Chemotherapy 1991;37:275-282
In vitro Activity of Sparfloxacin and Six Reference Antibiotics against Gram-Positive Bacteria Arnold Louie, Aldona L. Baltch, William J. Ritz, Raymond P. Smith Division of Infectious Diseases, Departments of Medicine and Pharmacology, Albany Department of Veterans Affairs Medical Center and the Albany Medical College, Albany, N.Y., USA
Key Words. Sparfloxacin • AT-4140 • Fluoroquinolones • Gram-positive bacteria
Introduction
The currently available fluoroquino lones possess potent activity against gram negative bacilli and have proven efficacy in the treatment of gram-negative bacte rial infections. Their activity against gram
positive cocci, however, is not high [1-3]. Consequently, failure of these antibiotics to eradicate gram-positive bacterial infec tions is not uncommon [4, 5]. Sparfloxacin (AT-4140, CI-978, PD 131501) is a new fluoroquinolone (fig. 1). Preliminary studies suggest that sparfloxa-
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Abstract. The in vitro activity of sparfloxacin, a new fluoroquinolone, was assessed against 234 gram-positive bacterial isolates by agar dilution (104 CFU/spot). Sparfloxacin activity was compared with that of ciprofloxacin and five other antibiotics. Sparfloxacin was the most active drug tested against methicillin-sensitive and methicillin-resistant Staphylococcus aureus (MRSA) and coagulase-negative staphylococci (MIC90, 0.125— 0.25 mg/1). Sparfloxacin was also the most active drug tested against Enterococcus faecalis (MIC90, 1 mg/1) and showed equal activity against gentamicin-susceptible and gen tamicin-resistant (MIC > 2,000 mg/1) enterococci. Sparfloxacin was the most active quinolone tested against Streptococcus pneumoniae and S. pyogenes (MIG#), 1 mg/1). Most Corynebacterium jeikeium showed exquisite susceptibility to sparfloxacin (MIC, 0.06-0.25 mg/1). For MRSA, time-kill curves showed sparfloxacin to be rapidly bacte ricidal at the MIC of the organism. Sparfloxacin showed greater and more sustained bactericidal activity than ciprofloxacin and vancomycin at lx and 2x the MIC. Reduc tion in the activity of sparfloxacin occurred with decreased agar pH (from 7.0 to 6.0) and increased bacterial inoculum. Sparfloxacin showed superior activity compared to refer ence drugs against most gram-positive bacteria.
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ment. Control strains were obtained from the Amer ican Type Culture Collection and included: 5. aureus ATCC 29213 and ATCC 25923, and E. faecalis ATCC 29212.
Fig. I. Chemical structure of sparfloxacin (AT-4140, CI-978, PD 131501).
cin has potent activity against a broad spectrum of microorganisms [6, 7]. We evaluated the in vitro activity of sparfloxacin and six reference antibiotics against 234 gram-positive bacterial iso lates: methicillin-sensitive (MSSA) and methicillin-resistant Staphylococcus aureus (MRSA), coagulase-negative staphylococ ci, gentamicin-sensitive (MIC < 2,000 mg/1) and high-level gentamicin-resistant (MIC > 2,000 mg/1) Enterococcus faecalis, Strep tococcus pyogenes, Streptococcus pneumo niae and Corynehacterium jeikeium. We al so examined the bactericidal activity of sparfloxacin, ciprofloxacin and vancomy cin against MRSA by the time-kill curve method. Lastly, we studied the effect of acidic pH and increased bacterial inocu lum on the susceptibility of MRSA and en terococci to sparfloxacin, ciprofloxacin and gentamicin.
Antimicrobial Agents Antibiotics were obtained from the following sources: sparfloxacin (AT-4140, CI-978, PD 131501), Parke-Davis; ciprofloxacin, Miles Inc.; ofloxacin, Ortho; vancomycin, Eli Lilly; erythromycin and pen icillin G, Sigma, and oxacillin, Bristol-Myers Squibb. Antibiotics were prepared as directed by the manu facturers' instructions, sterilized by filtration (0.45pm filter) and used immediately. Susceptibility Testing Antimicrobial susceptibilities were determined by the twofold serial agar dilution technique de scribed by Washington and Sutter [8], Employing a Steers replicator [9], a final inoculum of 10J CFU/ spot was delivered to the surface of antibiotic-con taining Mueller-Hinton agar (MHA; BBL). For the evaluation of antibiotic MICs for streptococcal, enterococcal and C. jeikeium isolates, MHA was sup plemented with 5% defibrinated sheep blood. For oxacillin and penicillin G susceptibility testing against MRSA, NaCl was added to MHA (final con centration 4%). All plates were incubated at 37°C, in air, and read 24 h later. The MIC was defined as the lowest antibiotic concentration at which not more than 1 colony was visible. A faint haze was dis regarded. The effect of acidic pH on drug activity against 15 MRSA and 16 gentamicin-susceptible (MIC < 2,000 mg/1) E. faecalis isolates was assessed by fol lowing the procedure described above. However, the agar pH was adjusted from 7.0 to 6.0 with 10 N HCI before the agar suspension was autoclaved. The inoculum effect was determined by delivering bacte rial concentrations of 102 and 104 CFU/spot onto the same antibiotic-containing agar plate. MICs were determined as described.
Bacterial Isolates All microorganisms were clinical isolates ob tained from the Albany Department of Veterans Af fairs Medical Center and the Bacteriology Labora tory of the Wadsworth Center for Laboratories and Research of the New York State Health Depart
Time-Kill Studies Time-kill curves were established for 2 clinical MRSA isolates against sparfloxacin, ciprofloxacin and vancomycin to assess and compare the bacte ricidal kinetics of these drugs. First, the MIC of the antibiotic for each bacterial isolate was determined by serial macrotubc dilution in Mueller-Hinton
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Materials and Methods
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C. jeikeium can be best appreciated graph ically (fig. 2). This graph shows two pop ulations of organisms, one that was highly sensitive to sparfloxacin and another which was resistant (MIC, 64 mg/l). Organisms that were resistant to sparflox acin demonstrated similar MICs for the other quinolones. Excluding the quinolone-resistant C. jeikeium, the MIC90 for sparfloxacin was 0.25 mg/l and for cipro floxacin 1.0 mg/l. Reduction in agar pH from 7.0 to 6.0 decreased the activity of sparfloxacin, ci profloxacin and gentamicin against each of the 15 MRSA isolates studied. The MICs increased by two tubes. Sparfloxacin and ciprofloxacin MICs also increased by Results two tubes for 10 of the 16 gentamicin-sus The MICs obtained for isolates tested ceptible entcrococcal isolates tested. How at a bacterial inoculum of 104 CFU/spot on ever, a four-tube rise in MICs was observ MHA at pH 7.0 are summarized in table 1. ed for all 16 enterococcal isolates when Sparfloxacin demonstrated potent activity they were tested against gentamicin under against MSSA, MRSA and coagulase-neg- similar conditions (data not shown). ative staphylococci. Sparfloxacin MICs Antibiotic activity for sparfloxacin, ci were 4 times lower than those of ciproflox profloxacin and gentamicin was reduced acin and ofloxacin against all staphylococ with increased MRSA or E. faecalis inocu ci. Gentamicin-sensitive (MIC 2,000 mg/l) E. faecalis were equal 10* CFU/spot, a one-tube increase in MIC ly susceptible to sparfloxacin (MICW of was seen against 9 of 15 MRSA isolates for 1 mg/l for both groups). Ciprofloxacin and sparfloxacin, ciprofloxacin and gentami the other reference compounds were less cin. For gentamicin-susceptible E. faecalis, active than sparfloxacin against the genta sparfloxacin, ciprofloxacin and gentamicin micin-susceptible and gentamicin-resistant showed an inoculum effect for 88, 38 and enterococci with MICgo of >2 mg/l. The 64%, respectively, of the 16 isolates exam susceptibilities of 5. pyogenes and S. pneu ined (data not shown). moniae were best to penicillin G, oxacillin Time-Kill Curx’es and erythromycin. However, among the quinolones, sparfloxacin was at least Ciprofloxacin and sparfloxacin were twice more active than ciprofloxacin and rapidly bactericidal against MRSA at drug concentrations > lx the MIC (fig. 3). Van ofloxacin. The activity of sparfloxacin against comycin was bacteriostatic at lx the MIC
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broth (BBL) supplemented with 25 mg/1 of Mg2+ and 50 mg/l of Ca:+ (MHB-S). The final bacterial in oculum was It)5 CFU/ml. The MIC was defined as the lowest antibiotic dilution at which no growth or haze was visible. Subsequently, bacteria in log phase growth were inoculated into flasks containing MHB-S and antibiotic at concentrations equal to 0.25. 0.5, 1 and 2x the MIC. The final bacterial con centration was 10s CFU/ml. An antibiotic-free flask containing MHB-S and 105 CFU/ml served as a growth control. Culture flasks were incubated at 37°C in a water shaker bath. Samples were taken from each flask at 0, I, 2. 4. 6 and 24 h, serially dilut ed with 0.9% NaCI and inoculated on antibiotic-free trypticase soy agar (BBL). Plates were incubated at 37 °C, and quantitative colony counts were deter mined 24 h later.
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Table 1. Activity of sparfloxacin and reference antibiotics against gram-positive bacteria Antibiotic
MIC. mg/1 range
50%
90%
MSSA (39)
sparfloxacin ciprofloxacin ofloxacin erythromycin vancomycin oxacillin penicillin G
0.06-0.25 0.25-1.0 0.12-1.0 0.25-> 4.0 0.5-2.Ö 0.25-1.0 ¿0.06-64.0
0.06 0.5 1.0 0.25 1.0 0.5 4.0
0.12 0.5 1.0 4.0 1.0 1.0 32.0
MRSA (41)
sparfloxacin ciprofloxacin ofloxacin erythromycin vancomycin oxacillin penicillin G
0.06-8.0 0.25-^64 0.25-32.0 2.0—> 512 1.0-2.0 8.0-512.0 4.0-128.0
0.06 0.5 0.5 >512.0 2.0 256.0 64.0
0.25 2.0 2.0 >512.0 2.0 256.0 64.0
Staphylococci (coagulasc negative, 40)
sparfloxacin ciprofloxacin ofloxacin erythromycin vancomycin oxacillin penicillin G
0.06-16.0 0.03-128 0.25-64 0.12—> 512 0.5-2.0 0.25-> 512 ¿0.01-256
0.12 0.25 0.5 1.0 1.0 1.0 0.5
0.25 2.0 2.0 >512 2.0 256.0 128.0
S. pyogenes (21)
sparfloxacin ciprofloxacin ofloxacin erythromycin vancomycin oxacillin penicillin G
0.5-4.0 0.25-4.0 1-16.0 0.03-0.06 0.5-1.0 0.03-0.25 ¿0.01-0.12
1.0 1.0 4.0 0.06 0.5 0.06 ¿0.01
2.0 4.0 8.0 0.06 0.5 0.06 0.03
S. pneumoniae (19)
sparfloxacin ciprofloxacin ofloxacin erythromycin vancomycin oxacillin penicillin G
0.12-1.0 Ö.5-2.0 0.5-2.0 ¿0.01-0.12 0.25-1.0 0.03-2.0 ¿0.01-0.25
0.5 1.0 2.0 ¿0.01 0.5 0.06 0.03
1.0 2.0 2.0 0.03 0.5 0.5 0.03
C. jeikeium (11)
sparfloxacin ciprofloxacin ofloxacin
0.06-64.0 0.06-64.0 0.25-32.0
0.25 1.0 1.0
0.5-64 1-64 32.0
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Organism
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279
Table 1 (continued) Organism
Antibiotic
MIC, mg/l range
C. jeikeium (II)
erythromycin vancomycin oxacillin penicillin G
¿0.01-512 0.5-1.0 0.25-> 512 0.03-> 512
E. faecalis (Gentamicin MIC, < 2.0(H) mg/l; 44)
Sparfloxacin ciprofloxacin ofloxacin erythromycin vancomycin oxacillin penicillin G
0.25-1.0 0.5-64.0 1-32.0 0.5-> 64.0 0.5-4.0 2—> 512 0.25-> 64.0
E. faecalis (Gentamicin MIC; >2,000 mg/l; 20)
Sparfloxacin ciprofloxacin ofloxacin erythromycin vancomycin oxacillin penicillin G
0.25-1.0 Ö.5-2.0 1.0-4.0 0.5—> 512 1.0-4.0 8.0-256 2.0-8.0
50%
90%
128.0
512.0
1.0
1.0
>512.0 >512.0
>512.0 >512.0
0.5 1.0
2.0 2.0 2.0 16.0 4.0
1.0
4.0 4.0 >64.0 2.0 64—> 512 64.0
0.5-1.0
1.0
1.0
2.0 4.0 >512.0 4.0 128.0 8.0
2.0 >512.0 2.0 32.0 4.0
Fig. 2. The activity of Sparfloxacin (a) and Ciprofloxacin (b) against C.jeikeium.
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Figures in parentheses indicate the numbers of isolates.
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and bactericidal at 2x the MIC, although the bactericidal effect required 24 h to be come apparent. At 24 h, Sparfloxacin dem onstrated a more sustained and greater bactericidal activity than ciprofloxacin and vancomycin for drug concentrations of 1 x and 2x the MIC. At 1x the MIC, bacterial counts for ciprofloxacin were similar to those of controls (l()y CFU/ml), while those for vancomycin were similar to the original inoculum (HP CFU/ml).
Fig. 3. Bactericidal activity at two different con centrations of Sparfloxacin (SPAR) and ciprofloxa cin (CIPRO; a), and Sparfloxacin and vancomycin (VANCO; b) against MRSA. The MICs were: Spar floxacin 0.25 mg/l. ciprofloxacin 0.5 mg/1 and vanco mycin 2 mg/1.
We found Sparfloxacin to have superior gram-positive activity compared to cipro floxacin and ofloxacin against all clinical isolates tested. Moreover, sparfloxacin was the most active of the seven antibiotics studied against MSSA, MRSA, the coagulase-negative staphylococci and the gen tamicin-sensitive and high-level gentami cin-resistant E. faecalis. The excellent activity of sparfloxacin against staphylococci is important. In many institutions, multiply antibioticresistant gram-positive bacteria, such as MRSA and coagulasc-negative staphylo cocci, are responsible for an increasing number of nosocomial infections. These organisms are commonly resistant to antistaphylococcal penicillins, cephalosporins and aminoglycosides. Vancomycin has be come the cornerstone of therapy for treat ing patients infected with these organisms. However, vancomycin must be adminis tered parenterally, is expensive and has certain side effects. Moreover, our timekill curves show that, against MRSA, it takes >6 h before the bactericidal activity of vancomycin becomes apparent. In con
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Discussion
trast, sparfloxacin is bactericidal within 2-4 h. The rapid bactericidal activity of an antibiotic may be crucial in the severely ill or neutropenic patient. Since staphylococ ci are markedly susceptible to sparfloxacin (MIG,,, of 0.125 mg/l for MSSA and 0.25 mg/l for MRSA and coagulase-negative staphylococci) and sparfloxacin is rap idly bactericidal at a concentration equal to the MIC, this drug may be an alterna tive to vancomycin for treating staphylo coccal infections. Sparfloxacin was also the most active drug tested against E.faecalis. Sparfloxa cin was equally active against gentamicinsusceptible and high-level gentamicin-re sistant isolates (MICyo of 1 mg/l for both strains). Ciprofloxacin demonstrated MIG,,) of 4 and 2 mg/l for these two groups, respectively. We believe that we arc the first to report the activity of spar floxacin and reference compounds against gentamicin-susceptible and high-level gen tamicin-resistant enterococci. This distinc tion is important because high-level gen tamicin-resistant organisms account for more than 50% of all enlcrococcal isolates in some institutions 110]. Sparfloxacin may expand the selection of antibiotics that are active against these organisms. We believe that we are also the first to report the susceptibility of C. jeikeium to sparfloxacin. Although some isolates were resistant to all quinolones (MICs of 3264 mg/l), most strains were very suscepti ble. For the susceptible organisms (7 of 11 isolates), the M IG) of sparfloxacin was ‘A of that of ciprofloxacin, with MIGhi of 0.25 and 1 mg/l, respectively. The high activity of sparfloxacin against quinolone-susceptiblc organisms should be evaluated fur ther in a clinical setting.
281
Although currently available quinoloncs have excellent gram-negative and good staphylococcal coverage, their low activity against streptococci, particularly S. pneumoniae, has curtailed their use as empiric therapy for community-acquired pneumonia, bronchitis, sinusitis and otitis media. Studies have reported up to a 33% treatment failure and relapse rate when ci profloxacin was prescribed to treat pneu mococcal pneumonia and bronchitis [5]. Cooper and Lawlor [II] reported a patient with documented bacteremia on the fifth day of ciprofloxacin for S. pneumoniae pneumonia. Our study demonstrates that the in vitro activity of sparfloxacin (MIC9„ of 1 mg/l) is twice that of ciprofloxacin against 5. pneumoniae. Kojima et al. [6] and Nakamura et al. [7] tested a similar number of isolates and reported even greater differences (MlCy,, of 0.2 and 3.13 mg/l for sparfloxacin and ciprofloxa cin, respectively). Pharmacokinetic studies reveal that, in animals, sparfloxacin con centrations in tissues are 1-11 times grea ter than scrum levels [12]. Moreover, when equal doses (mg/kg) of sparfloxacin and ciprofloxacin were given to rats, peak sparfloxacin tissue levels were 3-14 times greater than peak ciprofloxacin concentra tions [12], In animal models of lethal S. au reus and 5. pneumoniae sepsis, sparfloxa cin was more efficacious than ciprofloxa cin on a weight-by-weight basis [7]. The improved activity of sparfloxacin against gram-positive bacteria and its higher tissue concentrations compared to the currently available fluoroquinolones suggest that sparfloxacin might also be more efficacious in the treatment of com munity-acquired and nosocomial infec tions due to gram-positive bacteria.
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This work was supported by the Parkc-Davis Pharmaceutical Research Division, Division of War ner-Lambert, Ann Arbor. Mich., USA, and in part by the Medical Research Service of the Department of Veterans Affairs. We thank Judy Dean and Ste phanie Brennan for their excellent secretarial assis tance.
References 1 Ncu HC: New antibiotics: Areas of appropriate use. J Infect Dis 1987:155:403^17. 2 Phillip I, King A: Comparative activity of the 4-quinolones. Rev Infect Dis I988;10(suppl 1): S70-S76. 3 Fuchs PC: In vitro antimicrobial activity and sus ceptibility testing of ofloxacin: Current status. Am J Med 1987;87(suppl 6C):10S-13S. 4 Davies BI, Maesen FPV, Baur C: Ciprofloxacin in the treatment of acute exacerbations of chron ic bronchitis. Eur J Clin Microbiol 1986:5:226— 231. 5 Thys JP: Quinolones in the treatment of bron chopulmonary infections. Rev Infect Dis 1988; 10(suppl 1):S212—S217. 6 Kojima T, Inoue M, Mitsuhashi S: In vitro activ ity of AT-4140 against clinical bacterial isolates. Antimicrob Agents Chemother 1989;33:1980— 1988. 7 Nakamura S, Minami A, Nakata K, Kurobc N. Kouno K, Sakaguchi Y, Kashimoto S, Yoshida H, Kojima T, Oliue T, Fujimoto K, Nakamura M, Hashimoto M, Shimizu M: In vitro and in vivo antibacterial activities of AT-4140, a new broadspectrum quinolone. Antimicrob Agents Chemo ther 1989;33:1167-1173.
8 Washington JA, Sutter VL: Dilution susceptibil ity test: Agar and macro-broth dilution proce dures; in Lcnette E, Balows A, Hausier WJ, Tru ant JP (eds): Manual of Clinical Microbiology, ed 3. Washington, American Society for Micro biology. 1980, pp 453-458. 9 Steers E. Foltz EL. Graves BS. Ridens J: An inocula-rcplicating apparatus for routine testing of bacterial susceptibility to antibiotics. Antibiotics 1959;9:307-311. 10 Zcrvos MJ. Kauffman CA. Therasse PM, Berg man AG. Mikesell TS, Schaberg DR: Nosoco mial infection by gentamicin-resistant Strepto coccus faecalis: An epidemiologic study. Ann In tern Med 1987;106:687-691. 11 Cooper B, Lawlor M: Pneumococcal bacteremia during ciprofloxacin therapy for pneumococcal pneumonia. Am J Med 1989;87:475. 12 Nakamura S, Kurobc N. Ohue T, Hashimoto M, Shimizu M: Pharmacokinetics of a novel quino lone, AT-4140, in animals. Antimicrob Agents Chemother 1990;34:89-93.
Aldona L. Baltch, MD Chief, Infectious Disease VA Medical Center 113 Holland Avenue Albany, NY 12208 (USA)
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Acknowledgements
In vitro Activity of Sparfloxacin and Six Reference Antibiotics against Gram-Positive Bacteria Arnold Louie, Aldona L. Baltch, William J. Ritz, Raymond P. Smith Division of Infectious Diseases, Departments of Medicine and Pharmacology, Albany Department of Veterans Affairs Medical Center and the Albany Medical College, Albany, N.Y., USA
Key Words. Sparfloxacin • AT-4140 • Fluoroquinolones • Gram-positive bacteria
Introduction
The currently available fluoroquino lones possess potent activity against gram negative bacilli and have proven efficacy in the treatment of gram-negative bacte rial infections. Their activity against gram
positive cocci, however, is not high [1-3]. Consequently, failure of these antibiotics to eradicate gram-positive bacterial infec tions is not uncommon [4, 5]. Sparfloxacin (AT-4140, CI-978, PD 131501) is a new fluoroquinolone (fig. 1). Preliminary studies suggest that sparfloxa-
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Abstract. The in vitro activity of sparfloxacin, a new fluoroquinolone, was assessed against 234 gram-positive bacterial isolates by agar dilution (104 CFU/spot). Sparfloxacin activity was compared with that of ciprofloxacin and five other antibiotics. Sparfloxacin was the most active drug tested against methicillin-sensitive and methicillin-resistant Staphylococcus aureus (MRSA) and coagulase-negative staphylococci (MIC90, 0.125— 0.25 mg/1). Sparfloxacin was also the most active drug tested against Enterococcus faecalis (MIC90, 1 mg/1) and showed equal activity against gentamicin-susceptible and gen tamicin-resistant (MIC > 2,000 mg/1) enterococci. Sparfloxacin was the most active quinolone tested against Streptococcus pneumoniae and S. pyogenes (MIG#), 1 mg/1). Most Corynebacterium jeikeium showed exquisite susceptibility to sparfloxacin (MIC, 0.06-0.25 mg/1). For MRSA, time-kill curves showed sparfloxacin to be rapidly bacte ricidal at the MIC of the organism. Sparfloxacin showed greater and more sustained bactericidal activity than ciprofloxacin and vancomycin at lx and 2x the MIC. Reduc tion in the activity of sparfloxacin occurred with decreased agar pH (from 7.0 to 6.0) and increased bacterial inoculum. Sparfloxacin showed superior activity compared to refer ence drugs against most gram-positive bacteria.
276
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ment. Control strains were obtained from the Amer ican Type Culture Collection and included: 5. aureus ATCC 29213 and ATCC 25923, and E. faecalis ATCC 29212.
Fig. I. Chemical structure of sparfloxacin (AT-4140, CI-978, PD 131501).
cin has potent activity against a broad spectrum of microorganisms [6, 7]. We evaluated the in vitro activity of sparfloxacin and six reference antibiotics against 234 gram-positive bacterial iso lates: methicillin-sensitive (MSSA) and methicillin-resistant Staphylococcus aureus (MRSA), coagulase-negative staphylococ ci, gentamicin-sensitive (MIC < 2,000 mg/1) and high-level gentamicin-resistant (MIC > 2,000 mg/1) Enterococcus faecalis, Strep tococcus pyogenes, Streptococcus pneumo niae and Corynehacterium jeikeium. We al so examined the bactericidal activity of sparfloxacin, ciprofloxacin and vancomy cin against MRSA by the time-kill curve method. Lastly, we studied the effect of acidic pH and increased bacterial inocu lum on the susceptibility of MRSA and en terococci to sparfloxacin, ciprofloxacin and gentamicin.
Antimicrobial Agents Antibiotics were obtained from the following sources: sparfloxacin (AT-4140, CI-978, PD 131501), Parke-Davis; ciprofloxacin, Miles Inc.; ofloxacin, Ortho; vancomycin, Eli Lilly; erythromycin and pen icillin G, Sigma, and oxacillin, Bristol-Myers Squibb. Antibiotics were prepared as directed by the manu facturers' instructions, sterilized by filtration (0.45pm filter) and used immediately. Susceptibility Testing Antimicrobial susceptibilities were determined by the twofold serial agar dilution technique de scribed by Washington and Sutter [8], Employing a Steers replicator [9], a final inoculum of 10J CFU/ spot was delivered to the surface of antibiotic-con taining Mueller-Hinton agar (MHA; BBL). For the evaluation of antibiotic MICs for streptococcal, enterococcal and C. jeikeium isolates, MHA was sup plemented with 5% defibrinated sheep blood. For oxacillin and penicillin G susceptibility testing against MRSA, NaCl was added to MHA (final con centration 4%). All plates were incubated at 37°C, in air, and read 24 h later. The MIC was defined as the lowest antibiotic concentration at which not more than 1 colony was visible. A faint haze was dis regarded. The effect of acidic pH on drug activity against 15 MRSA and 16 gentamicin-susceptible (MIC < 2,000 mg/1) E. faecalis isolates was assessed by fol lowing the procedure described above. However, the agar pH was adjusted from 7.0 to 6.0 with 10 N HCI before the agar suspension was autoclaved. The inoculum effect was determined by delivering bacte rial concentrations of 102 and 104 CFU/spot onto the same antibiotic-containing agar plate. MICs were determined as described.
Bacterial Isolates All microorganisms were clinical isolates ob tained from the Albany Department of Veterans Af fairs Medical Center and the Bacteriology Labora tory of the Wadsworth Center for Laboratories and Research of the New York State Health Depart
Time-Kill Studies Time-kill curves were established for 2 clinical MRSA isolates against sparfloxacin, ciprofloxacin and vancomycin to assess and compare the bacte ricidal kinetics of these drugs. First, the MIC of the antibiotic for each bacterial isolate was determined by serial macrotubc dilution in Mueller-Hinton
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C. jeikeium can be best appreciated graph ically (fig. 2). This graph shows two pop ulations of organisms, one that was highly sensitive to sparfloxacin and another which was resistant (MIC, 64 mg/l). Organisms that were resistant to sparflox acin demonstrated similar MICs for the other quinolones. Excluding the quinolone-resistant C. jeikeium, the MIC90 for sparfloxacin was 0.25 mg/l and for cipro floxacin 1.0 mg/l. Reduction in agar pH from 7.0 to 6.0 decreased the activity of sparfloxacin, ci profloxacin and gentamicin against each of the 15 MRSA isolates studied. The MICs increased by two tubes. Sparfloxacin and ciprofloxacin MICs also increased by Results two tubes for 10 of the 16 gentamicin-sus The MICs obtained for isolates tested ceptible entcrococcal isolates tested. How at a bacterial inoculum of 104 CFU/spot on ever, a four-tube rise in MICs was observ MHA at pH 7.0 are summarized in table 1. ed for all 16 enterococcal isolates when Sparfloxacin demonstrated potent activity they were tested against gentamicin under against MSSA, MRSA and coagulase-neg- similar conditions (data not shown). ative staphylococci. Sparfloxacin MICs Antibiotic activity for sparfloxacin, ci were 4 times lower than those of ciproflox profloxacin and gentamicin was reduced acin and ofloxacin against all staphylococ with increased MRSA or E. faecalis inocu ci. Gentamicin-sensitive (MIC 2,000 mg/l) E. faecalis were equal 10* CFU/spot, a one-tube increase in MIC ly susceptible to sparfloxacin (MICW of was seen against 9 of 15 MRSA isolates for 1 mg/l for both groups). Ciprofloxacin and sparfloxacin, ciprofloxacin and gentami the other reference compounds were less cin. For gentamicin-susceptible E. faecalis, active than sparfloxacin against the genta sparfloxacin, ciprofloxacin and gentamicin micin-susceptible and gentamicin-resistant showed an inoculum effect for 88, 38 and enterococci with MICgo of >2 mg/l. The 64%, respectively, of the 16 isolates exam susceptibilities of 5. pyogenes and S. pneu ined (data not shown). moniae were best to penicillin G, oxacillin Time-Kill Curx’es and erythromycin. However, among the quinolones, sparfloxacin was at least Ciprofloxacin and sparfloxacin were twice more active than ciprofloxacin and rapidly bactericidal against MRSA at drug concentrations > lx the MIC (fig. 3). Van ofloxacin. The activity of sparfloxacin against comycin was bacteriostatic at lx the MIC
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broth (BBL) supplemented with 25 mg/1 of Mg2+ and 50 mg/l of Ca:+ (MHB-S). The final bacterial in oculum was It)5 CFU/ml. The MIC was defined as the lowest antibiotic dilution at which no growth or haze was visible. Subsequently, bacteria in log phase growth were inoculated into flasks containing MHB-S and antibiotic at concentrations equal to 0.25. 0.5, 1 and 2x the MIC. The final bacterial con centration was 10s CFU/ml. An antibiotic-free flask containing MHB-S and 105 CFU/ml served as a growth control. Culture flasks were incubated at 37°C in a water shaker bath. Samples were taken from each flask at 0, I, 2. 4. 6 and 24 h, serially dilut ed with 0.9% NaCI and inoculated on antibiotic-free trypticase soy agar (BBL). Plates were incubated at 37 °C, and quantitative colony counts were deter mined 24 h later.
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Table 1. Activity of sparfloxacin and reference antibiotics against gram-positive bacteria Antibiotic
MIC. mg/1 range
50%
90%
MSSA (39)
sparfloxacin ciprofloxacin ofloxacin erythromycin vancomycin oxacillin penicillin G
0.06-0.25 0.25-1.0 0.12-1.0 0.25-> 4.0 0.5-2.Ö 0.25-1.0 ¿0.06-64.0
0.06 0.5 1.0 0.25 1.0 0.5 4.0
0.12 0.5 1.0 4.0 1.0 1.0 32.0
MRSA (41)
sparfloxacin ciprofloxacin ofloxacin erythromycin vancomycin oxacillin penicillin G
0.06-8.0 0.25-^64 0.25-32.0 2.0—> 512 1.0-2.0 8.0-512.0 4.0-128.0
0.06 0.5 0.5 >512.0 2.0 256.0 64.0
0.25 2.0 2.0 >512.0 2.0 256.0 64.0
Staphylococci (coagulasc negative, 40)
sparfloxacin ciprofloxacin ofloxacin erythromycin vancomycin oxacillin penicillin G
0.06-16.0 0.03-128 0.25-64 0.12—> 512 0.5-2.0 0.25-> 512 ¿0.01-256
0.12 0.25 0.5 1.0 1.0 1.0 0.5
0.25 2.0 2.0 >512 2.0 256.0 128.0
S. pyogenes (21)
sparfloxacin ciprofloxacin ofloxacin erythromycin vancomycin oxacillin penicillin G
0.5-4.0 0.25-4.0 1-16.0 0.03-0.06 0.5-1.0 0.03-0.25 ¿0.01-0.12
1.0 1.0 4.0 0.06 0.5 0.06 ¿0.01
2.0 4.0 8.0 0.06 0.5 0.06 0.03
S. pneumoniae (19)
sparfloxacin ciprofloxacin ofloxacin erythromycin vancomycin oxacillin penicillin G
0.12-1.0 Ö.5-2.0 0.5-2.0 ¿0.01-0.12 0.25-1.0 0.03-2.0 ¿0.01-0.25
0.5 1.0 2.0 ¿0.01 0.5 0.06 0.03
1.0 2.0 2.0 0.03 0.5 0.5 0.03
C. jeikeium (11)
sparfloxacin ciprofloxacin ofloxacin
0.06-64.0 0.06-64.0 0.25-32.0
0.25 1.0 1.0
0.5-64 1-64 32.0
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Organism
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279
Table 1 (continued) Organism
Antibiotic
MIC, mg/l range
C. jeikeium (II)
erythromycin vancomycin oxacillin penicillin G
¿0.01-512 0.5-1.0 0.25-> 512 0.03-> 512
E. faecalis (Gentamicin MIC, < 2.0(H) mg/l; 44)
Sparfloxacin ciprofloxacin ofloxacin erythromycin vancomycin oxacillin penicillin G
0.25-1.0 0.5-64.0 1-32.0 0.5-> 64.0 0.5-4.0 2—> 512 0.25-> 64.0
E. faecalis (Gentamicin MIC; >2,000 mg/l; 20)
Sparfloxacin ciprofloxacin ofloxacin erythromycin vancomycin oxacillin penicillin G
0.25-1.0 Ö.5-2.0 1.0-4.0 0.5—> 512 1.0-4.0 8.0-256 2.0-8.0
50%
90%
128.0
512.0
1.0
1.0
>512.0 >512.0
>512.0 >512.0
0.5 1.0
2.0 2.0 2.0 16.0 4.0
1.0
4.0 4.0 >64.0 2.0 64—> 512 64.0
0.5-1.0
1.0
1.0
2.0 4.0 >512.0 4.0 128.0 8.0
2.0 >512.0 2.0 32.0 4.0
Fig. 2. The activity of Sparfloxacin (a) and Ciprofloxacin (b) against C.jeikeium.
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Figures in parentheses indicate the numbers of isolates.
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and bactericidal at 2x the MIC, although the bactericidal effect required 24 h to be come apparent. At 24 h, Sparfloxacin dem onstrated a more sustained and greater bactericidal activity than ciprofloxacin and vancomycin for drug concentrations of 1 x and 2x the MIC. At 1x the MIC, bacterial counts for ciprofloxacin were similar to those of controls (l()y CFU/ml), while those for vancomycin were similar to the original inoculum (HP CFU/ml).
Fig. 3. Bactericidal activity at two different con centrations of Sparfloxacin (SPAR) and ciprofloxa cin (CIPRO; a), and Sparfloxacin and vancomycin (VANCO; b) against MRSA. The MICs were: Spar floxacin 0.25 mg/l. ciprofloxacin 0.5 mg/1 and vanco mycin 2 mg/1.
We found Sparfloxacin to have superior gram-positive activity compared to cipro floxacin and ofloxacin against all clinical isolates tested. Moreover, sparfloxacin was the most active of the seven antibiotics studied against MSSA, MRSA, the coagulase-negative staphylococci and the gen tamicin-sensitive and high-level gentami cin-resistant E. faecalis. The excellent activity of sparfloxacin against staphylococci is important. In many institutions, multiply antibioticresistant gram-positive bacteria, such as MRSA and coagulasc-negative staphylo cocci, are responsible for an increasing number of nosocomial infections. These organisms are commonly resistant to antistaphylococcal penicillins, cephalosporins and aminoglycosides. Vancomycin has be come the cornerstone of therapy for treat ing patients infected with these organisms. However, vancomycin must be adminis tered parenterally, is expensive and has certain side effects. Moreover, our timekill curves show that, against MRSA, it takes >6 h before the bactericidal activity of vancomycin becomes apparent. In con
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Discussion
trast, sparfloxacin is bactericidal within 2-4 h. The rapid bactericidal activity of an antibiotic may be crucial in the severely ill or neutropenic patient. Since staphylococ ci are markedly susceptible to sparfloxacin (MIG,,, of 0.125 mg/l for MSSA and 0.25 mg/l for MRSA and coagulase-negative staphylococci) and sparfloxacin is rap idly bactericidal at a concentration equal to the MIC, this drug may be an alterna tive to vancomycin for treating staphylo coccal infections. Sparfloxacin was also the most active drug tested against E.faecalis. Sparfloxa cin was equally active against gentamicinsusceptible and high-level gentamicin-re sistant isolates (MICyo of 1 mg/l for both strains). Ciprofloxacin demonstrated MIG,,) of 4 and 2 mg/l for these two groups, respectively. We believe that we arc the first to report the activity of spar floxacin and reference compounds against gentamicin-susceptible and high-level gen tamicin-resistant enterococci. This distinc tion is important because high-level gen tamicin-resistant organisms account for more than 50% of all enlcrococcal isolates in some institutions 110]. Sparfloxacin may expand the selection of antibiotics that are active against these organisms. We believe that we are also the first to report the susceptibility of C. jeikeium to sparfloxacin. Although some isolates were resistant to all quinolones (MICs of 3264 mg/l), most strains were very suscepti ble. For the susceptible organisms (7 of 11 isolates), the M IG) of sparfloxacin was ‘A of that of ciprofloxacin, with MIGhi of 0.25 and 1 mg/l, respectively. The high activity of sparfloxacin against quinolone-susceptiblc organisms should be evaluated fur ther in a clinical setting.
281
Although currently available quinoloncs have excellent gram-negative and good staphylococcal coverage, their low activity against streptococci, particularly S. pneumoniae, has curtailed their use as empiric therapy for community-acquired pneumonia, bronchitis, sinusitis and otitis media. Studies have reported up to a 33% treatment failure and relapse rate when ci profloxacin was prescribed to treat pneu mococcal pneumonia and bronchitis [5]. Cooper and Lawlor [II] reported a patient with documented bacteremia on the fifth day of ciprofloxacin for S. pneumoniae pneumonia. Our study demonstrates that the in vitro activity of sparfloxacin (MIC9„ of 1 mg/l) is twice that of ciprofloxacin against 5. pneumoniae. Kojima et al. [6] and Nakamura et al. [7] tested a similar number of isolates and reported even greater differences (MlCy,, of 0.2 and 3.13 mg/l for sparfloxacin and ciprofloxa cin, respectively). Pharmacokinetic studies reveal that, in animals, sparfloxacin con centrations in tissues are 1-11 times grea ter than scrum levels [12]. Moreover, when equal doses (mg/kg) of sparfloxacin and ciprofloxacin were given to rats, peak sparfloxacin tissue levels were 3-14 times greater than peak ciprofloxacin concentra tions [12], In animal models of lethal S. au reus and 5. pneumoniae sepsis, sparfloxa cin was more efficacious than ciprofloxa cin on a weight-by-weight basis [7]. The improved activity of sparfloxacin against gram-positive bacteria and its higher tissue concentrations compared to the currently available fluoroquinolones suggest that sparfloxacin might also be more efficacious in the treatment of com munity-acquired and nosocomial infec tions due to gram-positive bacteria.
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This work was supported by the Parkc-Davis Pharmaceutical Research Division, Division of War ner-Lambert, Ann Arbor. Mich., USA, and in part by the Medical Research Service of the Department of Veterans Affairs. We thank Judy Dean and Ste phanie Brennan for their excellent secretarial assis tance.
References 1 Ncu HC: New antibiotics: Areas of appropriate use. J Infect Dis 1987:155:403^17. 2 Phillip I, King A: Comparative activity of the 4-quinolones. Rev Infect Dis I988;10(suppl 1): S70-S76. 3 Fuchs PC: In vitro antimicrobial activity and sus ceptibility testing of ofloxacin: Current status. Am J Med 1987;87(suppl 6C):10S-13S. 4 Davies BI, Maesen FPV, Baur C: Ciprofloxacin in the treatment of acute exacerbations of chron ic bronchitis. Eur J Clin Microbiol 1986:5:226— 231. 5 Thys JP: Quinolones in the treatment of bron chopulmonary infections. Rev Infect Dis 1988; 10(suppl 1):S212—S217. 6 Kojima T, Inoue M, Mitsuhashi S: In vitro activ ity of AT-4140 against clinical bacterial isolates. Antimicrob Agents Chemother 1989;33:1980— 1988. 7 Nakamura S, Minami A, Nakata K, Kurobc N. Kouno K, Sakaguchi Y, Kashimoto S, Yoshida H, Kojima T, Oliue T, Fujimoto K, Nakamura M, Hashimoto M, Shimizu M: In vitro and in vivo antibacterial activities of AT-4140, a new broadspectrum quinolone. Antimicrob Agents Chemo ther 1989;33:1167-1173.
8 Washington JA, Sutter VL: Dilution susceptibil ity test: Agar and macro-broth dilution proce dures; in Lcnette E, Balows A, Hausier WJ, Tru ant JP (eds): Manual of Clinical Microbiology, ed 3. Washington, American Society for Micro biology. 1980, pp 453-458. 9 Steers E. Foltz EL. Graves BS. Ridens J: An inocula-rcplicating apparatus for routine testing of bacterial susceptibility to antibiotics. Antibiotics 1959;9:307-311. 10 Zcrvos MJ. Kauffman CA. Therasse PM, Berg man AG. Mikesell TS, Schaberg DR: Nosoco mial infection by gentamicin-resistant Strepto coccus faecalis: An epidemiologic study. Ann In tern Med 1987;106:687-691. 11 Cooper B, Lawlor M: Pneumococcal bacteremia during ciprofloxacin therapy for pneumococcal pneumonia. Am J Med 1989;87:475. 12 Nakamura S, Kurobc N. Ohue T, Hashimoto M, Shimizu M: Pharmacokinetics of a novel quino lone, AT-4140, in animals. Antimicrob Agents Chemother 1990;34:89-93.
Aldona L. Baltch, MD Chief, Infectious Disease VA Medical Center 113 Holland Avenue Albany, NY 12208 (USA)
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Acknowledgements
