P. C. Appdbamn', S. K. Spangkr', E. Crotty* and M. R. Jacobs 1 '

Journal of Antimicrobial Chemotherapy (1989) 23, 509-516 Susceptibility of penicillin-sensitive and -resistant strains of Streptococcus pneumoniae to new antimicrobial agents, including daptomydn, teicoplanin, cefpodoxime and qninolones P. C. Appdbamn', S. K. Spangkr', E. Crotty* and M. R. Jacobs 1 ' 'Departments of Pathology (Clinical Microbiology). Hershey Medical Center, Hershey, PA 17033; 'University Hospitals of Cleveland, 47 Abington Road, Cleveland, OH 44106, USA The minimal inhibitory concentrations (MICs) of nine antibiotics were determined by agar dilution on 123 strains of Streptococcus pneumoniae (65 penicillin sensitive, 42 intermediate resistant and 16 resistant). The antimicrobial agents tested were penicillin G, clindamycin, trospectomycin, daptomydn, teicoplanin, cefpodoxime, dprofloxarin, ofloxacin and vancomycin. Of these, daptomydn, teicoplanin and vancomydn demonstrated the greatest in-vitro activity against penicillin-resistant strains (MIC^s < 025 mg/1). Gprofloxarin, ofloxacin and trospectomycin had equivalent activities unaffected by penicillin-susceptibility (MIC, 0 of both quinolones mg/1, and of trospectomycin 4-0 mg/1). Cefpodoxime was also active in vitro against all strains (MIC^o mg/1), but MICs increased with increasing penidllin-mics. Most penicillin-susceptible strains were susceptible to clindamycin, but many penicillin intermediate resistant and resistant strains were resistant to this drug. Results of this study indicate that several newly introduced and experimental antibiotics have potential in the treatment of infections caused by resistant strains of Str. pneumoniae. Introduction Streptococcus pneumoniae continues to be a significant cause of morbidity and mortality in humans, and is the leading cause of bacterial pneumonia as well as an important cause of meningitis, bacteraemia and otitis media. Although this organism was originally exquisitely sensitive to penicillin, the last two decades have witnessed the emergence of strains resistant to penicillin as well as other antimicrobials in many parts of the world (Ward & Koornhof, 1980; Tweardy, Jacobs & Speck, 1983; Appelbaum, 1987). Although non-meningitic infections due to Str. pneumoniae with intermediate and resistant penicillin MICs may, under certain circumstances, be successfully treated with high doses of antibiotics such as penicillin to which they are resistant in vitro, treatment may not always be successful. The high doses of potentially toxic antibiotics used may lead to unwanted side-effects (Ward & Koornhof, 1980). In contrast, clinical failure of penicillin in the treatment of meningitis caused by intermediate penicillin-resistant strains approaches 80%, and no cases of meningitis due to penicillin-resistant strains have responded to penicillin (Tweardy et al., 1983). The increased in-vitro activity of many new antimicrobial agents (Pallanza et al., 1983; Eliopoulos, Gardella & Moellering, 1984; Gombert & Aulicino, 1984; van 509 O3O5-7453/89/O4O5O9+O8 $02.00/0 1989 The Britiih Society for Antimicrobial Chemotherapy Downloaded from http://jac.oxfordjournals.org/ at Pennsylvania State University on March 3, 16

510 P. C. Appdbunn et al Caekenberghe & Pattyn, 1984; Vcrbist et at., 1984; Williams & Gruneberg, 1984; King, Shannon & Phillips, 1985; Fass & Helsel, 1986; Mandell & Ncu, 1986; Saito et at., 1987; Fass & Helse( 1988; Jones & Barry, 1988; Zurenko et at., 1988), together with substantial cerebrospinal fluid (CSF) penetration in some cases (Isaacs et at., 1986; Neihart et at., 1987), suggests that these drugs may play an increasing role in the treatment of mcningitic and non-meningitic infections with penicillin-resistant strains of Str. pnewnoniae. The aim of this in-vitro study was to determine the minimal inhibitory concentration (MIC) of two quinolones (ciprofloxacin, ofloxacin), trospectomycin, daptomycin (LY14O632), teicoplanin, cefpodoxime (U-76253A), clindamycin and vancomycin for 123 pneumococcal strains (65 penicillin-sensitive, 42 intermediate resistant and 16 fully resistant). Bacterial isolates Methods One hundred and twenty-three clinical isolates of S. pneumoniae, obtained from blood, CSF, nasopharynx or sputum, were used in this study. Sixty-five of the isolates were penicillin-sensitive strains (MIC < 01 mg/1) isolated at the University Hospitals of Cleveland. Forty-two of the isolates were classed as intermediate penicillin-resistant strains, with MICs between 01 and 10 mg/1, and 16 isolates were penicillin-resistant strains with MICs > 1 mg/1. The majority of intermediate strains and all resistant strains were isolated in Durban and Johannesburg, South Africa between 1977 and 1986. We have no evidence that the strains tested were epidemiologically related. South African strains were collected over a ten-year period from different carriers throughout the country, and represented prevalent resistant serotypes and antibiograms. Other strains were isolated from symptomatic patients in the USA and also were probably not epidemiologically related. Antimicrobial agents Antimicrobial agents used were ciprofloxacin (Miles Pharmaceuticals, West Haven, CT); ofloxacin (Ortho Pharmaceuticals, Raritan, NJ); trospectomycin, cefpodoxime, clindamycin (The Upjohn Co., Kalamazoo, MI); teicoplanin (Merrell-Dow Research Institute, Cincinnatti, OH); benzylpenicillin, vancomycin, daptomycin (Eli Lilly & Co., Indianapolis, IN). Agents were supplied as laboratory powders of known potency, and stock solutions were made as recommended by the manufacturers. Downloaded from http://jac.oxfordjournals.org/ at Pennsylvania State University on March 3, 16 MIC determination MICs were determined by the agar dilution method (Tweardy et al., 1983) in 15 x 100 plastic Petri dishes containing 25 ml Mueller-Hinton agar (BBL Microbiology Systems, Cockeysville, MD) supplemented with 5% sheep blood, incorporating the above antimicrobials in concentrations from 0-008 to 32 mg/1 in doubling dilutions. After pouring, plates were dried, wrapped in plastic and refrigerated at 4 C prior to use within seven days. For MIC testing, suspensions with a turbidity equivalent to that of a 0-5 McFarland standard were prepared by suspending growth from blood agar plates in 2 ml

In-Titro susceptibility of Str. pneumonia* 511 Mueller-Hinton broth. Suspensions were further diluted 1 : 100 to obtain a final inoculum of 10* organisms in 2 fd. Plates were inoculated using a Steers replicator with 3 mm inoculating pins, and incubated overnight at 37 C. The lowest concentration of antibiotic showing no growth was read as the MIC for each isolate against each antibiotic. Quality control organisms were included with each run: these included Staphylococcus aureus ATCC 29213, Escherichia coli ATCC 25922, Enterococcus faecalis ATCC 29212, Pseudomonas aeruginosa ATCC 27853, and a known penicillin-susceptible strain of Str. pnenmoniae (WRU 294). Results The MIC 90 of penicillin G was 003 mg/1 against penicillin-susceptible strains, 10 mg/1 against intermediate penicillin-resistant strains, and 40mg/1 against resistant strains (Table I). Comparison of the MIC 90 s of penicillin with the other drugs tested revealed that penicillin G was the most active agent in vitro against penicillin-sensitive strains. However, against intermediate and resistant strains, in-vitro activity, superior to that of penicillin, was demonstrated by vancomycin (MIC 90 ^ 0-25 mg/1), daptomycin (MIC 90 mg/1) and teicoplanin (MIC 90 mg/1). Ciprofloxacin, ofloxacin and trospectomycin showed similar MIC 90 s for sensitive, intermediate and resistant strains. Cefpodoxime was very active in vitro against penicillin-sensitive strains (MIC 90 003mg/1), but MIC 90 s increased with the penicillin MIC against intermediate and resistant strains ( mg/1 for both groups). Most penicillin-sensitive strains (91%) were susceptible to clindamycin (MIC <01 mg/1), but several intermediate and most resistant strains were resistant in vitro to this drug (MIC 90 >32 mg/1) (Table I). Most penicillin-susceptible, clindamycin-resistant strains of Str. pneumoniae were recent South African isolates (Klugman et al., 1986), as were all penicillin intermediate resistant and resistant strains which were also resistant to clindamycin. Discussion In recent studies, in-vitro activity superior to that of penicillin against penicillin-resistant strains of Str. pneumoniae has been demonstrated for cefotaxime, cefoperazone, imipenem, ceftriaxone, vancomycin, ciprofloxacin, coumermycin and novobiocin (Watanakunakorn & Glotzbecker, 1980; Ward & Moellering, 1981; Landesman et al., 1981; Tweardy et al., 1983; Gombert & Aulicino, 1984). Our studies add daptomycin and teicoplanin to this list. By comparison, ciprofloxacin, ofloxacin and trospectomycin had less in-vitro activity in our study (MIC 9O s 2-4 mg/1), but susceptibility was unaffected by the penicillin susceptibility status of pneumococcal strains. Data on the distribution of ciprofloxacin and ofloxacin in the body have been reported (Hooper & Wolfson, 1985; Wolfson & Hooper, 1985; Neu, 1987). For ciprofloxacin after a 500 mg oral dose, maximal serum concentrations of 1-9-2-9 mg/1 are obtained, with a terminal elimination half-life of 3-3-4-9 h. Corresponding values after a 600 mg oral dose of ofloxacin are 11 mg/1 and 7-0 h, respectively (Hooper & Wolfson, 1985). Although the overall in-vitro activity of both quinolones against resistant and sensitive pncumococci in our study approaches the susceptibility breakpoint for both drugs (Eliopoulos et al., 1984; King et al., 1985), a good response Downloaded from http://jac.oxfordjournals.org/ at Pennsylvania State University on March 3, 16

512 P. C. Appdbmmn et al Table L In-vitro susceptibility of 123 strains of Str. pneumoniae Antimicrobial agent MIC J0 (mg/1) MIC, 0 (mg/1) Penicillin susceptible intermediate resistant 0-015 0-25 0-03 1-0 4-0 Daptomycin P-intcr Teicoplanin Vancomycin Ciprofloxacin Ofloxacin Trospectomycin P-susccpt Cefpodoxime Clindamycin 0-125 0-016 003 0-06 sjo-25 s 0-25 <0-25 10 10 10 1-0 10 0015 1-0 0-06 006 0-25 0-125 <0-25 <0-25 <0-25 40 40 40 003 16 >32 >32 Downloaded from http://jac.oxfordjournals.org/ at Pennsylvania State University on March 3, 16 to ciprofloxacin has been reported in the treatment of respiratory tract infections, including those due to penicillin-resistant strains (Raoof, Wollschlager & Khan, 1986). Although ciprofloxacin has been used for treatment of P. aeruginosa ventriculitis (Isaacs et al., 1986), achievable levels in the uninflamed CSF (0-13-0-30 mg/1, compared to serum levels of 9-304mg/1) show that drug levels may not be adequate for Gram-positive, as opposed to Gram-negative, organisms (McClain, Rhoads & Krol, 1988). The equivalent drug penetration of ciprofloxacin and ofloxacin

In-vitro susceptibility of Str. pruumoniae 513 (Hooper & Wolfson, 1985; Wolfson & Hooper, 1985) suggests that similar therapeutic conclusions may be drawn for both drugs. Peak teicoplanin serum concentrations of 7 mg/1 have been observed 4 h after the first of a six dose intramuscular 0 mg regimen (three doses every 12 h followed by three every 24 h). The peak height after the sixth dose was 12 mg/1, also appearing after 4 h. Trough levels ranged between 5-4-7-3 mg/1 from days 2-6, with a terminal half-life >40h (Williams & Griineberg, 1984). Although the latter data predict that good penetration of teicoplanin into body tissue occurs, poor CSF penetration of this drug has been documented in patients with bacterial meningitis. Levels ranging from <0-3 mg/1 in six patients to 0-8-1-3 mg/1 in one patient were observed, compared with serum levels of 2-6-23-0 mg/1 (Stahl et al., 1987). However, low MICJOS ( mg/1) found in our study, taken together with achievable CSF levels of up to 1-3 mg/1 in the one patient mentioned above (Stahl et al., 1987), may reflect possible use of this drug in selected cases of resistant pneumococcal meningitis. Results of the current study indicate that the drug has potential for use in the treatment of non-meningitic infections caused by resistant pneumococci. Conclusions on these therapeutic aspects must await experimental and clinical studies. Trospectomycin is a novel spectinomycin analogue with broad-spectrum antibacterial activity. The drug is 4-32 times more active than spectinomycin against many bacterial species including streptococci. It is moderately active (compared to spectinomycin) against most species of Enterobacteriaceae and is generally cross-resistant with spectinomycin in these organisms (Zurenko et al., 1988). All pneumococcal strains in our study were sensitive to trospectomycin at a preliminary breakpoint of < 32 mg/1 (Zurenko et al., 1988). Phannacokinetic data on trospectomycin indicate potentially good tissue levels. For intramuscular administration, the peak blood level is 0-05 x the dose in mg, such that a 1000 mg dose will yield a peak blood level of 50 mg/1, and a 500 mg dose a level of 25 mg/1. With intravenous administration, the peak blood level is 0-1 x the dose in mg, such that a 500 mg dose will yield a peak level of 50 mg and a 250 mg dose a level of 25 mg/1 (Dr Donald Batts, personal communication). The mean serum half-life of a looog intramuscular dose of trospectomycin is 2-18 h (Zurenko et al., 1988). Preliminary studies show good CSF penetration, with CSF levels of 51 mg/1 in rabbits with inflamed meninges compared with 096mg/1 in normal controls (Neihart et al., 1987), and therapeutic efficacy in the treatment of experimental meningitis due to Str. pneumoniae. This, in conjunction with good in-vitro activity against resistant as well as sensitive pneumococci, reflects a possible role for this drug in the treatment of non-meningitic as well as meningjtic pneumococcal infection due to sensitive or resistant strains. Cefpodoxime is an oral cephalosporin with a broad in-vitro spectrum of activity, but not against strains of P. aeruginosa, Acinetobacter spp., enterococci or methiciuin-resistant staphylococci (Jones & Barry, 1988; Fass & Helsel, 1988). Although cefpodoxime MICs increased with those of penicillin, MIC 90 s for resistant strains were still < 2 mg/1. Susceptible breakpoints of 1, 2 and 4 mg/1 have been suggested for cefpodoxime, but a final decision on which one to use has not yet been made (Jones & Barry, 1988). Adequate levels of cefpodoxime are achieved for the potential therapy of infections caused by susceptible organisms. For a dose of 0 mg every 12 h, the peak serum level is 2-42 mg/1 (mean half-life 2-70 h) and for 400 mg every 12 h the level is 3-39 mg/1 (mean half-life 2-72 h). No data on CSF penetration Downloaded from http://jac.oxfordjournals.org/ at Pennsylvania State University on March 3, 16

514 P. C. Appdbamn et al are available (Dr Donald Batts, personal communication). Because of its proposed oral route of administration, cefpodoxime shows promise for outpatient therapy of infections such as otitis media in children. Initial clinical studies with daptomycin at doses of 2mg/kg/day did not show adequate clinical efficacy and further studies with higher doses (up to 6 mg/kg/day) are being planned (Sexton et al., 1988). A mean serum peak level in humans of approximately 16mg/l is achieved after intravenous administration of single doses of daptomycin at 1 mg/kg (Bush, Boscia & Kaye, 1988). Although daptomycin is well distributed in tissues, CSF levels in uninflamed meninges are negligible. However, no information is currently available on drug levels in inflamed meninges (Dr David Preston, personal communication). Lack of effective penetration of clindamycin into CSF precludes its use in pneumococcal meningitis. However, it may be used in other systemic infections caused by clindamycin-susceptible pneumococci, which comprise most isolates from the USA and Europe (Appelbaum, 1987). Results of this study indicate that there may be a place for all the antimicrobial agents investigated in the treatment of non-meningitic infection caused by penicillin resistant strains of Str. pneunwniae. However, only vancomycin and possibly trospectomycin penetrate well enough through the blood-brain barrier to indicate therapeutic efficacy in meningitis caused by these strains. Acknowledgements This study was funded in part by grants from Ortho Pharmaceuticals, Raritan, NJ, The Upjohn Company, Kalamazoo, MI and Miles Pharmaceuticals, West Haven, CT. We thank Professor H. J. Koornhof, South African Institute For Medical Research, for kind provision of some pneumococcal strains. References Appelbaum, P. C. (1987). World-wide development of antibiotic resistance in pneumococci. European Journal of Clinical Microbiology 6, 367-77. Bush, L. M., Boscia, J. A. & Kaye, D. (1988). Daptomycin (LY146032) treatment of experimental enterococcal endocarditis. Antimicrobial Agents and Chemotherapy 32, 877-81. Eliopoulos, G. M., Gardella, A. & Moellering, R. C. (1984). In vitro activity of ciprofloxacin, a new carboxyquinolone antimicrobial agent. Antimicrobial Agents and Chemotherapy 25, 331-5. Fass, R J. & Helsel, V. L. (1986). In vitro activity of LY146032 against staphylococci, streptococci, and enterococci. Antimicrobial Agents and Chemotherapy 30, 781-4. Fass, R. J. & Helsel, V. L. (1988). In vitro activity of U-76,252 (CS-807), a new oral cephalosporin. Antimicrobial Agents and Chemotherapy 32, 1082-5. Gombert, M. E. & Aulicino, T. M. (1984). Susceptibility of multiply antibiotic-resistant pneumococci to the new quinolone antibiotics, nalidixic acid, coumermycin, and novobiocin. Antimicrobial Agents and Chemotherapy 26, 933 4. Hooper, D. C. & Wolfson, J. S. (1985). The fluoroquinolones: pharmacology, clinical uses, and toxicities in humans. Antimicrobial Agents and Chemotherapy 28, 716-21. Isaacs, D., Slack, M. P. E., Wilkinson, A. R. & Westwood, A. W. (1986). Successful treatment of pseudomonas ventriculitis with ciprofloxacin. Journal of Antimicrobial Chemotherapy 17, 535-8. Jones, R. N. & Barry, A. L. (1988). Antimicrobial activity and disk diffusion susceptibility Downloaded from http://jac.oxfordjournals.org/ at Pennsylvania State University on March 3, 16

Io-vitro susceptibility of Str. pneumoniae 515 testing of U-76, 253A (R-3746), the active metabolite of the new cephalosporin ester, U-76, 252 (CS-807). Antimicrobial Agents and Chemotherapy 32, 443-9. King, A., Shannon, K. & Phillips, I. (1985). The in-vitro activities of enoxacin and ofloxacin compared with that of ciprofloxacin. Journal of Antimicrobial Chemotherapy 15, 551-8. Klugman, K. P., Koornhof, H. J., Kuhnle, V., Miller, S. D., Ginsburg, P. J. & Mauff, A. C. (1986). Meningitis and pneumonia due to novel multiply resistant pneumococci. British Medical Journal 292, 730. Landesman, S. H., Cummings, M., Gruarin, A. & Bemheimer, H. (1981). Susceptibility of multiply antibiotic-resistant pneumococci to the new beta-lactam drugs and rosaramicin. Antimicrobial Agents and Chemotherapy 19, 675-7. Mandell, W. & Neu, H. C. (1986). In vitro activity of CI-934, a new quinolone, compared with that of other quinolones and other antimicrobial agents. Antimicrobial Agents and Chemotherapy 29, 852-7. McClain, J. B., Rhoads, J. & Krol, G. (1988). Cercbrospinal fluid concentrations of ciprofloxacin in subjects with uninflamed meninges. Journal of Antimicrobial Chemotherapy 21, 808-9. Neihart, R. E., Lee, V. W., Downs, N., Harms, J., Hinthorn, D. R. & Uu, C. (1987). Trospectomycin penetration into CSF and comparative effectiveness in experimental meningitis. In Program and Abstracts of the Twenty-seventh Interscience Conference on Antimicrobial Agents and Chemotherapy, New York, 1987. Abstract 270, p. 143. American Society for Microbiology, Washington, DC. Neu, H. C. (1987). Clinical use of the quinolones. Lancet ii, 1319-22. Pallanza, R., Berti, M., Goldstein, B. P., Mapelli, E., Randisi, E., Scotti, R. et al. (1983). Teichomycin: in-vitro and in-vivo evaluation in comparison with other antibiotics. Journal of Antimicrobial Chemotherapy 11, 419-25. Raoof, S., Wollschlager, C. & Khan, F. (1986). Treatment of respiratory tract infections with ciprofloxacin. Journal of Antimicrobial Chemotherapy 18, Suppl. D, 139-45. Saito, H., Rolston, K. V. I., Ho, D. H., LeBlanc, B. & Bodey, G. P. (1987). In-vitro activity of LY146032 against Gram-positive isolates from cancer patients. Journal of Antimicrobial Chemotherapy, 497-503. Sexton, D., Brown, R., McCloslcey, R., Dowell, A. R. & the Daptomycin Study Group. (1988). The use of daptomycin, a lipopeptide antibiotic, in the treatment of gram positive infections in man. In Program and Abstracts of the Twenty-eighth Interscience Conference on Antimicrobial Agents and Chemotherapy, Los Angeles, CA, 1988. Abstract 932, p. 275. American Society for Microbiology, Washington, DC. Stahl, J. P., Croize, J., Wolff, M., Garaud, J. J., Ledercq, P., Vachon, F. et al. (1987). Poor penetration of teicoplanin into cerebrospinal fluid in patients with bacterial meningitis. Journal of Antimicrobial Chemotherapy, 141-2. Tweardy, D. J., Jacobs, M. R. & Speck, W. T. (1983). Susceptibility of penicillin-resistant pneumococci to eighteen antimicrobials: implications for treatment of meningitis. Journal of Antimicrobial Chemotherapy 12, 133-9. van Caekcnberghe, D. L. & Pattyn, S. R. (1984). In vitro activity of ciprofloxacin compared with those of other new fluorinated piperazinyl-substituted quinolone derivatives. Antimicrobial Agents and Chemotherapy 25, 518-21. Verbist, L., Tjandramaga, B., Hendrickx, B., van Hecken, A., van Melle, P., Verbesselt, R. et al. (1984). In vitro activity and human pharmacokinetics of teicoplanin. Antimicrobial Agents and Chemotherapy 26, 881-6. Ward, J. & Koornhof, H. (1980). Antibiotic-resistant pneumococci. In Current Clinical Topics in Infectious Diseases No. 1 (Remington, J. S. & Swartz, M. N., Eds), pp. 265-87. McGraw Hill, New York. Ward, J. I. & Moellering, R. C. (1981). Susceptibility of pneumococci to 14 beta-lactam agents: comparison of strains resistant, intermediate-resistant, and susceptible to penicillin. Antimicrobial Agents and Chemotherapy, 4-7. Watanakunakom, C. & Glotzbecker, C. (1980). Susceptibility of recent clinical isolates of Streptococcus pneumoniae to 17 antibiotics. Journal of Antimicrobial Chemotherapy 6, 83-9. Williams, A. H. & Gruneberg, R. N. (1984). Teicoplanin. Journal of Antimicrobial Chemotherapy 14, 441-5. Downloaded from http://jac.oxfordjournals.org/ at Pennsylvania State University on March 3, 16

516 P. C. Appdbamn et al Wolfson, J. S. & Hooper, D. C. (1985). The fluoroquinolones: structures, mechanisms of action and resistance, and spectra of activity in vitro. Antimicrobial Agents and Chemotherapy 28, 581-6. Zurcnko, G. E., Yagi, B. H., Vavra, J. J. & Wentworth, B. B. (1988). In vitro antibacterial activity of trospectomycin (U-63366F), a novel spectinomycin analog. Antimicrobial Agents and Chemotherapy 32, 216-23. {Received 16 August 1988; revised version accepted 18 November 1988) Downloaded from http://jac.oxfordjournals.org/ at Pennsylvania State University on March 3, 16