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International Journal of Antimicrobial Agents 13 (2000) 223-226 Short communication Bactericidal activity of levofloxacin and ciprofloxacin on clinical isolates of different phenotypes of Pseudomonas aeruginosa* Bernardetta Segatore a, Domenico Setaccia, Mariagrazia Perilli a, Nicola Franceschini a, Federico Marchetti b, Gianfranco Amicosante a,* a Department of Sciences and Biomedical Technologies, School of Medicine, University of L'Aquila, Via Vetoio, 67010 Coppito, L' Aquila, Italy b Medical Department, Glaxo Wellcome, Verona, Italy Received 18 May 1999; accepted 20 July 1999 Abstract Levofloxacin has been reported to have in vitro activity against both Gram-positive and Gram-negative bacteria. A recent survey carried out at our Institution showed clinical isolates of Pseudomonas aeruginosa to be more susceptible to levofloxacin than to ciprofloxacin. The in vitro activity of the two fluoroquinolones was evaluated further by looking at their bactericid al activity against two strains of each of the following antibio-phenotypes off. aeruginosa: levofloxacin- and ciprofloxacin-susceptible, levofloxacin-susceptible/ciprofloxacin-resistant, levofloxacin-susceptible/ciprofloxacin-susceptible and ceftazidime-resistant, (National Committee for Clinical Laboratory Standards susceptibility breakpoints were used). MIC and MBC values were measured and time-kill experiments were carried out. Drugs were used at susceptibility or resistance breakpoint concentrations in the time-kill experiments and results were recorded over 12 h in an attempt to link in vitro results with the clinical situation The polypeptide profiles of outer membrane preparations of the six strains were examined by gel electrophoresis. Levofloxacin was shown to be more bactericidal than ciprofloxacin in the time-kill experiments. No differences were observed between the outer membrane proteins of the six strains. Levofloxacin showed greater bactericidal activity against P. aeruginosa clinical isolates than ciprofloxacin. © 2000 Elsevier Science B.V. and International Society of Chemotherapy. All rights reserved. Keywords: Levofloxacin; Bactericidal activity; Time-killing 1. Introduction Levofloxacin has been reported to be active in vitro against both Gram-positive and Gram-negative bacteria [1]. Two major targets have been identified for levofloxacin activity, topoisomerase II (DNA-gyrase) in Gram-negative bacteria [2] and topoisomerase IV in Gram-positive bacteria [3,4]. Levofloxacin activity against Pseudomonas aeruginosa is mainly directed against DNA-gyrase rather than topoisomerase IV [5]. * These results were presented in part at the 8th International Congress of Microbiology and Infectious Diseases, Losanna, Switzer land, 25-28 May 1997. * Corresponding author. Tel.: + 39-862-433455; fax: + 39-862433433. E-mail address: [email protected] (G. Amicosante) There are three mechanisms by which levofloxacin and the 4-fluoroquinolones are known to exert activity, namely A, B and C. Briefly, mechanism A, which is linked to protein and RNA synthesis or cell division, is shared by all 4-fluoroquinolones and it is the sole activity of nalixidic acid [6]. Mechanism B is found in addition in some 4-fluoroquinolones such as levofloxacin and ciprofloxacin, but does not affect protein or RNA synthesis or cell division. Ciprofloxacin activity against Gram-positive bacteria does not involve mechanism B [7,8]. Mechanism C interferes with protein and RNA synthesis but not cell division, and has been described in norfloxacin [9]. Morissey and Smith reported that levofloxacin and ciprofloxacin produce in vitro bactericidal activity against P. aeruginosa by both mechanisms A and B, mechanism A occurring only at very low drug concentrations [10]. 0924-8579/00/$ - see front matter © 2000 Elsevier Science B.V. and International Society of Chemotherapy. All rights reserved. PII:S50924-8579(99)00119-3 224 B. Segatore et al. /International Journal of Antimicrobial Agents 13 (2000) 223-226 Table 1 MIC and MBC values for the six P. aeruginosa clinical isolates Strains Antibiotics MIC (rag/1) MBC (mg/1) CG 12 Levofloxacin 1 2 1 FBF 15 Ciprofloxacin Levofloxacin Ciprofloxacin 8 1 4 8 NA 8 VE 36 NO 12 NO 6 0.25 Levofloxacin Ciprofloxacin Levofloxacin Ciprofloxacin Levofloxacin Ciprofloxacin Ceftazidime Levofloxacin Ciprofloxacin Ceftazidime 1 2 8 1 8 1 1 32 16 >64 32 >256 2 1 64 4 8 4 8 >256 A recent survey carried out at our Institution showed levofloxacin to be more active than ciprofloxacin against clinical isolates of P. aeruginosa. Despite comparable MIC values, 47% of strains were clinically susceptible to levofloxacin compared with 17% for ciprofloxacin when the National Committee for Clinical Laboratory Standards (NCCLS) breakpoints were applied [11]. The in vitro activity of the two fluoroquinolones was studied using a qualitative evaluation of their bactericidal activity against P. aeruginosa. Six strains of P. aeruginosa isolated during a previous survey were selected. Two strains of each of the following antibio-phenotypes were used: levofloxacin- and ciprofloxacin-susceptible (levo-S/cipro-S), FBF15CG12; levofloxacin-susceptible/ciprofloxacin-resistant (levo-S/cipro-R), VE36-NA8; levofloxacin-susceptible/ ciprofloxacin-susceptible and ceftazidime-resistant (levo-S/cipro-S/caz-R), NO6-NO12. Susceptibility breakpoints used were those of the NCCLS [12]. Minimal inhibitory concentrations were performed using a macrodilution broth method in Mueller Hinton broth supplemented with calcium and magnesium to physiological concentrations [13]. The minimal bactericidal concentrations and the killing-curve tests for levofloxacin, ciprofloxacin and Ceftazidime were carried out according to the method of Schoenknecht et al. [14] at a concentration equal to the susceptibility (1 mg/1 for ciprofloxacin and 2 mg/1 for levofloxacin) or resistance (4 mg/1 for ciprofloxacin, 8 mg/1 for levofloxacin and 32 mg/1 for ceftazidime) breakpoints, and using a starting inoculum of about 5 x 10 5 colony forming units/ml in each tube; a tube without antibiotic was used as a growth control. The bactericidal activity was defined as a reduction of > 3 log10. One hundred microliters of each tube were sampled at times 0, 4, 8 and 12 h, and the results obtained after 18 h incubation at 35°C; the counts were plotted against time. P. aeruginosa outer membranes were prepared by sarkosyl treatment in the presence of ethylenediaminetetraacetate. The polypeptide profiles of the six outer membrane preparations were separated by polyacrylamide gel electrophoresis [15]. 2. Materials and methods Levofloxacin was kindly provided by Glaxo Wellcome (Verona, Italy). Ceftazidime and ciprofloxacin were purchased from Sigma (Milan, Italy) and Mast (Merseyside, UK), respectively. 3. Results The MIC and MBC showed both compounds were bactericidal (Table 1). The killing curves showed levofloxacin to be more bactericidal than ciprofloxacin at 4 h for susceptible strains (Fig. 1). Bactericidal activity of levofloxacin against levo-S/cipro-R strains (Fig. 2) and levo-S/cipro-S/caz-R strains (Fig. 3) did not occur for 8 h, and that of ciprofloxacin for 12h. In order to gain further information about the bacterial resistance patterns, the polypeptide profiles of the six outer membrane protein (OMP) preparations were analyzed using polyacrylamide gel electrophoresis. No differences were observed between the OMPs of the six strains (data not shown). 4. Discussion Experiments were carried out to look more closely at the in vitro activity of levofloxacin and ciprofloxacin against recent clinical isolates of P. aeruginosa. The bacteriostatic and bactericidal activity of these two fluoroquinolones were matched with three different antibio-phenotypes. After MIC determinations, the MBC values found confirmed the bactericidal activity of 4fluoroquinolones. The MBC gives only a single indication of the bactericidal activity [16], and therefore time-kill experiments were also performed. In an attempt to link the in vitro results with clinical significance, instead of MIC multiples, drug susceptibility and resistance breakpoint concentrations were introduced. The difference in the breakpoint values reflects the different pharmacokinetic and pharmacodynamic properties of the two drugs [12]. After a single dose of 500 mg, levofloxacin reaches a serum peak (C max) of 5.2 mg/1 and a half life of 7 h, giving an area under the serum curve (AUC) value of 47.7 mg/l/h for once-daily administration [17]. Ciprofloxacin, at the same dose, has a serum Cmax of 2.3 mg/1, a half-life of 3.2 and an 226 B. Segatore et al. /International Journal of Antimicrobial Agents 13 (2000) 223-226 AUC value of 9.9 mg/l/h, so that at least two administrations per day would be required to reach therapeutic efficacy [18]. The AUC/MIC ratio seems to be the best predictor for the clinical efficacy of levofloxacin and ciprofloxacin in P. aeruginosa infection and it would not be crucial whether these two drugs were administered once or twice daily [19]. Ciprofloxacin cannot be administered once daily, and so sampling was over a 12 h period. The clinical importance of in vitro late re growth is unclear, particularly if it occurs after the usual dosing interval [20]. Levofloxacin turned out to be highly bactericidal (decrease > 3 log 10) within 4-8 h, even against cipro-R and caz-R strains, while ciprofloxacin showed bactericidal activity against cipro-S strains after 8 h. Morissey and Smith [10] reported the B mechanism of action of 4-fluoroquinolones occurs in both levofloxacin and ciprofloxacin against P. aeruginosa within the range of drug concentrations tested. The bacterial targets of levofloxacin and ciprofloxacin in P. aeruginosa are essentially the same, i.e. DNA-gyrase and topoisomerase IV being the latter determinant for high-level quinolone resistance [5]. Molecular analysis to compare gyr and/or par genes would have probably helped clarify the difference of activity. These strains had no differences in their outer membrane profiles. In conclusion, despite shared mechanisms of action, in our experiments, levofloxacin showed greater bactericidal activity against clinical isolates of P. aeruginosa than ciprofloxacin. References [1] Fu KP, Lafredo SC, Foleno B, et al. In vitro and in vivo antibacterial activities of levofloxacin (L-ofloxacin), an optically active ofloxacin. Antimicrob Agents Chemother 1992;36(4):8606. 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The bactericidal activity of DR-3355, an optically active isorner of ofloxacin. J Med Microbiol 1989;30:227-31. (8] Tanaka M, Otsuki M, Nishino T. Bactericidal activities of ofloxacin and its optically active isomer (DR-3355) on non-growing cells of Escherichia coli and Pseudomonas aeruginosa. Chemotherapy 1992;38:21-7. [9] Lewin CS, Amyes SGB, Smith JT. Bactericidal activity of enoxacin and lomefloxacin against Escherichia coli KL 16. Eur J Clin Microbiol Infect Dis 1989:8:731-3. [10] Morrissey L, Smith JT. Activity of 4-fluoroquinolones against Pseudomonas aeruginosa. Arzneim Forsch I994;44:l 157-61. [11] Segatore B, Setacci D, Penelli M. et al. Italian survey on comparative levofloxacin susceptibility in 334 clinical isolates of Pseudomonas aeruginosa. Antimicrob Agents Chemother 1999;43:428-31. [12] National Committee for Clinical Laboratory Standards. Methods for dilution antimicrobial susceptibility tests for bacteria that grow aerobically, 4th ed., Approved standard M7-A4. 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The clinical pharmacokinetics of levofloxacin. Clin Pharmacokinet 1997;32(2):101-19. [18] Bergan T, Thorsteinsson SB, Solberg R, Bjornskau L, Kolstad IM, Jhonson S. Pharmacokinetics of ciprofloxacin: intravenous and increasing oral doses. Am J Med 1987;82(Suppl 4A):97- 102. [19] MacGowan AP, Wootton M, Holt HA. The antibacterial effi cacy of levofloxacin and ciprofloxacin against Pseudomonas aeruginosa assessed by combining antibiotic exposure and bacterial susceptibility. J Antimicrob Chemother 1999;43:345-9. [20] National Committee for Clinical Laboratory Standards. Meth ods for determining bactericidal activity of antimicrobial agents, Tentative guideline M26-T. Villanova, PA: National Committee for Clinical Laboratory Standard, 1992.