J. W. Mouton, H. P. Endtz, J. G. den Hollander, N. van den Braak and H. A. Verbrugh

Journal of Antimicrobial Chemotherapy (1997) 39, Suppl. A, 75 80 JAC In-vitro activity of quinupristin/dalfopristin compared with other widely used antibiotics against strains isolated from patients with endocarditis J. W. Mouton, H. P. Endtz, J. G. den Hollander, N. van den Braak and H. A. Verbrugh Department of Clinical Microbiology, Erasmus University Hospital Rotterdam, Dr Molewaterplein 40, 3015 gd Rotterdam, The Netherlands The activity of quinupristin/dalfopristin was compared with that of other widely used antibiotics against 355 strains isolated from patients with endocarditis. MICs were determined by a standard agar dilution method. Quinupristin/dalfopristin was inhibitory at 1 mg/l for all coagulase-negative staphylococci (n = 36) and for the majority of Staphylococcus aureus strains (n = 87). The activity of quinupristin/dalfopristin against 186 viridans streptococci was somewhat dependent on the species, with MIC 50 s ranging from 0.5 to 2 mg/l, being least active against Streptococcus bovis and most active against Streptococcus gordonii and Streptococcus mitis. For the staphylococci, quinupristin/dalfopristin was as active against erythromycin-susceptible as erythromycin-resistant strains. Viridans streptococci showed a slight but significant correlation between sensitivity to erythromycin and quinupristin/dalfopristin. It is concluded that quinupristin/dalfopristin has the potential to treat serious infections such as endocarditis caused by Gram-positive cocci. Introduction Quinupristin/dalfopristin, a recently developed streptogramin antibiotic, is the first of its class available clinically for intravenous use. Most Gram-positive bacteria are susceptible to quinupristin/dalfopristin, 1 3 and its chief use would be in patients with suspected or proven infections caused by Gram-positive microorganisms, such as soft tissue infections due to streptococci and staphylococci. Since the mechanism of action, and therefore the development of resistance, is distinct from that of presently used antibiotics, 4,5 it may be particularly useful for treating patients with serious infections due to multi-resistant microorganisms. 6 Another possible indication would be endocarditis due to Gram-positive cocci. These infections are still difficult to treat in many cases, and because of increasing resistance the responsible microorganisms, will be even more difficult to treat in the future. To determine the potential of quinupristin/dalfopristin as treatment for these infections, its activity was compared with that of other frequently used antibiotics against Gram-positive cocci isolated from patients with endocarditis. Materials and methods Strains Strains were isolated from patients with endocarditis in The Netherlands during the period 1 November 1986 to 1 November 1988. These strains have been described extensively. 7 In brief, 419 strains were collected and identified, 326 from native valve endocarditis and 93 from prosthetic valve endocarditis. Of these, 64 were not Gram-positive cocci, or could not be recovered. The remaining 355 strains were used. Strains were identified by standard procedures, 8 as described by van der Meer et al. 7 All viridans streptococci were re-identified using the RAPID ID32 Strep system (biomérieux, Marcyl Etoile, France). Antibiotics Antibiotics were supplied as reference powders by their respective manufacturers in The Netherlands: quinupristin/dalfopristin (Rhône-Poulenc Rorer, Amstel- Tel: 31-10-4633511; Fax: 31-10-4633875. 1997 The British Society for Antimicrobial Chemotherapy 75

J. W. Mouton et al. veen), vancomycin (Eli Lilly, Nieuwegein), rifampicin, penicillin and teicoplanin (Yamanouchi Pharma, Leiderdorp), clindamycin (Upjohn, Amstelveen), clarithromycin and erythromycin (Abbott, Amstelveen), amoxycillin and flucloxacillin (Smith-Kline Beecham, Rijswrijk), cefuroxime (Glaxo, Zeist), cefotaxime (Hoechst-Marion- Roussel, Hoevelaken), gentamicin (Schering-Plough, Amstelveen), minocycline (Wyeth-Lederle, Hoofddorp). Stock solutions were prepared according to the recommendations of the National Committee for Clinical Laboratory Standards. 9 MIC determination MICs were determined by a standard agar dilution method. 10 Freshly prepared, serial two-fold dilutions of each antibiotic were incorporated in Mueller Hinton (Difco, Amsterdam, The Netherlands). Overnight Mueller Hinton broth cultures of the isolates were adjusted to a turbidity equivalent to a 0.5 McFarland standard and an inoculum of 10 4 cfu was delivered to the surface of the agar plates with a multipoint inoculator. The MIC was defined as the lowest concentration of each antibiotic which inhibited visible growth after incubation for 18 h at 37 C. Three control strains, Staphylococcus aureus ATCC 29213, Streptococcus pneumoniae ATCC 49619 and Enterococcus faecalis ATCC 29212, were included as a control in each batch. Results were analysed using the SAS computer program package. 11 Results The MIC 50 and MIC 90 and MIC range of quinupristin/ dalfopristin and comparative agents for the bacteria tested are shown in Tables I and II. For staphylococci, quinupristin/dalfopristin was inhibitory at 1 mg/l for all coagulase-negative staphylococci and for the majority of S. aureus strains. Quinupristin/dalfopristin was more active than vancomycin and teicoplanin for the coagulasenegative staphylococci. For -haemolytic streptococci, the activity of quinupristin/dalfopristin was similar to that of vancomycin and slightly less than that of teicoplanin. The two macrolides studied, clarithromycin and erythromycin, were both more active than quinupristin/dalfopristin against these strains. The activity of quinupristin/dalfopristin was not very high for the E. faecalis, the MIC 50 being 8 mg/l; vancomycin and teicoplanin were more active against these isolates. The activity of quinupristin/ dalfopristin against the 186 strains of Streptococcus bovis and viridans streptococci was slightly dependent on the species (Table II) with MIC 50 s ranging from 0.5 to 2 mg/l. Quinupristin/dalfopristin was least active against S. bovis and most active against Streptococcus gordonii and Streptococcus mitis. However, for all species, the range of activity within each species was quite large, indicating much interstrain variability. Except for S. bovis, activity was similar to that of vancomycin, while being less than that of teicoplanin. Quinupristin/dalfopristin was less active than erythromycin and clarithromycin. Since there is a common target for streptogramins and macrolides, 12 potential cross-resistance between erythromycin and quinupristin/dalfopristin was examined amongst staphylococci and the viridans streptococci (Table III). For the staphylococci, there appeared to be no cross-resistance. The majority of both erythromycinsusceptible and erythromycin-resistant isolates were inhibited by quinupristin/dalfopristin at concentrations of 1 mg/l. In contrast, erythromycin-resistant strains of viridans streptococci were slightly less sensitive than erythromycin-sensitive strains. Correlation analysis between susceptibility of erythromycin and quinupristin/ dalfopristin showed statistical significance (r 0.174; P 0.02). Discussion This study showed that quinupristin/dalfopristin is active against staphylococci and streptococci isolated from patients with endocarditis, the only exception being E. faecalis, which appeared only moderately susceptible. The majority of the strains were susceptible to concentrations achieved in vivo, 13 and quinupristin/dalfopristin could therefore be a suitable agent for treating infections due to staphylococci and streptococci. The somewhat lower activity against E. faecalis has been found by other workers. 14 The range of activity was large, however, and there might be occasions when the drug could be used. The alternative agents, vancomycin and teicoplanin, have similar activity, with teicoplanin being slightly more active. One of the disadvantages of the glycopeptides for treating endocarditis, however, is that these molecules are large, and their penetration into vegetations is not optimal. 15 However, in a rabbit model of aortic valve endocarditis, vancomycin was slightly more active than quinupristin/ dalfopristin. 16 For clinical use quinupristin/ dalfopristin may be an alternative in patients with endocarditis due to Gram-positive cocci that cannot be treated with standard therapy with -lactam antibiotics. Although we tested no methicillin-resistant isolates in this study, other studies have shown similar activity of quinupristin/ dalfopristin against methicillin- and susceptible-resistant staphylococci. 2,17,18 The streptogramins, lincosamides and macrolides have a common target, namely the 23S ribosomal RNA. 12 Different mechanisms of resistance have been described. 19 21 To determine whether there was cross-resistance between quinupristin/dalfopristin and the macrolides, we looked at the susceptibility of quinupristin/dalfopristin in erythromycin-susceptible and erythromycin-resistant strains. For staphylococci, we found no difference between these 76

Quinupristin/dalfopristin against endocarditis isolates Table I. Comparative in-vitro activity of quinupristin/dalfopristin and other antibiotics against Gram-positive cocci isolated from patients with endocarditis Species (number of strains) MIC 50 MIC 90 MIC range and antibiotic (mg/l) (mg/l) (mg/l) S. aureus (87) penicillin 2 20.0 0.03 2 quinupristin/dalfopristin 1 10.0 0.25 2 rifampicin 0.03 0.03 0.008 128 vancomycin 1 10.0 0.5 2 teicoplanin 1 20.0 0.5 2 flucloxacillin 0.25 0.50 0.125 1 cefuroxime 1 20.0 0.5 2 clindamycin 0.125 0.25 0.03 16 clarithromycin 0.25 0.25 0.125 32 erythromycin 0.25 0.50 0.06 32 Coagulase-negative staphylococci (36) penicillin 0.5 20.0 0.03 2 quinupristin/dalfopristin 0.5 10.0 0.25 1 rifampicin 0.03 0.03 0.008 0.25 vancomycin 2 20.0 0.5 4 teicoplanin 1 40.0 0.125 16 flucloxacillin 0.25 20.0 0.06 8 cefuroxime 0.5 40.0 0.125 128 clindamycin 0.125 160.0 0.03 16 clarithromycin 0.25 320.0 0.06 32 erythromycin 0.25 320.0 0.06 32 E. faecalis (35) amoxycillin 0.5 40.0 0.06 32 quinupristin/dalfopristin 8 160.0 0.5 16 minocycline 1 320.0 0.25 64 vancomycin 2 40.0 1 8 teicoplanin 0.5 10.0 0.125 1 clindamycin 16 640.0 0.125 64 clarithromycin 1 320.0 0.25 32 erythromycin 1 320.0 0.25 32 gentamicin 8 320.0 1 1024 -Haemolytic streptococci (11) penicillin 0.06 0.06 0.03 0.06 amoxycillin 0.03 0.06 0.015 0.5 quinupristin/dalfopristin 0.5 10.0 0.5 2 vancomycin 0.5 10.0 0.5 1 teicoplanin 0.25 0.25 0.06 0.25 cefuroxime 0.25 10.0 0.125 1 cefotaxime 0.03 0.03 0.015 0.03 clindamycin 0.06 0.50 0.06 0.5 clarithromycin 0.03 0.06 0.015 0.06 erythromycin 0.06 0.125 0.03 0.25 77

J. W. Mouton et al. Table II. Comparative in-vitro activity of quinupristin/dalfopristin and other antibiotics against viridans streptococci isolated from patients with endocarditis Species (number of strains) MIC 50 MIC 90 MIC range and antibiotic (mg/l) (mg/l) (mg/l) S. bovis (19) penicillin 0.06 10.00 0.03 2 amoxycillin 0.06 0.250 0.06 8 quinupristin/dalfopristin 2 160.00 0.015 16 vancomycin 0.5 10.00 0.5 1 teicoplanin 0.25 0.500 0.125 1 cefuroxime 0.125 0.125 0.03 0.125 cefotaxime 0.125 0.250 0.125 0.25 clindamycin 0.125 160.00 0.015 16 clarithromycin 0.125 320.00 0.125 32 erythromycin 0.5 320.00 0.125 32 gentamicin 4 320.00 1 32 S. gordonii (23) penicillin 0.03 0.500 0.015 16 amoxycillin 0.06 0.500 0.015 16 quinupristin/dalfopristin 1 20.00 0.5 8 vancomycin 1 10.00 0.5 2 teicoplanin 0.25 20.00 0.06 8 cefuroxime 0.03 0.030 0.03 0.25 cefotaxime 0.03 0.030 0.03 0.25 clindamycin 0.03 0.060 0.015 0.25 clarithromycin 0.125 0.125 0.125 32 erythromycin 0.125 0.125 0.125 32 gentamicin 2 40.00 0.25 16 S. mitis (28) penicillin 0.06 20.00 0.015 4 amoxycillin 0.06 0.500 0.015 1 quinupristin/dalfopristin 1 40.00 0.015 8 vancomycin 1 20.00 0.5 4 teicoplanin 0.125 0.250 0.03 1 cefuroxime 0.06 0.500 0.03 4 cefotaxime 0.06 0.500 0.03 1 clindamycin 0.03 0.030 0.015 0.06 clarithromycin 0.125 10.00 0.125 4 erythromycin 0.125 0.125 0.125 0.25 gentamicin 2 80.00 1 32 Streptococcus mutans (18) penicillin 0.03 0.060 0.015 0.25 amoxycillin 0.03 0.125 0.015 0.25 quinupristin/dalfopristin 0.5 40.00 0.015 8 vancomycin 1 20.00 0.5 2 teicoplanin 0.125 10.00 0.015 2 cefuroxime 0.03 0.250 0.03 0.25 cefotaxime 0.03 0.125 0.03 0.125 clindamycin 0.015 160.00 0.015 16 clarithromycin 0.125 320.00 0.125 32 erythromycin 0.125 320.00 0.125 32 gentamicin 2 40.00 0.25 8 78

Quinupristin/dalfopristin against endocarditis isolates Table II. Continued Species (number of strains) MIC 50 MIC 90 MIC range and antibiotic (mg/l) (mg/l) (mg/l) Streptococcus oralis (32) penicillin 0.03 0.250 0.015 8 amoxycillin 0.03 0.125 0.015 0.5 quinupristin/dalfopristin 1 20.00 0.5 16 vancomycin 1 20.00 0.06 2 teicoplanin 0.25 0.250 0.03 4 cefuroxime 0.125 0.125 0.03 5 cefotaxime 0.06 0.125 0.03 0.25 clindamycin 0.03 0.060 0.015 0.125 clarithromycin 0.125 0.125 0.125 32 erythromycin 0.125 0.125 0.125 32 gentamicin 8 160.00 0.5 32 Viridans streptococci, other (66) penicillin 0.06 0.500 0.015 1 amoxycillin 0.06 10.00 0.015 16 quinupristin/dalfopristin 1 40.00 0.015 32 vancomycin 1 20.00 0.25 4 teicoplanin 0.25 0.500 0.03 16 cefuroxime 0.06 0.250 0.03 0.5 cefotaxime 0.06 0.125 0.03 2 clindamycin 0.03 0.060 0.015 16 clarithromycin 0.125 20.00 0.125 32 erythromycin 0.125 0.125 0.125 32 gentamicin 4 160.00 0.5 32 Table III. Cross-resistance to erythromycin and quinupristin/dalfopristin in staphylococci and viridans streptococci Quinupristin/dalfopristin (mg/l) Isolates (number) MIC 50 MIC 90 range S. aureus erythromycin-susceptible (n 83) 0.5 10. 0.5 2 erythromycin-resistant (n 3) 0.5 10. 0.5 1 Coagulase-negative staphylococci erythromycin-susceptible (n 8) 0.5 10. 0.25 1 erythromycin-resistant (n 28) 0.5 0.5 0.25 0.5 Viridans streptococci erythromycin-susceptible (n 173) 10. 40. 0.015 32 erythromycin-resistant (n 13) 20. 160. 0.5 16 two groups, indicating the MLS B type of resistance. Hence quinupristin/dalfopristin may be used in treating infections due to erythromycin-resistant strains, 2 despite known cross-resistance between erythromycin and quinupristin. For the viridans streptococci, there was a difference between erythromycin-resistant and erythromycin-susceptible strains with respect to susceptibility to quinupristin/dalfopristin, shown both by the difference in susceptibility between the two groups and a correlation between susceptibility to erythromycin and to quinupristin/dalfopristin. The same was true for clarithromycin (results not shown). This suggests that there is some form of cross-resistance between these agents, probably dependent on the mechanism of resistance of 79

J. W. Mouton et al. the individual strain (as indicated by the low correlation (r 0.174), significant only because of the large number of strains tested). It may be concluded that quinupristin/dalfopristin is a potential therapeutic agent for serious infections, such as endocarditis, due to Gram-positive cocci. Although crossresistance to macrolides in viridans streptococci is low, susceptibility of individual strains should be taken into account when considering its use in the treatment of infections. Further clinical studies should be undertaken to determine its role in the treatment of patients with endocarditis due to Gram-positive cocci. References 1. Fass, R. (1991). In vitro activity of RP 59500, a semisynthetic injectable pristinamycin, against staphylococci, streptococci and enterococci. Antimicrobial Agents and Chemotherapy 35, 553 9. 2. Brumfitt, W., Hamilton-Miller, J. M. T. & Shah, S. (1992). In-vitro activity of RP 59500, a new semi-synthetic streptogramin antibiotic, against Gram-positive bacteria. Journal of Antimicrobial Chemotherapy 30, Suppl. A, 29 38. 3. Verbist, L. & Verhaegen, J. (1992). Comparative in-vitro activity of RP 59500. Journal of Antimicrobial Chemotherapy 30, Suppl. A, 39 44. 4. Vasquez, D. (1967). Mechanism of action. In The Strepto - gramin Family of Antibiotics, Vol. 1 (Gottlieb, D. & Shaw, D., Eds), pp. 387 403. Springer Verlag, NY. 5. Aumercier, M., Bouhallab, S., Capmau, M.-L. & Le Goffic, F. (1992). RP 59500: a new proposed mechanism for its bacterial activity. Journal of Antimicrobial Chemotherapy 30, Suppl. A, 9 14. 6. Pechere, J. C. (1992). In-vitro activity of RP 59500, a semisynthetic streptogramin, against staphylococci and streptococci. Jour - nal of Antimicrobial Chemotherapy 30 Suppl. A, 15 8. 7. van der Meer, J. T. M., van Vianen, W., Hu, E., van Leeuwen, W., Valkenburg, H. A., Thompson, J. & Michel, M. F. (1991). Distribution, antibiotic susceptibility and tolerance of bacterial isolates in culture-positive cases of endocarditis in the Netherlands. Euro - pean Journal of Clinical Microbiology and Infectious Diseases 10, 728 34. 8. Facklam, R. R. & Carey, R. B. (1985). Streptococci and aerococci. In Manual of Clinical Microbiology (Lennette, E. H., Balows, A., Hausler, W. J. & Shadomy, H. J., Eds), pp. 54 175. American Society for Microbiology, Washington, DC. 9. National Committee for Clinical Laboratory Standards. (1990). Methods for Dilution Antimicrobial Susceptibility Tests for Bacteria that Grow Aerobically, 2nd edn; Approved Standard M7-A2. NCCLS, Villanova, PA. 10. Sahm, D. F. & Washington, J. A. (1991). Antibacterial susceptibility tests: dilution methods. In Manual of Clinical Micro - biology (Balows, A., Hausler, W. J., Hermann, K. L., Isenberg, H. D. & Shadomy, H. J., Eds.), pp. 1105 16. American Society for Microbiology, Washington, DC. 11. SAS Institute Inc. (1990). SAS User s Guide. SAS Institute, Inc., Cary, NC. 12. Weisblum, B. (1985). Inducible resistance to macrolides, lincosamides and streptogramin type B antibiotics: the resistance phenotype, its biological diversity and structural elements that regulate expression a review. Journal of Antimicrobial Chemotherapy 16, Suppl. A, 63 90. 13. Etienne, S. D., Maontay, G., Le Liboux, A., Frydman, A. & Garaud, J. J. (1992). A phase I, double-blind, placebo controlled study of the tolerance and pharmacokinetic behaviour of RP 59500. Journal of Antimicrobial Chemotherapy 30, Suppl. A, 123 31. 14. Neu, H. C., Chin, N. & Gu, J. (1992). The in vitro activity of new streptogramins, RP 59500, RP 57669, and RP 54476, alone and in combinations. Journal of Antimicrobial Chemotherapy 30, Suppl. A, 83 94. 15. Cremieux, A. C., Maziere, B., Vallois, J. M., Ottaviani, M., Azancot, A., Raffoul, H. et al. (1989). Evaluation of antibiotic diffusion into cardiac vegetations by quantitative autoradiography. Journal of Infectious Diseases 159, 938 44. 16. Chambers, H. F. (1992). Studies of RP 59500 in vitro and in a rabbit model of aortic valve endocarditis caused by methicillinresistant Staphylococcus aureus. Journal of Antimicrobial Chemotherapy 30, Suppl. A, 117 20. 17. Soussy, C. J., Acar, J. F., Cluzel, R., Courvalin, P., Duval, J., Fleurette, J. et al. (1992). A collaborative study of the in-vitro sensitivity to RP 59500 of bacteria isolated in seven hospitals in France. Journal of Antimicrobial Chemotherapy 3 0, Suppl. A, 5 3 8. 18. Hoban, D. J., Weshnoweski, B., Palatnick, L., Zhanel, L. & Davidson, R. L. (1992). In-vitro activity of streptogramin RP 59500 against staphylococci, including bacterial kinetic studies. Journal of Antimicrobial Chemotherapy 30, Suppl. A, 59 65. 19. Duval, J. (1985). Evolution and epidemiology of MLS resistance. Journal of Antimicrobial Chemotherapy 16, Suppl. A, 137 49. 20. Le Goffic, F., Capmeau, M.-L., Bonnet, D., Cerceau, C., Soussy, C., Dublanchet, A. et al. (1977). Plasmid-mediated pristinamycin resistance. PAC IIA a new enzyme which modifies pristinamycin IIA. Journal of Antibiotics 30, 665 9. 21. Leclerc, R. & Courvalin, P. (1991). Intrinsic and unusual resistance to macrolide, lincosamide, and streptogramin antibiotics in bacteria. Antimicrobial Agents and Chemotherapy 35, 1273 6. 80