Activity of levofloxacin and ciprofloxacin against urinary pathogens

Abstract

This study compares the antibacterial activities of levofloxacin and ciprofloxacin against recently isolated urinary tract pathogens, by evaluating their MICs and MBCs in accordance with NCCLS susceptibility tests, time–kill curves and interference with bacterial adhesion to uroepithelial cells. A total of 200 clinical isolates was tested, including the species Escherichia coli, Klebsiella pneumoniae, Proteus mirabilis, Proteus vulgaris, Providencia rettgeri, Pseudomonas aeruginosa, Enterococcus faecalis, Staphylococcus aureus and Staphylococcus epidermidis. All E. coli isolates were susceptible to levofloxacin and only one was resistant to ciprofloxacin, and there were no differences between β-lactamase-positive and -negative strains. K. pneumoniae strains resistant to ciprofloxacin were also resistant to levofloxacin. Methicillin-resistant S. aureus seemed to be less susceptible than methicillin-susceptible strains to these quinolones. S. epidermidis strains were susceptible to levofloxacin and ciprofloxacin, with the exception of two isolates. Incubation of S. aureus and E. coli with subinhibitory antimicrobial concentrations reduced their capacity to adhere to uroepithelial cells; this was statistically significant at 0.25 × MIC with respect to controls (P < 0.05). Inhibition of adhesion ranged from 36 to 43% when bacteria were incubated in the presence of 0.25 × MIC of levofloxacin and ciprofloxacin, and from 10 to 27% at 0.125 × MIC. These findings suggest that levofloxacin is an effective alternative to ciprofloxacin in the treatment of urinary tract infections and that sub-inhibitory concentrations may contribute to efficacy.

Introduction

Urinary tract infections (UTIs) are among the most common bacterial infections. Microorganisms frequently isolated include Escherichia coli, other Enterobacteriaceae and some Gram-positive strains. Antibiotics used in therapy are usually able to reach high urinary concentrations, which are likely to be clinically effective. The fluoroquinolones have assumed an important role in the therapy of these infections, since they have a broad spectrum of activity, including Gram-positive and in particular Gram-negative bacteria.^1–3 Among recently developed fluoroquinolones, levofloxacin is widely used in clinical practice because of its established efficacy and safety.^4,5 levofloxacin shows better activity against Gram-positive bacteria and is less likely to select resistant strains compared with older quinolones.^6–8

Microbial attachment to the epithelial surface has been implicated in the initial stages of several infections.^9,10 Adherence is a pathogenic factor whereby organisms impair the integrity of the mucosal barrier and cause disease. Sublethal concentrations of various antibiotics markedly impair bacterial adhesion to human cells, by affecting the adherence properties of microorganisms.^11,12 Intermittent antibiotic doses generally cause oscillations in drug concentrations at an infection site, exceeding MIC values for a limited period only. After this, antibiotic concentrations may fall below the MIC and hence are less likely to eliminate bacteria, although they may affect bacterial virulence: this may occur after therapy has been discontinued but does not occur during quinolone therapy of UTIs. Knowledge of the pharmacodynamic effect of subinhibitory concentrations and drug pharmacokinetic profiles is a useful basis for optimal therapy.

The present study evaluates the antibacterial activity of levofloxacin and ciprofloxacin against recently isolated UTI pathogens, by studying MICs, MBCs, time–kill curves and interference by the two drugs with bacterial adhesion to uroepithelial cells.

Materials and methods

Microorganisms

A total of 200 strains isolated during 1999–2000 at ‘L. Sacco’ Teaching Hospital of Milan (Italy) were tested. They included 27 E. coli [10 producers and 17 non-producers of extended-spectrum β-lactamases (ESBLs)], 24 Klebsiella pneumoniae, 25 Proteus mirabilis, 16 Proteus vulgaris, 10 Providencia rettgeri, 22 Pseudomonas aeruginosa, 26 Enterococcus faecalis, 20 methicillin-susceptible Staphylococcus aureus (MSSA), 10 methicillin-resistant Staphylococcus aureus (MRSA) and 20 Staphylococcus epidermidis. These strains were isolated from patients presenting with UTI; only one isolate per patient was used in order to avoid strain duplication.

S. aureus strains were evaluated for methicillin resistance (oxacillin disc test, Becton Dickinson, Cockeysville, MD, USA), and E. coli strains were assayed for production of ESBLs by the Vitek automatic system (bioMérieux, Marcy-l'Étoile, France); confirmation by ceftazidime/ clavulanic acid and cefotaxime/clavulanic acid test was in accordance with NCCLS procedures.¹³S. aureus ATCC 29213, S. aureus ATCC 43300 and E. coli ATCC 25922 were used as controls for methicillin resistance and ESBL production.¹³

Antimicrobial agents

Stock solutions of levofloxacin (Aventis Pharma, Lainate, Italy) prepared at a concentration of 1280 mg/L in NaOH 0.05 mol/L and ciprofloxacin (Bayer SpA, Milan, Italy) dissolved in sterile phosphate buffer at a concentration of 2000 mg/L were stored at –20°C until use.

Determination of MICs and MBCs

Determinations of MICs and MBCs by the microdilution broth method were carried out according to NCCLS approved standards: an adjusted inoculum of the test organism was inoculated into Mueller–Hinton broth (Oxoid, Basingstoke, UK) containing two-fold serial dilutions of an initial antibiotic solution, so that each well contained approximately 5 × 10⁵ cfu/mL.^13,14 Results were observed after 18 h incubation at 37°C and the MIC was defined as the lowest concentration to inhibit visible growth. The MBC was determined by plating 0.010 mL from the wells showing no visible growth on to Mueller– Hinton agar plates (Oxoid) and incubating for 18–24 h. The MBC was defined as the concentration at which there was a 99.9% reduction in cfu compared with the original inoculum. For each analytical series, quality controls were carried out with E. coli ATCC 25922 strain: expected MIC ranges were 0.008–0.06 mg/L for levofloxacin and 0.004– 0.015 mg/L for ciprofloxacin.¹⁴

Time–kill curves

Bactericidal activity was evaluated by performing time–kill curves on the following strains: ESBL-positive E. coli (Ec 2068), ESBL-negative E. coli (Ec 2648), P. mirabilis (Pm 2912), MRSA (Sa 211) and MSSA (Sa 1492). MICs of each antibiotic for these strains are reported in Table I.

Each strain was grown to logarithmic phase in Mueller– Hinton broth. levofloxacin and ciprofloxacin sterile solutions (0.1 mL) were added to 19.9 mL of the broth cultures (10⁵–10⁶ cfu/mL), to give final drug concentrations equivalent to 0.5 × MIC, 1 × MIC, 2 × MIC and 4 × MIC. Antibiotic-free growth controls were also included. Flasks were incubated aerobically at 37°C with mechanical agitation. Viable counts were performed at 0, 3, 6 and 24 h after addition of antimicrobial agents, following washings by centrifugation to avoid antibiotic carry-over and serial 10-fold dilution in phosphate-buffered saline pH 7.3 (PBS; Oxoid). Colonies were counted on Mueller–Hinton plates after 24 h incubation in air at 37°C. The killing rate was determined by plotting total number of viable cells (mean of cfu) on a logarithmic scale against time.

Interference with adhesion to uroepithelial cells

Strains of E. coli (Ec 2648) and S. aureus (Sa 1492) tested previously for their capacity to adhere to uroepithelial cells, were examined; their MICs are reported in Table I. Adhesion assays were performed as described previously.^15,16 Briefly, human uroepithelial cells collected from healthy females (1 × 10⁵ cells/mL) were incubated with bacteria (1 × 10⁸ cfu/mL) previously grown with or without sub-inhibitory concentrations of levofloxacin or ciprofloxacin (0.25 × and 0.125 × MIC). Each sample was prepared in duplicate. After 45 min, non-adherent bacteria were eliminated by three centrifugations. The final pellet was then resuspended in a small quantity of PBS and placed either on a glass slide, dried and Gram stained, or on round microscope coverslips and dried for scanning electron microscopy (SEM). Coverslips were fixed in 2.5% glutaraldehyde (Sigma, St Louis, MO, USA) and postfixed with 1% osmium tetroxide (Sigma) for 90 min. Specimens were then dehydrated with ethanol and acetone, and, after critical point drying (Emitech), covered with gold and examined by SEM.

The mean number of adhered bacteria per cell was determined by counting the number of bacteria adhering to 40 uroepithelial cells. Each test was performed in triplicate. Inhibition of adherence (IA) was calculated using this formula:

\[IA\ (\%)\ {=}\ 100\ {\mbox{--}}\ [(mean\ number\ of\ adhering\ bacteria\ in\ presence\ of\ antibiotic/\]

\[mean\ number\ of\ adhering\ bacteria\ without\ antibiotic)\ {\times}\ 100]\]

Statistical analysis

Interference with adhesion to epithelial cells was compared by means of one-way analysis of variance. Differences were considered statistically significant when P ≤ 0.05.

Results

Antibacterial activity

The antibacterial activities of levofloxacin and ciprofloxacin are summarized in Tables II and III, which report the MIC and MBC values for all the test microorganisms. Quality control results fell within the accepted ranges for ciprofloxacin and levofloxacin with a mean value of 0.03 mg/L for levofloxacin and 0.013 mg/L for ciprofloxacin. MICs and MBCs of levofloxacin and ciprofloxacin were similar for most of the microorganisms tested, with differences of less than two dilutions. However, MIC₉₀ and MIC₅₀ values of levofloxacin were four-fold higher than those of ciprofloxacin for P. vulgaris, P. rettgeri and P. aeruginosa. Using NCCLS breakpoints of ≤2 and ≤1 mg/L for susceptibility to levofloxacin and ciprofloxacin,¹⁴ all E. coli isolates were susceptible to levofloxacin and only one was resistant to ciprofloxacin, with no remarkable differences between ESBL-positive and -negative strains. The K. pneumoniae strain resistant to ciprofloxacin was also resistant to levofloxacin, while P. mirabilis classified as intermediate for ciprofloxacin was susceptible to levofloxacin. One isolate of P. rettgeri and one of P. aeruginosa were susceptible to ciprofloxacin but intermediate for levofloxacin. None of the MSSA strains were resistant to levofloxacin, while four isolates were intermediate. By contrast, resistance to ciprofloxacin occurred in four of the 20 MSSA strains tested. MRSA seemed to be less susceptible than MSSA to levofloxacin; this difference seemed less marked for ciprofloxacin. S. epidermidis strains were generally susceptible to levofloxacin and ciprofloxacin, with the exception of two resistant strains, which were responsible for the high MIC and MBC values.

Time–kill curves

The bactericidal kinetics of levofloxacin and ciprofloxacin against the test strains are shown in Figures 1–3. The two antimicrobials were considered to have bactericidal activity when a decrease of at least 3 logs from the starting inoculum was observed. levofloxacin was bactericidal against E. coli at 2 × and 4 × MIC after 24 h, while ciprofloxacin caused a 3 log decrease in bacterial count after 24 h only at 4 × MIC, with a similar trend for the β-lactamase producer and non-producer. The bactericidal activity of levofloxacin against S. aureus was similar for methicillin-susceptible and methicillin-resistant strains. In contrast, ciprofloxacin was bactericidal for the MSSA strain at 4 × MIC, and bacteriostatic against the MRSA strain. There was a marked decrease in P. mirabilis count in the presence of levofloxacin after 24 h, while the action of ciprofloxacin was less sustained.

Impaired adhesion to uroepithelial cells

Table IV illustrates the interference by levofloxacin and ciprofloxacin subinhibitory concentrations with adhesion of E. coli and S. aureus to uroepithelial cells. For both strains, incubation with subinhibitory concentrations of the two drugs reduced adherence to uroepithelial cells. In particular, a statistically significant reduction in the mean number of adherent bacteria/cell was observed at 0.25 × MIC with both drugs. Bacterial adhesion to uroepithelial cells was also reduced when bacteria were incubated with 0.125 × MIC levofloxacin, although there was no statistical difference. Consequently, inhibition of adhesion ranged from 36 to 43% when bacteria were incubated with 0.25 × MIC of levofloxacin and ciprofloxacin, and from 10 to 27% at 0.125 × MIC. SEM photographs (Figure 4) show the typical appearance of urinary cells incubated with E. coli in the presence or absence of antimicrobial compounds.

Discussion

Fluoroquinolones are appropriate therapy for UTI because of their high urine concentrations and wide spectrum of activity. Our results confirm the broad antibacterial spectrum against both Gram-positive and -negative bacteria reported by other authors.^2,3,17,18 Other authors have evaluated levofloxacin antibacterial activity independently against ciprofloxacin-resistant and -susceptible strains,^19,20 but we chose to test strains irrespective of ciprofloxacin susceptibility. As a consequence, MIC₉₀ values for some species were higher than those reported previously.²¹

Ciprofloxacin MICs were generally lower than those of levofloxacin, but since the established breakpoints for ciprofloxacin are about half of those for levofloxacin, the effective potency of the two compounds was comparable. Although only a few MRSA strains were tested, they seemed less susceptible than MSSA to levofloxacin, confirming data reported by other authors.^18,22

Production of ESBLs did not influence levofloxacin and ciprofloxacin antibacterial activity against E. coli, which was markedly susceptible to these quinolones; the only ciprofloxacin-resistant E. coli strain was ESBL negative. In vitro activity against P. aeruginosa indicated that MICs of ciprofloxacin are generally lower than those of levofloxacin, although the pattern of susceptibility to the two drugs remains quite similar.

Comparison of the killing kinetics showed more sustained activity against the MRSA and P. mirabilis strains by levofloxacin compared with ciprofloxacin, which was not bactericidal against the strains even at 4 × MIC.

Although it has been reported that exposure to quinolones has caused a decline in the susceptibility of urinary isolates to quinolones,^2,22 in the present study levofloxacin and ciprofloxacin showed excellent activity against the urinary pathogens tested. Most of these strains were susceptible to both the test drugs, but resistance to ciprofloxacin was generally more frequent than to levofloxacin.

Levofloxacin and ciprofloxacin significantly affected the adherence properties of E. coli and S. aureus grown in the presence of subinhibitory concentrations of the two drugs. This effect has been observed for older and newer quinolones (oxolinic acid, ciprofloxacin, pefloxacin, enoxacin, lomefloxacin and rufloxacin) by several authors, who reported significant decreases in the adhesion of E. coli and S. aureus.^{15,20,21,23,24} Mechanisms involved include inhibition of synthesis or expression of adhesins, synthesis or release of cellular surface components, alterations in adhesion structures, modification of bacterial shape, all leading to the inability of bacteria to interact with host receptors.^16,25 Pefloxacin and enoxacin limit bacterial adhesion by inhibiting synthesis or expression of adhesins,^23,26 ciprofloxacin causes cell elongation in encapsulated K. pneumoniae strains,²⁷ ofloxacin interferes heavily with expression of E. coli P fimbriae.²⁸ Adhesion of S. aureus is mediated by specific adhesins,^29,30 while in E. coli other mechanisms, including the production of fimbriae, are involved. Since, to the best of our knowledge, the mechanisms by which levofloxacin affects bacterial adhesion are not fully elucidated, it may be hypothesized that subinhibitory concentrations of this drug might interfere with some of these structures, thus reducing bacterial adhesion.

In conclusion, levofloxacin activity against a wide range of Gram-positive and Gram-negative bacteria was confirmed. However, further studies on the effects of low concentrations of levofloxacin on virulence factors are needed to clarify the role played by subinhibitory concentrations of levofloxacin in modifying interactions between host and pathogen.

Table I.

MIC and MBC values (mg/L) of strains used for time–kill studies

	LVX		CIP
Microorganism	MIC	MBC	MIC	MBC
LVX, levofloxacin; CIP, ciprofloxacin.
E. coli 2068	0.03	0.06	0.004	0.004
E. coli 2648	0.016	0.06	0.008	0.008
P. mirabilis 2912	0.06	0.06	0.016	0.016
S. aureus 211	0.125	0.5	0.5	1
S. aureus 1492	0.125	0.25	0.5	0.5

Table II.

In vitro antibacterial activity of levofloxacin and ciprofloxacin against urinary pathogens

		Number of strains having MIC (mg/L) of
Microorganism	Drug	≥8	4	2	1	0.5	0.25	0.125	0.062	0.031	0.016	0.008	0.004
LVX, levofloxacin; CIP, ciprofloxacin.
aMIC90 value.
bMIC50 value.
E. coli (n = 27)	LVX			1		2			2a	6	8b	5	3
	CIP		1				1		1	12a,b	4	3	5
K. pneumoniae (n = 24)	LVX	1			2a		4	4	8b	5
	CIP	1			1	1a	3	3	2	4b	5	4
P. mirabilis (n = 25)	LVX				4a	3	3	2	13b
	CIP			4a	1	3	3	1	1	5b	7	1
P. vulgaris (n = 16)	LVX						1	1	7a,b	4	2	1
	CIP									2	9a,b	4	1
P. rettgeri (n = 10)	LVX		1	2a	4b	1		1	1
	CIP				1	4a	3b	1		1
P. aeruginosa (n = 22)	LVX	1	1	1a	3	7b	6	1		2
	CIP	2				4a	2	2	8b	2	2
E. faecalis (n = 26)	LVX	3a		3	20b
	CIP	3a		4	5	14b
MSSA (n = 20)	LVX		4a			2	4	10b
	CIP	4a				8b	2	2	4
MRSA (n = 10)	LVX	2a		2	2b			4
	CIP	2a			4b	2	1	1
S. epidermidis (n = 20)	LVX	2a				4	4	10b
	CIP	2a				5	9b	4

Table III.

In vitro antibacterial activity of levofloxacin and ciprofloxacin against urinary pathogens

		Number of strains having MBC (mg/L) of
Microorganism	Drug	≥8	4	2	1	0.5	0.25	0.125	0.062	0.031	0.016	0.008	0.004
LVX, levofloxacin; CIP, ciprofloxacin.
aMIC90 value.
bMIC50 value.
E. coli (n = 27)	LVX		1			2			4a	5	8b	5	2
	CIP		1				1		2a	13b	3	2	5
K. pneumoniae (n = 24)	LVX	1			2a		4	4	8b	5
	CIP	1			1	1a	3	3	2	4b	5	4
P. mirabilis (n = 25)	LVX		1	3a	2	1	4	3b	11
	CIP		4a	1	2	2	2	1	2b	4	7
P. vulgaris (n = 16)	LVX						1	2a	6b	4	2	1
	CIP								1	8a,b	4	2	1
P. rettgeri (n = 10)	LVX		1	2a	4b	1		1	1
	CIP				1	5a,b	2	1		1
P. aeruginosa (n = 22)	LVX	1	1	2a	8b	3	4	1		2
	CIP	2				4a	4	4b	5	2	1
E. faecalis (n = 26)	LVX	3a	3	8b	12b
	CIP	3a	3	10b	4	6
MSSA (n = 20)	LVX	2	2a			1	10b	5
	CIP	4a			3	7b	2	2	2
MRSA (n = 10)	LVX	2a		2	2b		1	3
	CIP	2a		2	2b	2	1	1
S. epidermidis (n = 20)	LVX	2a				4	5b	9
	CIP	2a				9b	6	3

Table IV.

Effect of sub-inhibitory concentrations of levofloxacin and ciprofloxacin on bacterial adherence to uroepithelial cells

		0.25 × MIC (bacteria/cell)		IA (%)		0.125 × MIC (bacteria/cell)		IA (%)
	Control (bacteria/cell)	LVX	CIP	LVX	CIP	LVX	CIP	LVX	CIP
LVX, levofloxacin; CIP, ciprofloxacin.
IA (%), inhibition of adherence (see text).
Data are expressed as mean ± s.d. of three experiments.
aP < 0.05 versus control.
S. aureus	81 ± 12	51 ± 4.6a	47 ± 16a	37 ± 11	43 ± 11	60 ± 26.6	73 ± 16	27 ± 21	10 ± 6.1
E. coli	76 ± 11	43 ± 17a	46 ± 10a	43 ± 20	38 ± 15	57 ± 7.2	61 ± 12	24 ± 8.7	19 ± 14

Figure 1.

Time–kill curves of levofloxacin and ciprofloxacin against E. coli. (a) levofloxacin versus ESBL-negative E. coli; (b) levofloxacin versus ESBL-positive E. coli; (c) ciprofloxacin versus ESBL-negative E. coli; (d) ciprofloxacin versus ESBL-positive E. coli. ♦, 4 × MIC; ▪, 2 × MIC; ▴, 1 × MIC; ×, 0.5 × MIC; •, control.

Figure 2.

Time–kill curves of levofloxacin and ciprofloxacin against S. aureus. (a) levofloxacin versus MSSA; (b) levofloxacin versus MRSA; (c) ciprofloxacin versus MSSA; (d) ciprofloxacin versus MRSA. ♦, 4 × MIC; ▪, 2 × MIC; ▴, 1 × MIC; ×, 0.5 × MIC; •, control.

Figure 3.

Time–kill curves of levofloxacin (a) and ciprofloxacin (b) against P. mirabilis. ♦, 4 × MIC; ▪, 2 × MIC; ▴, 1 × MIC; ×, 0.5 × MIC; •, control.

Figure 4.

SEMs of urinary cells incubated with bacteria. (a) Control: E. coli grown with no drug; (b) E. coli incubated with 0.25 × MIC of levofloxacin; (c) E. coli incubated with 0.125 × MIC of levofloxacin.

*

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This work was partially supported by Aventis Pharma, Lainate, Italy.