihowing the effect of concentration of RP 59500 on rate of killing over 8 h of MRSA, 5. aureus and enterococci. Values are meanj±s.E.M. for six strains of MRSA (OX four strains of S. aureus ( • ) and four strains of enterococci (D)-
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The usual finding in studies of killing kinetics with S. aureus was a slow progressive killing of bacteria by concentrations in excess of the MIC (maximally 2 to 2-5 log,0 in 8 h). Little or no additional killing occurred during the next 16 h, except at the highest antibiotic concentration. There was a trend for more rapid killing to occur with higher antibiotic concentrations (Figure). On some occasions, regrowth was observed overnight, but the bacteria isolated from these flasks remained sensitive to RP 59500. Enterococci were not killed so readily as other bacteria and, up to 8 h, killing was no more rapid with the higher antibiotic concentrations (Figure). Regrowth usually
In-Titro activity of RP 59500
35
Table V. Post-antibiotic effect of RP 59500 Microorganism S. aureus Enterococci
Number of strains tested 10 3
Duration (h) of PAE x 1 MIC x 3 MIC x10 MIC 2-4±0-67* 2-4±0*
4-4±l >5-5
>5-5 >5-5
•Mean value+S.D.
occurred at all antibiotic concentrations less than 4xMIC, and again the bacteria isolated under these circumstances were found to be sensitive to RP 59500.
A striking property of RP 59500 is that it produces a long PAE. We tested ten strains of S. aureus (four erythromycin-sensitive, four with induciblc resistance and two with constitutive resistance), and three enterococci (two erythromycin-sensitive and one resistant). A mean PAE of 2-4 h was found after 1 h exposure to 1 x MIC of RP 59500 for both species, and increasing the antibiotic concentration lengthened the duration of the PAE (Table V). With antibiotic concentrations of 10 x MIC, the PAE was too long to be measured by our methodology. There was no correlation between erythromycin susceptibility status and the duration of the PAE.
Discussion The uniform activity of RP 59500 against such commonly-resistant bacteria as MRSA, CNS and enterococci is encouraging. New therapeutic agents to counter the increasing resistance in these species are urgently needed, and of the novel parenteral agents currently being assessed, only daptomycin (Benson, Beaudette & Trenholm, 1987) seems to have such a broad spectrum. RP 59500 was found to be active against all the strains tested, irrespective of their erythromycin susceptibility status. This shows that all the erythromycin resistance in this population is due to the MLS,, type of resistance (Duval, 1985), caused by methylation of adenine in the 23S fraction of ribosomal RNA. Constitutively-resistant strains are resistant to type B streptogramins (in this case, the modified pristinamycin LA moiety), but retain sensitivity to RP 59500 because of the activity of the type A component (the modified pristinamycin IIA molecule) and synergy between the components. It has been suggested (Chabbert & Courvalin, 1971; Gibert, Domart & Chastre, 1985) that caution should be exercised in the use of pristinamycin against strains that show erythromycin resistance. The incidence of resistance to pristinamycin in staphylococci in France has remained low, despite this antibiotic being in general use. Figures of 1-5% and 0-5% have been reported (Modai, 1985; Thabaut, Meyran & Huerre, 1985). Resistance appears to be more common in MRSA than in methicillin-sensitive strains (Duval, 1985), but we have found no resistance to RP 59500 in more than 150 strains of MRSA tested. This type of resistance to pristinamycin is plasmid-mediated (Le Goffic et al., 1977; Duval, 1985), and involves acetylation of the antibiotic.
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Post-antibiotic effect
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W. Bramfitt et *L
Acknowledgement Part of this work was presented as a poster at the 30th Interscience Conference on Antimicrobial Agents and Chemotherapy, Atlanta, 1990. References Barrierc, J. C , Bouanchaud, D. H., Harris, N. V., Paris, J. M., Rolin, O. & Smith, C. (1990). The design, synthesis and properties of RP 59500 and related semi-synthetic strcptogramin antibiotics. Abstracts of 30th Interscience Conference on Antimicrobial Agents and Chemotherapy, Atlanta. Abstract 768. Benson, C. A., Beaudette, F. & Trenhohn, G. (1987). Comparative in-vitro activity of LY146032 a new peptolide with vancomycin and eight other agents against Gram-positive organisms. Antimicrobial Agents and Chemotherapy 20, 191-6. Chabbert, Y. & Courvatin, P. (1971). Synergic des composants des antibiotiques du groupe de la streptogramine. Pathologie Biologie 19, 613-9. Duval, J. (1985). Evolution and epidemiology of MLS resistance. Journal of Antimicrobial Chemotherapy 16, Suppl. A. 137-49. Gibert, C , Domart, Y. & Chastre, J. (1985). Pathologie intra hospitaliere due aux staphylocoques resistants a la meticiline. Presse Mtdicale 14, 2081-3. Janbon, N., Brunei, D., Bertrand, A. & Micbel-Briand, Y. (1962). La pristinamycin (7293 RP), antibiotique antistaphylococcique. Etude comparative in-vitro, premiere experience clinique. Semaine Thirapeutique 38, 25-33. Le Goffic, F., Capmau, M.-L., Bonnet, D., Cerceau, C , Soussy, C , Dublanchet, A. A Duval, J. (1977). Plasmid-mediatcd pristinamycin resistance. PAC HA a new enzyme which modifies pristinamycin IIA. Journal of Antibiotics 30, 665-9. Maple, P. A. C , Hamilton-Miller, J. M. T. & Brumfitt, W. (1989). World-wide antibiotic resistance in methicillin-rcsistant Staphylococcus aureus. Lancet i, 537-40. Maskell, J. P., Sefton, A. M., Yong, Chi, S. J. & Williams, J. D. (1988). Comparative in-vitro activity of erythromycin, vancomycin and pristinamycin. Infection 16, 365-70. Modal, J. (1985). Comparative evaluation of macroh'des, lincosamides and streptogramins in staphylococcal infections. Journal of Antimicrobial Chemotherapy 16, Suppl. A, 195-7.
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Entcrococci are resistant to the A component of streptogramins, and thus one would expect that erythromydn-resistant strains showing the MLSB pattern would be resistant to pristinamycin and to RP 59500. However, we did not find this to be the case for RP 59500, mirroring the experience of Maskell et al. (1988) with pristinamycin. The long PAE found here confirms the findings of Nougayrcde, Berthaud & Bouanchaud (1992), who suggested that this might be explained by the high stability of the complex between RP 59500 and the bacterial ribosome. Agents that inhibit protein synthesis usually show a PAE of not more than 4 h against Gram-positive cocci (Odenholt-Tornqvist, 1989), so our figures of > 6 h for RP 59500 and the values > 7 h reported by Nougayrede et al. (1992) emphasize a remarkable property of this antibiotic. Besides the obvious pharmacodynamic implications, the long PAE has an important practical application: when viable counting of cultures exposed to RP 59500 is being carried out, an incubation period of at least 40 h should be used, otherwise a false impression of killing rates will be obtained. Our results make it clear that the development of a water-soluble derivative of pristinamycin, which can be given by the intravenous route, is important. Seriously ill patients can be started on treatment with intravenous RP 59500, and therapy can be continued with oral pristinamycin once the patient's condition has improved sufficiently for oral antibiotic administration to be possible. Precise dosage regimens await results of pharmacokinctic investigations.
In-vttro activity of RP 59500
37
Nougayrede, A., Berthaud, N. & Bouanchaud, D. H. (1990). Post-antibiotic effects of RP 59500 with Staphylococcus aureus. Journal of Antimicrobial Chemotherapy 30, Suppl. A. 101-106. Odenholt-Tomqvist, I. (1989). Phannacodynamics of beta-lactam antibiotics. Scandinavian Journal of Infectious Diseases Suppl. 58, 1-55. Thabaut, A., Meyran, M. & Huerre, M. (1985). Evolution and present situation of Staphylococcus aureus sensitivity to MLS in hospital (1975-83). Journal of Antimicrobial Chemotherapy, Suppl. A, 16, 205-7.
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