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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
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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
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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.
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