Antimicrobial Activity of Ciprofloxacin against Pseudomonas

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1 ANTMCROBAL AGNTS AND CHMOTHRAPY, Aug. 1985, p Vol. 28, No /85/ $2./ Copyright C 1985, American Society for Microbiology Antimicrobial Activity of Ciprofloxacin against Pseudomonas aeruginosa, scherichia coli, and Staphylococcus aureus Determined by the Killing Curve Method: Antibiotic Comparisons and Synergistic nteractions L. J. CHALKLY* AND H. J. KOORNHOF Department of Medical Microbiology, School of Pathology, University of the Witwatersrand, and the South African nstitute for Medical Research, Johannesburg, South Africa Received 3 October 1984/Accepted 29 May 1985 A derivative of quinolinecarboxylic acid, ciprofloxacin (BAY o 9867) was found to be an effective bactericidal agent against Pseudomonas aeruginosa and scherichia coli. A bactericidal effect was achieved immediately after the addition of ciprofloxacin. At a concentration of.5,ug/ml, culture viability was reduced from 5 x 1 to about S x 13 CFU/ml within 15 min, and at.1,ug/ml, a >1-fold reduction in viability resulted during the first hour after exposure. This bactericidal activity observed during the lag phase in Mueller-Hinton broth was also demonstrated in a nongrowing system. The antibiotics used in comparative studies, i.e., tobramycin, aztreonam, cefotaxime, and azlocillin, did not show this initial bactericidal activity, and ciprofloxacin prevented culture regrowth at lower concentrations. Staphylococcus aureus was not as susceptible to ciprofloxacin; killing occurred at a concentration of.5,g/ml only after the onset of exponential growth in the control culture. Synergistic interactions were observed wtih ciprofloxacin in combination with tobramycin and azlociilin against P. aeruginosa and with cefotaxime and tobramycin against. coli. nterest in new quinoline derivatives norfloxacin (8, 9, 13) and, more recently, enoxacin C-919 (1, 11) as an alternate group of antibacterial agents has been expanded with the introduction of ciprofloxacin (BAY o 9867). Ciprofloxacin was shown to have activity against a wide range of bacteria and was particularly effective against the nterobacteriaceae (2, 6, 16, 17). Many of the strains tested by Wise et al. (17) were resistant to aminoglycosides or P-lactam antibiotics but sensitive to ciprofloxacin. The standard MC method for determining antibacterial activity provides no information on initial killing kinetics. The present investigation was designed to include the killing curve method to obtain additional information on the antimicrobial properties of ciprofloxacin in vitro, particularly with regard to its initial bactericidal activity against selected organisms. MATRALS AND MTHODS Strains and antibiotics. The susceptible culture collection strains Pseudomonas aeruginosa ATCC 27853, scherichia coli ATCC 25922, and Staphylococcus aureus ATCC were used. The following antimicrobial agents of known potency were evaluated: ciprofloxacin and azlocillin (Bayer AG, Wuppertal, Federal Republic of Germany), tobramycin (li Lilly & Co., ndianapolis, nd.), aztreonam (. R. Squibb & Sons, Princeton, N.J.), and cefotaxime (Roussel Laboratories, Wembley, ngland). Antibiotics were dissolved in water at 1,,ug/ml. All subsequent dilutions were made in cation-supplemented Mueller-Hinton broth (12; Difco Laboratories, Detroit, Mich.) and prepared fresh for each experiment. MC and MBC determinations. MCs were determined by the broth dilution method described by the National Com- * Corresponding author. mittee for Clinical Laboratory Standards (12) in cationsupplemented Mueller-Hinton broth (ph 7.2). Twofold antibiotic dilutions were prepared in 1 ml of broth. The inoculum was adjusted from an exponential-phase culture to give an initial concentration of 5 x 15 CFU/ml. After 18 h of incubation at 35 C, the MC was determined as the lowest concentration that completely inhibited growth. Samples (1,ul) were then transferred from tubes showing no growth onto blood agar plates which were incubated for 18 h at 35 C. The MBC was read as the lowest concentration of antibiotic which produced '99.9% killing of the inoculum (five colonies or fewer for 1,ul of subculture). Killing curve method. Time-kill curves were constructed by previously described techniques (1) with the following specifications. The medium was cation-supplemented Mueller-Hinton broth (ph 7.2). The stationary-phase inoculum was prepared by inoculating 1 ml of broth and incubating it for 2 h at 37 C. After 2 h, cell numbers were determined from an optical density-cfu standard curve, and dilutions of the culture were made in fresh medium to Antibacterial activities of ciprofloxacin and selected agents Organism Agent MC (kig/m) MBC (plg/ml) TABL 1. P. aeruginosa Ciprofloxacin.16.3 Azlocillin 8 16 Aztreonam.6.6 Tobramycin.5 1. coli Ciprofloxacin Cefotaxime.6.6 Aztreonam Tobramycin 1 1 S. aureus Ciprofloxacin

2 332 CHALKLY AND KOORNHOF ANTMCROB. AGNTS CHMOTHR. (A) P. augna (B).coli u. w ~~~~~~~~~~~~~~~ s x1O939P (C) S. aureus TMM (h) 13' i 2 3 {t-24 FG. 1. Activity of ciprofloxacin (in micrograms per milliliter) against the following organisms. (A) P. aeruginosa:,.5;,.1;, control. (B). coli:,.5; *,.5;,.1; O, control. (C) S. aureus:,.5;,.25;, control.

3 VOL. 28, 1985 ANTBACTRAL ACTVTY OF CPROFLOXACN 333 L- U. - w -J m 16 (C) S. aureus Downloaded from o3 Z-o on September 1, 218 by guest i FG. 2. Activity of ciprofloxacin (in micrograms per milliliter) in phosphate-buffered saline against the following organisms. (A) P. aeruginosa:,.5;,.1; O, control. (B). coli:,.1;,.5; O, control. (C) S. aureus:, 1.;,.5; O, control. give an initial inoculum of about 5 x i5 CFU/ml. Tests were carried out in 25-ml flasks containing 1 ml of culture with the required concentrations of antibiotics. The cultures were incubated at 37 C and 15 rpm on an orbital environmental shaker (New Brunswick Scientific Co., nc., dison, N.J.). Samples were taken at regular intervals and suitably diluted for viable count estimations either by the method of Miles and Misra (2,ul delivered on each of five spots) or spread plate (1,ul on each of three plates). All tests, unless otherwise indicated, were performed on stationary-phase cells. For the experiment using exponential-phase cells, the inoculum was prepared from 3-h cultures. For determinations of antibacterial activity in a basically nongrowing system, stationary-phase cells and ciprofloxacin were diluted in and added to phosphate-buffered saline (ph 7.2) instead of broth.

4 334 CHALKLY AND KOORNHOF ANTMCROB. AGNTS CHMOTHR. U- m U o 1 FG. 3. Activities of ciprofloxacin in the presence and absence of 1%o (vol/vol) horse serum.. coli: A,.5,ug of ciprofloxacin per ml; A,.5,ug of ciprofloxacin per ml plus horse serum; V, controls. P. aeruginosa:,.1,ug ciprofloxacin per ml;,.1,ug of ciprofloxacin per ml plus horse serum; x, controls. S. aureus: *,.5 p.g of ciprofloxacin per ml; O,.5,ug of ciprofloxacin per ml plus horse serum; V, controls. RSULTS The MCs and MBCs of ciprofloxacin and selected agents used for antibiotic ccomparisons and synergistic interactions by the killing curve method are shown in Table 1. The killing curves shown are representative curves; experinients were performed in duplicate. For neat sampling (viable counts below 5 x 13 CFU/ml), some antibiotic carry-over was observed with ciprofloxacin at.5,ug/ml against. coli and P. aeruginosa. Samples (1 ml) were therefore filtered through membrane filters (.45-pum pore diameter; Millipore Corp., Bedfordi Mass.), and cells were washed on the filter with 1 ml of Ringer solution (one-fourth strength). Antibiotic carry-over did not occur with ciprofloxacin concentrations of <.5,ug/ml, with the other antibiotics at the concentrations used, or with the ciprofloxacin levels used against S. aureus. From hours to 4, ciprofloxacin was bactericidal at concentrations of.5 and.1 jig/ml against P. aeruginosa,.5,.1, and.5 jig/ml against. coli, and.5,ug/ml against S. aureus (Fig. 1). Regrowth of the culture after 24 h did not occur with P. aeruginosa containing the above-mentioned concentrations of ciprofloxacin or with ciprofloxacin at.5,ug/ml against. coli; culture viability remained at <12 CFU/ml. Partial regrowth was observed in. coli cultures with ciprofloxacin at.5,ug/ml, with culture viability at 24 h being 1.5 x 14 CFU/ml. P. aeruginosa and. coli cultures were extremely sensitive to ciprofloxacin, which caused an i

5 VOL. 28, 1985 ANTBACTRAL ACTVTY OF CPROFLOXACN 335 ULL -J J w - L) l_ FG. 4. Comparison of ciprofloxacin, tobramycin, aztreonam, and cefotaxime activities against. coli. Symbols: *,.5,ug of ciprofloxacin per ml; O, 1.,ug of tobramycin per ml; /, 1.,ug of aztreonam per ml; V,.1,ug of cefotaxime per ml; V,.1,g of cefotaxime per ml; x, control. immediate bactericidal effect, reducing viability from 5 x lo, to 4.7 x 13 and 4.3 x 13 CFU/ml, respectively, within 15 min, i.e., before completion of the lag phase. Against S. aureus; killing by ciprofloxacin only occurred after a period of about 1.5 h, which corresponded to the onset of exponential growth in the control culture. The effect of ciprofloxacin on essentially nongrowing cells was also investigated in phosphate-buffered saline (Fig. 2). The activity of ciprofloxacin was only slightly reduced in this system, and rapid initial killing was still evident in P. aeruginosa and. coli cultures. However, the bactericidal activity of ciprofloxacin was considerably reduced against S. aureus in phosphate-buffered saline, which did not support exponential growth. The addition of 1%o (vol/vol) horse serum (Fig. 3) slightly decreased antibacterial activity against. coli and P. aeruginosa, but killing was comparable after 4 h against. coli and after 2 h against P. aeruginosa. The rates of killing of S. aureus with and without horse serum were similar, but the onset of killing in the presence of serum was delayed. The activities of ciprofloxacin, tobramy-

6 336 CHALKLY AND KOORNHOF ANTMCROB. AGNTS CHMOTHR. 5x19;. <18! 17 T 1 6l a 15 \ _ v 14 ) FG. 5. Comparison of ciprofloxacin, tobramycin, aztreonam, and azlocillin activities against P. aeruginosa. Symbols:,.1,ug of ciprofloxacin per ml; *, 5.,ug of tobramycin per ml; O, 1.,ug of tobramycin per ml; A, 5.,ug of aztreonam per ml; A, 1.,ug of aztreonam per ml; V, 1.,g of azlocillin per ml; x, control. cin, aztreonam, and cefotaxime against. coli were compared (Fig. 4). The immediate bactericidal effect shown by ciprofloxacin was not demonstrated with the other agents. Ciprofloxacin produced a rate of killing similar to that of the other agents tested, but it killed at a lower concentration. Similarly, ciprofloxacin compared favorably with tobramycin, aztreonam, and azlocillin against P. aeruginosa (Fig. 5). As stationary-phase cells were more susceptible to ciprofloxacin than to the other agents tested, a comparison against the exponential cells of P. aeruginosa was also made (Fig. 6). With exponential cells as the initial inoculum, ciprofloxacin at.5,ug/ml produced activity similar to that of tobramycin and aztreonam at 1. p.g/ml during the first 5-h period. There was still a delay of 1 h before aztreonanm produced a bactericidal effect in this system. Tobramycin at 1.,ug/ml, although causing a slightly greater kill initially, failed to reduce viability below 12 CFU/ml by hour 5, and subsequent outgrowth occurred by 24 h. These results were prevented by concentrations of ciprofloxacin and aztreonam at.5 and 1.,ug/ml, respectively. Based on a difference of _2 log1 CFU at 24 h between the results of combinations and the results of the most active of

7 VOL. 28, 1985 ANTBACTRAL ACTVTY OF CPROFLOXACN 337 L- w -J Downloaded from on September 1, 218 by guest FG. 6. Activities of ciprofloxacin, tobramycin, and aztreonam against exponential cells of P. aeruginosa. Symbols:,.5,ug of ciprofloxacin per ml;,.1,ug of ciprofloxacin per ml; O, 1.,ug of tobramycin per ml; A, 1.,ug of aztreonam per ml; x, control. the two individual components as defined by Hallander et al. (7), synergy was observed when ciprofloxacin was combined with tobramycin or azlocillin against P. aeruginosa (Fig. 7 and 8) and with cefotaxime against. coli (Fig. 9). A _2 log1o reduction in viability between the combination and its most active constituent during the first 6-h period was shown with other concentrations and combinations of the antibiotics (Fig. 9 and 1). With a combination of tobramycin and ciprofloxacin against. coli (Fig. 1), a >2 log1o reduction in viability was observed by 4 h but not at 24 h as outgrowth of this culture occurred. Ciprofloxacin in combination with aztreonam showed an increase in bactericidal activity over

8 338 CHALKLY AND KOORNHOF ANTMCROB. AGNTS CHMOTHR.., 17 L w O3 //2 12Lf FG. 7. Activities of ciprofloxacin and tobramycin in combination against P. aeruginosa. Symbols: *,.25,ug of ciprofloxacin plus 1.,ug of tobramycin per ml;,.25,ug of ciprofloxacin plus.5,ug of tobramycin per ml;,.25,ug of ciprofloxacin plus.25 plg of tobramycin per ml; *, 1.,ug of tobramycin plus.1,ug of ciprofloxacin per ml; A,.25,ug of ciprofloxacin per ml; V, 1.,ug of tobramycin per ml; V,.5,ug of tobramycin per ml; A,.1,ug of ciprofloxacin per ml; x,.25,ug of tobramycin per ml and control; _, all cultures at 24 h unless otherwise indicated. either agent alone against. coli and P. aeruginosa, although synergy as defined was not effected (results not shown). Antagonism did not occur with the combinations tested at the concentrations (of the agents) used. DSCUSSON The quinoline compounds have emerged as an interesting and potentially important group of chemotherapeutic agents. Ciprofloxacin as evaluated here is particularly effective against gram-negative bacteria at lower concentrations than the other agents with similar antibacterial spectra. t is, however, far less active against S. aureus. t has been proposed that as ciprofloxacin has a spectrum of activity similar to that of nalidixic acid, it probably has a mode of action similar to that of nalidixic acid (17). The latter agent acts on the A subunit of DNA gyrase, which inhibits and affects DNA supercoiling, resulting in inhibition of DNA replication and possibly transcription, depending on the promoter (4, 5). Bacterial isolates resistant to norfloxacin have been found to be resistant to nalidixic acid also (15). However, most nalidixic acid-resistant isolates were not

9 VOL. 28, 1985 ANTBACTRAL ACTVTY OF CPROFLOXACN 339 U- - -J w -J D1 C... Downloaded from FG. 8. Activities of ciprofloxacin and azlocillin in combination against P. aeruginosa. Symbols:,.25,ug of ciprofloxacin plus 5.,ug of azlocillin per ml;,.25,g of ciprofloxacin plus 2.5,ug of azlocillin per ml; *, 1.,ug of azlocillin plus.1,ug of ciprofloxacin per ml; A,.25 Rg of ciprofloxacin per ml;, 1.,g of azlocillin per ml; A,.1,ug of ciprofloxacin per ml; V, 5.,ug of azlocillin per mi; x, 2.5,ug of aziocillin per ml and control; _, all cultures at 24 h unless otherwise indicated. on September 1, 218 by guest concurrently resistant to norfloxacin. The frequency of resistance to ciprofloxacin was shown to be generally low (2). Wise et al. (17) found that strains which were less susceptible to nalidixic acid and norfloxacin tended to be less susceptible to ciprofloxacin, although other workers (16) found no cross resistance between nalidixic acid and ciprofloxacin. Nalidixic acid has been shown to be active only on exponentially growing cultures (3), whereas we found that ciprofloxacin was also bactericidal during the lag phase. n phosphate-buffered saline, an essentially nonreplicating system, ciprofloxacin was still effective, being able to penetrate and kill cells in their maintenance state. Although ciprofloxacin is bactericidal, regrowth of cultures did occur at some concentrations. nitially, a large proportion of the bacterial population may be affected by ciprofloxacin, with resultant death, but some cells may remain viable because (i) they are in a different growth stage and less susceptible; (ii) the concentration of ciprofloxacin is not sufficient to reach the intracellular levels required to effect the death of all the cells in the culture; and (iii) resistance occurs. These cells with reasonable doubling times, e.g., 3 min, will account for the culture viability increasing from <12 to >19 CFU/ml during overnight incubation. Crumplin et al. (5) showed that

10 34 CHALKLY AND KOORNHOF 11 ANTMCROB. AGNTS CHMOTHR T D 17 LL - 'j 16 w -J 2 4 FG. 9. Activities of ciprofloxacin and cefotaxime in combination against. coli. Symbols:,.25,ug of ciprofloxacin plus.5,ug of cefotaxime per ml;,.25,ug of ciprofloxacin plus.25,ug of cefotaxime per ml; *,.5 jig of cefotaxime plus.1,ug of ciprofloxacin per ml; V,.5,ug of cefotaxime per ml; A,.25 jig of ciprofloxacin per ml; V,.25 jig of cefotaxime per ml; A,.1 jig of ciprofloxacin per ml; x, control; _, all cultures at 24 h unless otherwise indicated. norfloxacin causes a rapid reduction in culture viability, but the mechanism of this initial bactericidal action is unknown. With nalidixic acid, dilution and plating of exposed cells onto an antibiotic-free medium removes the inhibition, and DNA replication can continue unimpaired (3). The initial bactericidal effect observed with ciprofloxacin may, therefore, be due in part to a greater affinity for the target site, resulting in inhibition being maintained even after transfer of the organisms to fresh medium. t is also possible that ciprofloxacin binds to an additional subcomponent of DNA gyrase. Because synergy would enhance the therapeutic effect of ciprofloxacin, experiments were designed to test the effects of various drug combinations. We defined synergy as a difference of '2 log1 CFU/ml at 6 and 24 h between the results of a combination of agents compared with the results of the most active of the two agents alone. Other workers (7, 1) have tended to determine synergy by the combined effect after 24 h, as they have found this period to be optimal for correlation with the checkerboard method. However, to study initial synergistic interactions, conventional methods

11 VOL. 28, 1985 ANTBACTRAL ACTVTY OF CPROFLOXACN 341 T17-16 a: cl.~~~~~~~~~~~~< FG. 1. Activities of ciprofloxacin and tobramycin in combination against. coli. Symbols: *, 1. jig of tobramycin plus.1,ug of ciprofloxacin per ml;,.25,ug of ciprofloxacin plus.25 ±Lg of tobramycin per ml;,.25,ug of ciprofloxacin plus.1,ug of tobramycin per ml; V, 1. plg of tobramycin per ml; A,.25,ug of ciprofloxacin per ml; V,.25,g of tobramycin per ml; A,.1 p.g of ciprofloxacin per ml; x,.1,ug of tobramycin and control; _, all cultures at 24 h. based on levels after 24 h are not appropriate. n a shaking system, an early synergistic effect based on killing curves can easily be masked by outgrowth after overnight incubation (Fig. 9 and 1). The basis of synergy between ciprofloxacin and other drugs is at present not known. However, ciprofloxacin is able to efficiently penetrate P. aeruginosa and. coli cells, and various degrees of interaction may therefore be expected when ciprofloxacin is combined with 1-lactam antibiotics. Synergy between ciprofloxacin and tobramycin may be explained on the basis of an increase in tobramycin bactericidal activity caused by the effect of ciprofloxacin on cell replication, with subsequent metabolic imbalance. However, combinations of ciprofloxacin with chloramphenicol were shown to be antagonistic (14). Ciprofloxacin shows some interesting bactericidal properties and is potentially a valuable agent for the treatment of urinary tract infections (R. Ziegler, K.-H. Graefe, W. Wingender, W. Gau, H.-J. Zeiler, U. Leitz, and P. Schact, Program Abstr. 23rd ntersci. Conf. Antimicrob. Agents Che-

12 342 CHALKLY AND KOORNHOF mother., abstr. no. 851, 1983). t may also be useful for the treatment of systemic infections subject to in vivo investigations. n a human trial on healthy volunteers (Ziegler et al., 23rd CAAC), the peak level of ciprofloxacin in plasma was found to be 1.58,ug/ml at 1 h after a single oral dose of 25 mg. This concentration is probably just within the therapeutic range for the treatment of systemic infections. The findings of excellent bactericidal activity at achievable levels against major bacterial pathogens provide good grounds for the clinical evaluation of ciprofloxacin and related compounds in systemic infections. LTRATUR CTD 1. Chartrand, S. A., R. K. Scribner, A. H. Weber, D. F. Welch, and M.. Marks n vitro activity of C-919 (AT-2266), an oral antipseudomonal compound. Antimicrob. Agents Chemother. 23: Chin, N.-X., and H. C. Neu Ciprofloxacin, a quinolone carboxylic acid compound active against aerobic and anaerobic bacteria. Antimicrob. Agents Chemother. 25: Cook, T. M., W. H. Deitz, and W. A. Goss Mechanism of action of nalidixic acid on scherichia coli. V. ffects on the stability of cellular constituents. J. Bacteriol. 91: Cozzarelli, N. R DNA gyrase and the supercoiling of DNA. Science 27: Crumplin, G. C., M, Kenwright, and T. Hirst nvestigations into the mechanism of action of the antibacterial agent norfloxacin. J. Antimicrob. Chemother. 13(Suppl. B): liopoulos, G. M., A. Gardelia, and R. C. Moeliering, Jr n vitro activity of ciprofloxacin, a new carboxyquinoline antimicrobial agent. Antimicrob. Agents Chemother. 25: Hallander, H.., K. Dornbusch, L. Gazelius, K. Jacobson, and. Karlsson Synergism between aminoglycosides and cephalosporins with antipseudomonal activity: interaction index and killing curve method. Antimicrob. Agents Chemother. 22: ANTMCROB. AGNTS CHMOTHR. 8. to, A., K. Hirai, M. noue, H. Koga, S. Suzue, T. rikura, and S. Mitsuhashi n vitro antibacterial activity of AM-715, a new nalidixic acid analog. Antimicrob. Agents Chemother. 17: Khan, M. Y., R. P. Gruninger, S. M. Nelsonf, and R.. Klicker Comparative in vitro activity of norfloxacin (MK-366) and ten other oral antimicrobial agents against urinary bacterial isolates. Antimicrob. Agents Chemother. 21: Krogstadi D. J., and R. C. Moeliering, Jr Combinations of antibiotics: mechanisms of interaction against bacteria, p. 35. n V. Lorian (ed.), Antibiotics in laboratory medicine. The Williams & Wilkins Co., Baltimore. 11. Nakamura, S., A. Minami, H. Katae, S. noue, J. Yamagishi, Y. Takase, and M. Shimizu n vitro antibacterial properties of AT-2266, a new pyridonecarboxylic acid. Antimicrob. Agents Chemother. 23: National Committee for Clinical Laboratory Standards Methods for dilution antimicrobial susceptibility test for bacteria that grow aerobically. M7-TC. National Committee for Clinical Laboratory Standards, Villanova, Pa. 13. Neu, H. C., and P. Labthalikal n vitro activity of norfloxacin, a quinolinecarboxylic acid, compared with that of 3-lactams, aminoglycosides, and trimethoprim. Antimicrob. Agents Chemother. 22: Reeves, D. S., M. J. Bywater, H. A. Holt, and L.. White n vitro studies with ciprofloxacin, a new 4-quinoline compound. J. Antimicrob. Chemother. 13: Shungu, D. L.,. Weinberg, and H. H. Gadebusch Tentative interpretive standards for disk diffusion susceptibility testing with norfloxacin (MK-366, AM-715). Antimicrob. Agents Chemother. 23: Van Caekenberghe, D. L., and S. R. Pattyn n vitro activity of ciprofloxacin compared with those of other new fluorinated piperazinyl-substituted quinoline derivatives. Antimicrob. Agents Chemother. 25:518-52i. 17. Wise, R., J. M. Andrews, and L. J. dwards n vitro activity of BAY 9867, a new quinoline derivative, compared with those of other antimicrobial agents. Antimicrob. Agents Chemother. 23: