Antimicrobial Activity of Ciprofloxacin against Pseudomonas

ANTIMICROBIAL AGENTS AND CHEMOTHERAPY, Aug. 1985, p. 331-342 Vol. 28, No. 20066-4804/85/080331-12$02.00/0Copyright C 1985, American Society for Microbiology

Antimicrobial Activity of Ciprofloxacin against Pseudomonasaeruginosa, Escherichia coli, and Staphylococcus aureus

Determined by the Killing Curve Method: Antibiotic Comparisonsand Synergistic InteractionsL. J. CHALKLEY* AND H. J. KOORNHOF

Department of Medical Microbiology, School of Pathology, University of the Witwatersrand, and the South AfricanInstitute 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 bactericidalagent against Pseudomonas aeruginosa and Escherichia coli. A bactericidal effect was achieved immediatelyafter the addition of ciprofloxacin. At a concentration of 0.5 ,ug/ml, culture viability was reduced from 5 x 10to about S x 103 CFU/ml within 15 min, and at 0.1 ,ug/ml, a >10-fold reduction in viability resulted during thefirst hour after exposure. This bactericidal activity observed during the lag phase in Mueller-Hinton broth wasalso 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 ciprofloxacinprevented culture regrowth at lower concentrations. Staphylococcus aureus was not as susceptible tociprofloxacin; killing occurred at a concentration of 0.5 ,Ig/ml only after the onset of exponential growth in thecontrol culture. Synergistic interactions were observed wtih ciprofloxacin in combination with tobramycin andazlociilin against P. aeruginosa and with cefotaxime and tobramycin against E. coli.

Interest in new quinoline derivatives norfloxacin (8, 9, 13)and, more recently, enoxacin CI-919 (1, 11) as an alternategroup of antibacterial agents has been expanded with theintroduction of ciprofloxacin (BAY o 9867). Ciprofloxacinwas shown to have activity against a wide range of bacteriaand was particularly effective against the Enterobacteria-ceae (2, 6, 16, 17). Many of the strains tested by Wise et al.(17) were resistant to aminoglycosides or P-lactam antibiot-ics but sensitive to ciprofloxacin. The standard MIC methodfor determining antibacterial activity provides no informa-tion on initial killing kinetics. The present investigation wasdesigned to include the killing curve method to obtainadditional information on the antimicrobial properties ofciprofloxacin in vitro, particularly with regard to its initialbactericidal activity against selected organisms.

MATERIALS AND METHODS

Strains and antibiotics. The susceptible culture collectionstrains Pseudomonas aeruginosa ATCC 27853, Escherichiacoli ATCC 25922, and Staphylococcus aureus ATCC 25923were used.The following antimicrobial agents of known potency were

evaluated: ciprofloxacin and azlocillin (Bayer AG, Wup-pertal, Federal Republic of Germany), tobramycin (Eli Lilly& Co., Indianapolis, Ind.), aztreonam (E. R. Squibb & Sons,Princeton, N.J.), and cefotaxime (Roussel Laboratories,Wembley, England).

Antibiotics were dissolved in water at 1,000 ,ug/ml. Allsubsequent dilutions were made in cation-supplementedMueller-Hinton broth (12; Difco Laboratories, Detroit,Mich.) and prepared fresh for each experiment.MIC and MBC determinations. MICs were determined by

the broth dilution method described by the National Com-

* Corresponding author.

mittee for Clinical Laboratory Standards (12) in cation-supplemented Mueller-Hinton broth (pH 7.2). Twofold anti-biotic dilutions were prepared in 1 ml of broth. The inoculumwas adjusted from an exponential-phase culture to give aninitial concentration of 5 x 105 CFU/ml. After 18 h ofincubation at 35°C, the MIC was determined as the lowestconcentration that completely inhibited growth. Samples (10,ul) were then transferred from tubes showing no growth ontoblood agar plates which were incubated for 18 h at 35°C. TheMBC was read as the lowest concentration of antibioticwhich produced '99.9% killing of the inoculum (five colo-nies or fewer for 10 ,ul of subculture).

Killing curve method. Time-kill curves were constructedby previously described techniques (10) with the followingspecifications. The medium was cation-supplementedMueller-Hinton broth (pH 7.2). The stationary-phaseinoculum was prepared by inoculating 10 ml of broth andincubating it for 20 h at 37°C. After 20 h, cell numbers weredetermined from an optical density-CFU standard curve,and dilutions of the culture were made in fresh medium to

TABLE 1. Antibacterial activities of ciprofloxacin and selectedagents

Organism Agent MIC (kig/mI) MBC (pLg/ml)P. aeruginosa Ciprofloxacin 0.016 0.03

Azlocillin 8 16Aztreonam 0.06 0.06Tobramycin 0.5 1

E. coli Ciprofloxacin 0.016 0.016Cefotaxime 0.06 0.06Aztreonam 0.125 0.25Tobramycin 1 1

S. aureus Ciprofloxacin 0.25 2

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332 CHALKLEY AND KOORNHOF

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(A) P. augna

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~~~~~~~~~~~~~~~103

10213 4 24s 920 1 24TIME (h) TIMIME (h)

4x1O939P

(C) S. aureus

103'

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FIG. 1. Activity of ciprofloxacin (in micrograms per milliliter) against the following organisms. (A) P. aeruginosa: 0, 0.5; 0, 0.1; 0,

control. (B) E. coli: 0, 0.5; *, 0.05; 0, 0.01; O, control. (C) S. aureus: 0, 0.5; 0, 0.25; 0, control.

(B) E.coli

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ANTIBACTERIAL ACTIVITY OF CIPROFLOXACIN 333

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FIG. 2. Activity of ciprofloxacin (in micrograms per milliliter) in phosphate-buffered saline against the following organisms. (A) P.aeruginosa: 0, 0.5; 0, 0.1; O, control. (B) E. coli: 0, 0.1; 0, 0.05; O, control. (C) S. aureus: 0, 1.0; 0, 0.5; O, control.

give an initial inoculum of about 5 x i05 CFU/ml. Tests werecarried out in 25-ml flasks containing 10 ml of culture withthe required concentrations of antibiotics. The cultures wereincubated at 37°C and 150 rpm on an orbital environmentalshaker (New Brunswick Scientific Co., Inc., Edison, N.J.).Samples were taken at regular intervals and suitably dilutedfor viable count estimations either by the method of Milesand Misra (20 ,ul delivered on each of five spots) or spread

plate (100 ,ul on each of three plates). All tests, unlessotherwise indicated, were performed on stationary-phasecells. For the experiment using exponential-phase cells, theinoculum was prepared from 3-h cultures. For determina-tions of antibacterial activity in a basically nongrowingsystem, stationary-phase cells and ciprofloxacin were di-luted in and added to phosphate-buffered saline (pH 7.2)instead of broth.

(C) S. aureus

Z-o

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FIG. 3. Activities of ciprofloxacin in the presence and absence of 10%o (vol/vol) horse serum. E. coli: A, 0.05 ,ug of ciprofloxacin per ml;A, 0.05 ,ug of ciprofloxacin per ml plus horse serum; V, controls. P. aeruginosa: 0, 0.1 ,ug ciprofloxacin per ml; 0, 0.1 ,ug of ciprofloxacinper ml plus horse serum; x, controls. S. aureus: *, 0.5 p.g of ciprofloxacin per ml; O, 0.5 ,ug of ciprofloxacin per ml plus horse serum; V,controls.

RESULTS

The MICs and MBCs of ciprofloxacin and selected agentsused for antibiotic ccomparisons and synergistic interactionsby the killing curve method are shown in Table 1.The killing curves shown are representative curves; ex-

perinients were performed in duplicate. For neat sampling(viable counts below 5 x 103 CFU/ml), some antibioticcarry-over was observed with ciprofloxacin at 0.5 ,ug/mlagainst E. coli and P. aeruginosa. Samples (1 ml) weretherefore filtered through membrane filters (0.45-pum porediameter; Millipore Corp., Bedfordi Mass.), and cells werewashed on the filter with 10 ml of Ringer solution (one-fourthstrength). Antibiotic carry-over did not occur with cipro-

floxacin concentrations of <0.5 ,ug/ml, with the other anti-biotics at the concentrations used, or with the ciprofloxacinlevels used against S. aureus.From hours 0 to 4, ciprofloxacin was bactericidal at

concentrations of 0.5 and 0.1 jig/ml against P. aeruginosa,0.5, 0.1, and 0.05 jig/ml against E. coli, and 0.5 ,ug/ml againstS. aureus (Fig. 1). Regrowth of the culture after 24 h did notoccur with P. aeruginosa containing the above-mentionedconcentrations of ciprofloxacin or with ciprofloxacin at 0.5,ug/ml against E. coli; culture viability remained at <102CFU/ml. Partial regrowth was observed in E. coli cultureswith ciprofloxacin at 0.05 ,ug/ml, with culture viability at 24h being 1.5 x 104 CFU/ml. P. aeruginosa and E. coli cultureswere extremely sensitive to ciprofloxacin, which caused an


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ANTIBACTERIAL ACTIVITY OF CIPROFLOXACIN

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FIG. 4. Comparison of ciprofloxacin, tobramycin, aztreonam, and cefotaxime activities against E. coli. Symbols: *, 0.05 ,ug ofciprofloxacin per ml; O, 1.0 ,ug of tobramycin per ml; /, 1.0 ,ug of aztreonam per ml; V, 0.1 ,ug of cefotaxime per ml; V, 0.01 ,g of cefotaximeper ml; x, control.

immediate bactericidal effect, reducing viability from 5 x lo,to 4.7 x 103 and 4.3 x 103 CFU/ml, respectively, within 15min, i.e., before completion of the lag phase. Against S.aureus; killing by ciprofloxacin only occurred after a periodof about 1.5 h, which corresponded to the onset of exponen-tial 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 thissystem, and rapid initial killing was still evident in P.

aeruginosa and E. coli cultures. However, the bactericidalactivity of ciprofloxacin was considerably reduced against S.aureus in phosphate-buffered saline, which did not supportexponential growth. The addition of 10%o (vol/vol) horseserum (Fig. 3) slightly decreased antibacterial activityagainst E. coli and P. aeruginosa, but killing was comparableafter 4 h against E. coli and after 2 h against P. aeruginosa.The rates of killing of S. aureus with and without horseserum were similar, but the onset of killing in the presence ofserum was delayed. The activities of ciprofloxacin, tobramy-

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TIME (h)FIG. 5. Comparison of ciprofloxacin, tobramycin, aztreonam, and azlocillin activities against P. aeruginosa. Symbols: 0, 0.1 ,ug of

ciprofloxacin per ml; *, 5.0 ,ug of tobramycin per ml; O, 1.0 ,ug of tobramycin per ml; A, 5.0 ,ug of aztreonam per ml; A, 1.0 ,ug of aztreonamper ml; V, 10.0 ,g of azlocillin per ml; x, control.

cin, aztreonam, and cefotaxime against E. coli were com-pared (Fig. 4). The immediate bactericidal effect shown byciprofloxacin was not demonstrated with the other agents.Ciprofloxacin produced a rate of killing similar to that of theother agents tested, but it killed at a lower concentration.Similarly, ciprofloxacin compared favorably with tobramy-cin, aztreonam, and azlocillin against P. aeruginosa (Fig. 5).As stationary-phase cells were more susceptible to cipro-floxacin than to the other agents tested, a comparison againstthe exponential cells of P. aeruginosa was also made (Fig.6). With exponential cells as the initial inoculum,

ciprofloxacin at 0.05 ,ug/ml produced activity similar to thatof tobramycin and aztreonam at 1.0 p.g/ml during the first 5-hperiod. There was still a delay of 1 h before aztreonanmproduced a bactericidal effect in this system. Tobramycin at1.0 ,ug/ml, although causing a slightly greater kill initially,failed to reduce viability below 102 CFU/ml by hour 5, andsubsequent outgrowth occurred by 24 h. These results wereprevented by concentrations of ciprofloxacin and aztreonamat 0.5 and 1.0 ,ug/ml, respectively.Based on a difference of _2 log10 CFU at 24 h between the

results of combinations and the results of the most active of

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TIME (h)FIG. 6. Activities of ciprofloxacin, tobramycin, and aztreonam against exponential cells of P. aeruginosa. Symbols: 0, 0.05 ,ug of

ciprofloxacin per ml; 0, 0.01 ,ug of ciprofloxacin per ml; O, 1.0 ,ug of tobramycin per ml; A, 1.0 ,ug of aztreonam per ml; x, control.

the two individual components as defined by Hallander et al.(7), synergy was observed when ciprofloxacin was combinedwith tobramycin or azlocillin against P. aeruginosa (Fig. 7and 8) and with cefotaxime against E. coli (Fig. 9). A _2log1o reduction in viability between the combination and itsmost active constituent during the first 6-h period was shown

with other concentrations and combinations of the antibiot-ics (Fig. 9 and 10). With a combination of tobramycin andciprofloxacin against E. coli (Fig. 10), a >2 log1o reduction inviability was observed by 4 h but not at 24 h as outgrowth ofthis culture occurred. Ciprofloxacin in combination withaztreonam showed an increase in bactericidal activity over

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TIME (h)FIG. 7. Activities of ciprofloxacin and tobramycin in combination against P. aeruginosa. Symbols: *, 0.025 ,ug of ciprofloxacin plus 1.0

,ug of tobramycin per ml; 0, 0.025 ,ug of ciprofloxacin plus 0.5 ,ug of tobramycin per ml; E, 0.025 ,ug of ciprofloxacin plus 0.25 pLg oftobramycin per ml; *, 1.0 ,ug of tobramycin plus 0.01 ,ug of ciprofloxacin per ml; A, 0.025 ,ug of ciprofloxacin per ml; V, 1.0 ,ug of tobramycinper ml; V, 0.5 ,ug of tobramycin per ml; A, 0.01 ,ug of ciprofloxacin per ml; x, 0.25 ,ug of tobramycin per ml and control; _, all culturesat 24 h unless otherwise indicated.

either agent alone against E. coli and P. aeruginosa, al-though synergy as defined was not effected (results notshown). Antagonism did not occur with the combinationstested at the concentrations (of the agents) used.

DISCUSSION

The quinoline compounds have emerged as an interestingand potentially important group of chemotherapeutic agents.Ciprofloxacin as evaluated here is particularly effectiveagainst gram-negative bacteria at lower concentrations than

the other agents with similar antibacterial spectra. It is,however, far less active against S. aureus.

It has been proposed that as ciprofloxacin has a spectrumof activity similar to that of nalidixic acid, it probably has amode of action similar to that of nalidixic acid (17). The latteragent acts on the A subunit of DNA gyrase, which inhibitsand affects DNA supercoiling, resulting in inhibition ofDNAreplication and possibly transcription, depending on thepromoter (4, 5). Bacterial isolates resistant to norfloxacinhave been found to be resistant to nalidixic acid also (15).However, most nalidixic acid-resistant isolates were not


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FIG. 8. Activities of ciprofloxacin and azlocillin in combination against P. aeruginosa. Symbols: 0, 0.025 ,ug of ciprofloxacin plus 5.0 ,ugof azlocillin per ml; 0, 0.025 ,g of ciprofloxacin plus 2.5 ,ug of azlocillin per ml; *, 10.0 ,ug of azlocillin plus 0.01 ,ug of ciprofloxacin per ml;A, 0.025 Rg of ciprofloxacin per ml; , 10.0 ,g of azlocillin per ml; A, 0.01 ,ug of ciprofloxacin per ml; V, 5.0 ,ug of azlocillin per mi; x, 2.5,ug of aziocillin per ml and control; _, all cultures at 24 h unless otherwise indicated.

concurrently resistant to norfloxacin. The frequency ofresistance to ciprofloxacin was shown to be generally low(2). Wise et al. (17) found that strains which were lesssusceptible to nalidixic acid and norfloxacin tended to be lesssusceptible to ciprofloxacin, although other workers (16)found no cross resistance between nalidixic acid andciprofloxacin.

Nalidixic acid has been shown to be active only onexponentially growing cultures (3), whereas we found thatciprofloxacin was also bactericidal during the lag phase. Inphosphate-buffered saline, an essentially nonreplicating sys-tem, ciprofloxacin was still effective, being able to penetrate

and kill cells in their maintenance state. Althoughciprofloxacin is bactericidal, regrowth of cultures did occurat some concentrations. Initially, a large proportion of thebacterial population may be affected by ciprofloxacin, withresultant 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 reachthe intracellular levels required to effect the death of all thecells in the culture; and (iii) resistance occurs. These cellswith reasonable doubling times, e.g., 30 min, will account forthe culture viability increasing from <102 to >109 CFU/mlduring overnight incubation. Crumplin et al. (5) showed that

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1010

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FIG. 9. Activities of ciprofloxacin and cefotaxime in combination against E. coli. Symbols: 0, 0.025 ,ug of ciprofloxacin plus 0.05 ,ug ofcefotaxime per ml; 0, 0.025 ,ug of ciprofloxacin plus 0.025 ,ug of cefotaxime per ml; *, 0.05 jig of cefotaxime plus 0.01 ,ug of ciprofloxacinper ml; V, 0.05 ,ug of cefotaxime per ml; A, 0.025 jig of ciprofloxacin per ml; V, 0.025 jig of cefotaxime per ml; A, 0.01 jig of ciprofloxacinper ml; x, control; _, all cultures at 24 h unless otherwise indicated.

norfloxacin causes a rapid reduction in culture viability, butthe mechanism of this initial bactericidal action is unknown.With nalidixic acid, dilution and plating of exposed cells ontoan antibiotic-free medium removes the inhibition, and DNAreplication can continue unimpaired (3). The initial bacteri-cidal effect observed with ciprofloxacin may, therefore, bedue in part to a greater affinity for the target site, resulting ininhibition being maintained even after transfer of the orga-nisms to fresh medium. It is also possible that ciprofloxacinbinds to an additional subcomponent of DNA gyrase.

Because synergy would enhance the therapeutic effect ofciprofloxacin, experiments were designed to test the effectsof various drug combinations. We defined synergy as adifference of '2 log10 CFU/ml at 6 and 24 h between theresults of a combination of agents compared with the resultsof the most active of the two agents alone. Other workers (7,10) have tended to determine synergy by the combined effectafter 24 h, as they have found this period to be optimal forcorrelation with the checkerboard method. However, tostudy initial synergistic interactions, conventional methods

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ANTIBACTERIAL ACTIVITY OF CIPROFLOXACIN 341

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102 .cl.~~~~~~~~~~~~<02 I

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FIG. 10. Activities of ciprofloxacin and tobramycin in combination against E. coli. Symbols: *, 1.0 jig of tobramycin plus 0.01 ,ug ofciprofloxacin per ml; 0, 0.025 ,ug of ciprofloxacin plus 0.25 ±Lg of tobramycin per ml; 0, 0.025 ,ug of ciprofloXacin plus 0.1 ,ug of tobramycinper ml; V, 1.0 pLg of tobramycin per ml; A, 0.025 ,ug of ciprofloxacin per ml; V, 0.25 ,g of tobramycin per ml; A, 0.01 p.g of ciprofloxacinper ml; x, 0.1 ,ug of tobramycin and control; _, all cultures at 24 h.

based on levels after 24 h are not appropriate. In a shakingsystem, an early synergistic effect based on killing curvescan easily be masked by outgrowth after overnight incuba-tion (Fig. 9 and 10).The basis of synergy between ciprofloxacin and other

drugs is at present not known. However, ciprofloxacin isable to efficiently penetrate P. aeruginosa and E. coli cells,and various degrees of interaction may therefore be ex-pected when ciprofloxacin is combined with 1-lactam anti-biotics. Synergy between ciprofloxacin and tobramycin may

be explained on the basis of an increase in tobramycinbactericidal activity caused by the effect of ciprofloxacin oncell replication, with subsequent metabolic imbalance. How-ever, combinations of ciprofloxacin with chloramphenicolwere shown to be antagonistic (14).

Ciprofloxacin shows some interesting bactericidal proper-ties and is potentially a valuable agent for the treatment ofurinary tract infections (R. Ziegler, K.-H. Graefe, W. Win-gender, W. Gau, H.-J. Zeiler, U. Leitz, and P. Schact, Pro-gram Abstr. 23rd Intersci. Conf. Antimicrob. Agents Che-

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mother., abstr. no. 851, 1983). It may also be useful for thetreatment of systemic infections subject to in vivo investi-gations. In a human trial on healthy volunteers (Ziegler etal., 23rd ICAAC), the peak level of ciprofloxacin in plasmawas found to be 1.58 ,ug/ml at 1 h after a single oral dose of250 mg. This concentration is probably just within thetherapeutic range for the treatment of systemic infections.The findings of excellent bactericidal activity at achievablelevels against major bacterial pathogens provide goodgrounds for the clinical evaluation of ciprofloxacin andrelated compounds in systemic infections.

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2. Chin, N.-X., and H. C. Neu. 1984. Ciprofloxacin, a quinolonecarboxylic acid compound active against aerobic and anaerobicbacteria. Antimicrob. Agents Chemother. 25:319-326.

3. Cook, T. M., W. H. Deitz, and W. A. Goss. 1966. Mechanism ofaction of nalidixic acid on Escherichia coli. IV. Effects on thestability of cellular constituents. J. Bacteriol. 91:774-779.

4. Cozzarelli, N. R. 1980. DNA gyrase and the supercoiling ofDNA. Science 207:953-960.

5. Crumplin, G. C., M, Kenwright, and T. Hirst. 1984. Investiga-tions into the mechanism of action of the antibacterial agentnorfloxacin. J. Antimicrob. Chemother. 13(Suppl. B):9-23.

6. Eliopoulos, G. M., A. Gardelia, and R. C. Moeliering, Jr. 1984.In vitro activity of ciprofloxacin, a new carboxyquinoline anti-microbial agent. Antimicrob. Agents Chemother. 25:331-335.

7. Hallander, H. 0., K. Dornbusch, L. Gazelius, K. Jacobson, andI. Karlsson. 1982. Synergism between aminoglycosides andcephalosporins with antipseudomonal activity: interaction indexand killing curve method. Antimicrob. Agents Chemother.22:743-752.

8. Ito, A., K. Hirai, M. Inoue, H. Koga, S. Suzue, T. Irikura, andS. Mitsuhashi. 1980. In vitro antibacterial activity of AM-715, anew nalidixic acid analog. Antimicrob. Agents Chemother.17:103-108.

9. Khan, M. Y., R. P. Gruninger, S. M. Nelsonf, and R. E. Klicker.1982. Comparative in vitro activity of norfloxacin (MK-0366)and ten other oral antimicrobial agents against urinary bacterialisolates. Antimicrob. Agents Chemother. 21:848-851.

10. Krogstadi D. J., and R. C. Moeliering, Jr. 1980. Combinations ofantibiotics: mechanisms of interaction against bacteria, p. 305.In V. Lorian (ed.), Antibiotics in laboratory medicine. TheWilliams & Wilkins Co., Baltimore.

11. Nakamura, S., A. Minami, H. Katae, S. Inoue, J. Yamagishi, Y.Takase, and M. Shimizu. 1983. In vitro antibacterial propertiesof AT-2266, a new pyridonecarboxylic acid. Antimicrob. AgentsChemother. 23:641-648.

12. National Committee for Clinical Laboratory Standards. 1983.Methods for dilution antimicrobial susceptibility test for bacte-ria that grow aerobically. M7-TC. National Committee forClinical Laboratory Standards, Villanova, Pa.

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14. Reeves, D. S., M. J. Bywater, H. A. Holt, and L. 0. White. 1984.In vitro studies with ciprofloxacin, a new 4-quinoline com-pound. J. Antimicrob. Chemother. 13:325-331.

15. Shungu, D. L., E. Weinberg, and H. H. Gadebusch. 1983.Tentative interpretive standards for disk diffusion susceptibilitytesting with norfloxacin (MK-0366, AM-715). Antimicrob.Agents Chemother. 23:256-260.

16. Van Caekenberghe, D. L., and S. R. Pattyn. 1984. In vitroactivity of ciprofloxacin compared with those of other newfluorinated piperazinyl-substituted quinoline derivatives. Anti-microb. Agents Chemother. 25:518-52i.

17. Wise, R., J. M. Andrews, and L. J. Edwards. 1983. In vitroactivity of BAY 09867, a new quinoline derivative, comparedwith those of other antimicrobial agents. Antimicrob. AgentsChemother. 23:559-564.


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Antimicrobial Activity of Ciprofloxacin against Pseudomonas