Conditions Required for the Bactericidal Activity of Fleroxacin and Pefloxacin Against Escherichia Cob KL 16

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J. Med. Microbiol. - Vol. 32 (1990), 83-86 0022-261 5/90/00324083/$10.00 0 1990 The Pathological Society of Great Britain and Ireland Conditions required for the bactericidal activity of fleroxacin and pefloxacin against Escherichia cob KL 16

C. S. LEWIN and S. G. 6. AMYES

Department of Bacteriology, Medical School, University of Edinburgh, Teviot Place, Edinburgh EH8 9AG

Summary. Pefloxacin and fleroxacin showed biphasic bactericidal activity against Escherichia coli KL16 in nutrient broth. Bacteriostatic concentrations of chloramphenicol, an inhibitor of protein synthesis, and rifampicin, an inhibitor of RNA synthesis, could not completely abolish the bactericidal activity of either drug. Pefloxacin and fleroxacin were also active against non-dividing E. coli KL16. Therefore, pefloxacin and fleroxacin are able to kill bacteria which are not dividing nor actively synthesising protein or RNA.

Introduction KL16. We chose this laboratory strain because of Pefloxacin and fleroxacin have low minimum its consistent use in previous investigations of the inhibitory concentrations (MICs) against a wide bactericidal mechanisms of the 4-quinolone~.~*~~~-~ range of clinical isolates. 1-3 The 4-quinolones do not merely inhibit bacterial multiplication, they Materials and methods can kill bacteria at concentrations above the MIC4,’ and other 4-quinolones have been found to kill Bacterial strain bacteria under different conditions. Protein synthe- E. coli KL16, a prototroph4 obtained from Professor sis, RNA synthesis and bacterial division are J.T. Smith, was used in this study. The strain was kept needed for bactericidal activity in older 4-quino- on drug-free nutrient-agar plates and sub-cultured every lones such as nalidixic a~id.~-~One explanation is 10 days. Colonies taken from drug-free nutrient-agar that the older 4-quinolones kill bacteria by a single plates were used to prepare the overnight cultures used in mechanism, termed A, for which bacterial protein these experiments. and RNA synthesis are pre-req~isites.~~’However, all the modern fluorinated 4-quinolones that have been investigated are active against non-dividing Antibacterial preparation perhaps because of a bactericidal Fleroxacin (Roche) was dissolved in 0.5 M NaOH mechanism additional to‘A.495 (0.02 ml/mg) then made up to the appropriate concentra- The fluorinated 4-quinolones can be divided into tion with sterile distilled water. Pefloxacin (Rhone- two groups. The first, which includes ciprofloxacin, Poulenc), chloramphenicol (Parke-Davis) and rifampicin (Ciba) were dissolved directly in sterile distilled water. DR-3355, lomefloxacin and oflo~acin,~~’98 contains those whose second bactericidal mechanism, termed B, does not require bacterial protein and Determination of the antibacterial eflects of the RNA synthesis. The second group contains norflox- 4-quinolones acin and enoxacin, which are bactericidal against The killing activity against E. coli KL16 of the 4- non-dividing bacteria but still require protein and quinolones at drug concentrations from 0-15 to 90 mg/L RNA synthesis to be able to kill ba~teria.~”This was determined in nutrient broth at 37°C over a 3-h mechanism, first found in norfloxacin, has been period. Survival was estimated by serial dilution in termed C.9 nutrient broth followed by viable counting on nutrient MICs are indices of the inhibition of the agar as described by Lewin et al. ’ ’ multiplication and not of the killing of bacteria. The rate of kill of bacteria treated with pefloxacin and Therefore, we decided to study the mechanisms by fleroxacin in nutrient broth or phosphate-buffered saline which pefloxacin and fleroxacin kill Escherichia coli (PBS) was measured at 30-min intervals over a 4-h period by viable counting on nutrient agar as described by Received 20 Oct. 1989; accepted 31 Oct. 1989. Lewin and Smith.’ 83 84 C. S. LEWIN AND S. G. B. AMYES

Results bacteriostatic concentration of chloramphenicol of (20 mg/L), which inhibits protein synthesis, to Studies the bactericidal activity of pefloxacin or and fleroxacin, separately, at a range of concentra- pefloxacin fleroxacin 1.5 mg/L also did not tions from 0.15 to 90 mg/L against E. coli KL16 in completely abolish the bactericidal activity of these 3 4-quinolones against E. coli KL16 in nutrient broth nutrient broth over h showed a biphasic effect 2 3). against this species characteristic of the bactericidal (figs. and Hence pefloxacin and fleroxacin activity of the 4-quinolones (fig. 1). The bactericidal seem to exert mechanism B which does not require bacterial protein or RNA synthesis. The abolition activity of each increased with concentration until or RNA the most, or optimal, bactericidal concentration of protein synthesis did significantly was reached. Higher concentrations became pro- reduce the bactericidal activity of pefloxacin and or fleroxacin, which confirms that these 4-quinolones gressively less bactericidal. The most, optimum, A. bactericidal concentration of pefloxacin or fleroxa- possess mechanism When the bactericidal E. coli activity of pefloxacin or fleroxacin 1.5 mg/L against cin against KL16 in nutrient broth was E. coli 1.5 mg/L. KL16 were investigated in PBS, in which The bactericidal mechanisms of pefloxacin and bacteria are unable to divide, the 4-quinoloneswere fleroxacin were then studied. Both pefloxacin and bactericidal confirming the presence of mechanism fleroxacin may be assumed to exert mechanism A B. since this is common to all 4-quinolones. Pefloxacin and fleroxacin (1 -5 mg/L) were still significantly Discussion E. coli bactericidal against KL16 in nutrient broth of of When the bactericidal activities fleroxacin even in the presence a bacteriostatic concentra- E. coli tion of rifampicin (1 60 mg/L) which inhibits RNA and pefloxacin against KL16 were investi- synthesis (figs. 2 and 3). The addition of a gated in nutrient broth, both drugs showed a biphasic effect in common with all other 4- quinolones inve~tigated.~~~*’**The most (or optimal) bactericidal concentrations of pefloxacin and fleroxacin were 1.5 mg/L; hence the potency of these two drugs, at least against E. coli, is similar. The optimum killing concentrations of pefloxacin

0 60 120 180 2 40 0 0.1 1 10 100 Time (mid 4-quinolone concentration (mg/ L) Fig. 2. Survival of E. coli KL16 in nutrient broth at 37°C with pefloxacin 1.5 mg/L, 0-0; pefloxacin 1.5 mg/L + chlor- Fig. 1. Survival of E. coli KL16 after exposure for 3 h to amphenicol 20 mg/L A-A; pefloxacin 1.5 mg/L + rif- pefloxacin (0-0) and fleroxacin (0-0) in nutrient ampicin 160 mg/L 0-0 ; chloramphenicol 20 mg/L or

broth at 37°C. rifampicin 160 mg/L (each 8 ~ 0 ). FLEROXACIN AND PEFLOXACIN AGAINST E. COLZ 85

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0.01-(, 0.01-(, 1 I I 0 60 120 180 210 60 120 160 210 Time (min) Time (min) Fig. 3. Survival of E. coli KL16 in nutrient broth at 37°C with Fig. 4. Survival of E. coli KL16 in PBS at 37°C (0-0) and fleroxacin 1.5 mg/L (0-0); fleroxacin 1.5 mg/L + chlor- with pefloxacin 1.5 mg/L (0-0) or fleroxacin 1.5 mg/L amphenicol 20 mg/L (A-A); fleroxacin 1.5 mg/L + (0-0). rifampicin 160 mg/L (U-U); chloramphenicol 20 mg/L or rifampicin 160 mg/L (each 0- 0 1. and fleroxacin are higher than those reported for B displayed by lomefloxacin which appears to be ciprofloxacin (0.15 mg/L), DR-3355 (0.5 mg/L) and much weaker? ofloxacin (0*9mg/L) but the same as that of Therefore, both pefloxacin and fleroxacin, like norfloxacin and lower than that of enoxacin (3- other modern fluorinated 4-quinolones that have 5 mg/L).4y7v8It is also much lower than the figure of been investigated, display an additional bacteri- 90 mg/L for the oldest 4-quinolone in clinical use, cidal mechanism against E. coli KL16 as well as nalidixic acid. 5-6 The most bactericidal concentra- mechanism A and so are active against non- tion of pefloxacin and fleroxacin, although above dividing bacteria. For both drugs this additional those reported for some of the other fluorinated 4- mechanism was the mechanism known as B quinolones, is still within the clinically achievable described by Smith and Lewin.’ Hence these two serum and urine levels of either 3914 4-quinolones are in the class of modern fluorinated Both pefloxacin and fleroxacin exert mechanism 4-quinolones that includes ciprofloxacin, ofloxacin B as well as mechanism A; they did not require and DR-3355 which do not require bacterial protein bacterial protein synthesis and RNA synthesis for or RNA synthesis or bacteria capable of division bactericidal activity. The mechanism B displayed to exert their lethal action. by pefloxacin and fleroxacin was active, killing 90% of the bacteria during 4 h. This is similar to the mechanism B displayed by ofloxacin, ciprofloxacin We are grateful to the Scottish Home and Health Department and DR-33555*7and contrasts with the mechanism for financial support for C.S.L.

REFERENCES 3. Gonzalez J P, Henwood J M.Pefloxacin: a review of its antibacterial activity, pharmacokinetic properties and 1. Phillips I, King A, Shannon K. In vitro properties of the therapeutic use. Drugs 1989; 37:628-668. quinolones. In: Andriole V T (ed) The quinolones. 4. Smith J T. Awakening the slumbering potential of the 4- London, Academic Press. 1988: 83-1 18. quinolone antibacterials. Pharm J 1984; 233 : 299-305. 2. Paganoni R, Herzog C, Braunsteiner A, Hohl P. Fleroxacin: 5. Smith J T, Lewin C S. Chemistry and mechanisms of action in-vitro activity worldwide against 20,807 clinical of the quinolone antibacterials. In: Andriole V T (ed) isolates and comparison to ciprofloxacin and norfloxa- The quinolones. London, Academic Press. 1988 : 23- cin. J Antimicrob Chemother 1988; 22 Suppl D: 3-17. 82. 86 C. S. LEWIN AND S. G. B. AMYES

6. Dietz W H, Cook T M, Goss W A. Mechanism of action of 10. Zeiler H-J, Grohe K. The in vitro and in vivo activity of nalidixic acid on Escherichia coli: 111. Conditions ciprofloxacin. Eur J Clin Microbioll984; 3:339-343. required for lethality. J Bacterioll966;61 : 768-773. 11. Lewin C S, Howard B M A, Ratcliffe N T, Smith J T. 4- 7. Lewin C S, Amyes S G B. Bactericidal activity of DR-3355 quinolones and the SOS response. J Med Microbiol an optically active isomer of ofloxacin. J Med Microbiol 1989; 29: 139-144. 1989; 30:227-231. 12. Lewin C S, Smith J T. Bactericidal mechanismsof ofloxacin. J Antimicrob Chemother 1988; 22: Suppl C: 1-8. 8. Lewin C S, Amyes S G B, Smith J T. Bactericidal activity 13. Bergan T. Pharmacokinetics of fluorinated quinolones. In of enoxacin and lomefloxacin against Escherichia coli Andriole V T (ed) The quinolones. London, Academic KL16. Eur J Clin Microbwl Infect Dis 1989; 8: 731-733. Press. 1988: 23-82. 9. Ratcliffe N T, Smith J T. Norfloxacin has a novel 14. Weidekamm E, Portmann R, Partos C, Dell D. Single and bactericidal mechanism unrelated to that of other 4- multiple dose pharmacokinetics of fleroxacin. J Anti- quinolones. J Pharm PharmacoIl985 ; 37 : 92. microb Chemother 1988; 22 Suppl D: 145-154.