Abstract

Levofloxacin was investigated against viridans group streptococci in vitro and in rats with experimental aortic endocarditis. The MIC90s of levofloxacin and ciprofloxacin for 20 independent isolates of such bacteria were 1 and 8 mg/L, respectively. Rats were infected with two types of organism: either fully susceptible to levofloxacin MIC ≤ 0.5 mg/L) or borderline susceptible (MIC 1–2 mg/L). Fully levofloxacin-susceptible bacteria comprised one penicillin-susceptible (MIC 0.004 mg/L) Streptococcus gordonii, and one penicillin-tolerant as well as one intermediate penicillin-resistant (MIC 0.125 mg/L) isogenic strains. Borderline levofloxacin-susceptible bacteria comprised one penicillin-susceptible Streptococcus sanguis and one highly penicillin-resistant Streptococcus mitis (MIC 2 mg/L). Rats were treated for 5 days with drug dosages simulating the following treatments in humans: (i) levofloxacin 500 mg orally once a day (q24 h), (ii) levofloxacin 500 mg orally twice a day (q12 h), (iii) levofloxacin 1 g orally q24 h, (iv) ciprofloxacin 750 mg orally q12 h, and (v) ceftriaxone 2 g iv q24 h. Levofloxacin was equivalent or superior to ceftriaxone, and was successful in treating experimental endocarditis irrespective of penicillin resistance. Nevertheless, standard levofloxacin treatment equivalent to 500 mg q24 h in human was less effective than twice daily 500 mg or once daily 1 g doses against borderline-susceptible organisms. Ciprofloxacin, used as a negative control, was ineffective and selected for resistant isolates. This underlines the importance of MIC determinations when treating severe streptococcal infection with quinolones. In the case of borderline-susceptible pathogens, total daily doses of 1 g of levofloxacin should be considered.

Introduction

Levofloxacin is a new quinolone with improved efficacy against Gram-positive pathogens, including staphylococci and streptococci.1,2,3,4 Both species are potentially important targets for such a new compound because they are responsible for severe infections in the hospital and in the community. In the hospital, coagulase-negative staphylococci and Staphylococcus aureus are the leading cause of nosocomial bacteraemia5,6 and viridans group streptococci have become a major cause of severe sepsis in neutropenic cancer patients.7,8 In the community, S. aureus and viridans group streptococci are the major cause of infective endocarditis, and account together for c. 90% of all cases.9 This is in addition to many other community-acquired infections caused by staphylococci and streptococci, which may be less dramatic than endocarditis or severe sepsis.

Classical treatment of such severe infections requires prolonged administration ofβ -lactams or vancomycin given by parental routes.10,11,12 In contrast, newer quinolones are well absorbed by the gastrointestinal tract, and have a bioavailability of≥ 90% after oral administration.13 If such compounds proved to be effective against severe staphylococcal and streptococcal infections, they could greatly simplify the treatment of these diseases, by decreasing the technical needs of parenteral therapy and their complications, and by lowering the global cost of patient care.

Recently, experiments in rats demonstrated that simulation of pharmacokinetics produced by oral administration of levofloxacin 350 mg/day to humans was equivalent or more effective than parenteral flucloxacillin or vancomycin against experimental S. aureus endocarditis.14 These experiments also indicated that levofloxacin, like other newer quinolones, was less prone than the earlier drug, ciprofloxacin, to select for quinolone-resistant staphylococci both in vitro and in vivo.14,15 This is important, because acquisition of quinolone resistance by Gram-positive pathogens has been an ongoing problem ever since these molecules have been used in human medicine.16 These good therapeutic results against experimental S. aureus endocarditis raised the question as to whether this drug might also be active against severe viridans group streptococcal infections. This specific issue needs to be addressed, because precedents with sparfloxacin and trovafloxacin indicated that these new compounds were less effective than standard β-lactam therapy against experimental streptococcal endocarditis, in spite of the fact that they had very low MICs for the infecting bacteria.15,17

The present work attempts to provide an answer to this question. Specifically, the efficacy of the equivalent of human dosages of oral levofloxacin of 500 mg/day and 1 g/day was investigated in rats with experimental viridans group streptococcal endocarditis. To make the study representative, it was important to use test organisms that expressed the most frequent antibiotic-susceptibility phenotypes encountered in the clinical environment. Therefore, two types of streptococci were studied. The first type was fully susceptible to levofloxacin (MIC ≤ 0.5 mg/L), and comprised a penicillin-susceptible Streptococcus gordonii, and one penicillin-tolerant as well as one intermediate penicillin-resistant strain of this organism. The second type was borderline susceptible to levofloxacin (MIC 1–2 mg/L), and comprised one penicillin-susceptible clinical isolate of Streptococcus sanguis, and one fully penicillin-resistant (MIC 2 mg/L) clinical isolate of Streptococcus mitis. The therapeutic efficacy of levofloxacin was compared with that of ceftriaxone. Ciprofloxacin was used as a negative treatment control in certain experiments, in order to compare levofloxacin with an earlier quinolone with less activity against Gram-positive pathogens.

Materials and methods

Bacteria and growth conditions

Five isolates of viridans group streptococci displaying either full levofloxacin susceptibility (MIC ≤ 0.5 mg/L), or borderline levofloxacin susceptibility (MIC 1–2 mg/L), were used for the experiments with animals (Table I). Bacteria fully susceptible to levofloxacin comprised a previously described penicillin-susceptible S. gordonii Challis, as well as one penicillin-tolerant (Tol+) and one intermediate penicillin-resistant (IPR) strain of this organism.18,19 Bacteria with borderline susceptibility to levofloxacin comprised one penicillin-susceptible clinical isolate of S. sanguis and one fully penicillin-resistant (MIC 2 mg/L) isolate of S. mitis. Both organisms were also described in previous experiments with animals.17,19 In addition, a panel of 17 clinical isolates of viridans group streptococci recovered from patients with bacteraemia were tested for their susceptibilities to levofloxacin and ciprofloxacin. Unless otherwise stated, all bacteria were grown at 35°C, either in brain heart infusion (BHI; Difco Laboratories, Detroit, MI, USA) without aeration, or on Columbia agar plates (Becton Dickinson Microbiology Systems, Cockeysville, MD, USA) supplemented with 3% blood. Bacterial stocks were kept at –70°C in BHI supplemented with 10% (v/v) glycerol.

Antibiotics

Levofloxacin was provided by Roussel Uclaf (Romainville, France), ciprofloxacin was purchased from Bayer AG (Wuppertal, Germany) and ceftriaxone was purchased from Roche Pharma (Reinach, Switzerland).

Antibiotic susceptibility testing and bactericidal activity

The MICs were determined by a standard broth macrodilution method,20 with a final inoculum of 105–106 cfu/mL. The MIC was defined as the lowest antibiotic concentration that inhibited visible bacterial growth after 24 h of incubation at 35°C. The MBCs of the drugs were measured by subculturing 0.01 mL samples from the MIC tubes showing no turbidity on to agar plates, as described previously.19 MBC was determined after incubation of the plates for 48 h at 35°C and defined as the lowest antibiotic concentration resulting in ≥99.9% killing of the initial inoculum.

The bactericidal kinetics of the drugs were determined in broth cultures as follows. Series of flasks containing fresh prewarmed medium were inoculated with c. 106 cfu/mL (final concentration) from an overnight culture of bacteria and the flasks were further incubated at 35°C. Immediately after inoculation, the antibiotics were added to the flasks to give final concentrations approximating to the peak drug levels produced in human serum after administration of therapeutic doses of the compounds (see Results). These concentrations were: (i) 8 and 10 mg/L for levofloxacin given at 500 mg or 1 g per day, respectively, (ii) 4 mg/L for ciprofloxacin and (iii) 200 mg/L for ceftriaxone. In certain experiments, bacterial kinetic studies were performed with relatively low levofloxacin concentrations, equivalent to −3, 43 and 8 3 MIC of the drug for the organisms. Viable counts were determined just before and at various times after the addition of the antibiotics by plating adequate dilutions of the cultures on to agar plates. Antibiotic carry-over was minimized by taking the following precautions. (i) For levofloxacin and ciprofloxacin, 0.5 mL samples of the cultures were transferred from the flasks into microcentrifuge tubes, and the bacteria were spun and resuspended twice in antibiotic-free medium to remove residual drug before plating. (ii) For ceftriaxone, the samples were plated on to blood agar supplemented with 0.2% (v/v) of Bacillus cereus 569/H9 β-lactamase (penicillin-aminoβ-lactam hydrolase; Genzyme Diagnostics, Kent, UK).

Production of endocarditis and infusion pump installation

The production of catheter-induced aortic vegetations in the rats and the installation of the programmable infusion pump (Pump 44; Harvard Apparatus, Inc., South Natick, MA, USA) to deliver the antibiotics were performed under general anaesthesia as previously described.21,22 Infectious endocarditis was induced 24 h after catheterization by iv challenge of the animals with 0.5 mL of saline containing 107 cfu of either of the test organisms. This inoculum was 100 times greater than the minimum inoculum producing endocarditis in 90% of the untreated rats. No animal was left untreated for more than 24 h. Fewer than 10% of the rats died as a result of the procedure, either as a consequence of the anaesthesia or from catheter-induced cardiac arrhythmia. In therapeutic experiments rats were checked twice a day by one of the investigators and killed whenever signs of suffering were observed. Animals were killed by CO2 inhalation.

Antibiotic treatment of experimental endocarditis

Therapy was started 24 h after bacterial inoculation, and lasted for 5 days. The antibiotics were administered at changing flow rates with the pump described above, in order to simulate the drug kinetics in human serum during treatment with either (i) 500 mg of levofloxacin orally q24 h,23 (ii) 500 mg of levofloxacin orally q12 h,24 (iii) 1 g of levofloxacin orally q24 h,25 (iv) 750 mg of ciprofloxacin orally q12 h,26 or 2 g of ceftriaxone iv q24 h.27 This required total amounts of antibiotics of levofloxacin, 92 and 195 mg/kg q24 h for the 500 mg and 1 g daily regimens, respectively; ciprofloxacin, 40.8 mg/kg q12 h, and 537 mg/kg of ceftriaxone q24 h.

Pharmacokinetic studies

Concentrations of antibiotic in serum were determined on day 2 of therapy in groups of six to nine uninfected or infected rats. The concentrations of drug in the infected animals were determined in internal controls of therapeutic experiments, in which adequate drug delivery was tested routinely. Blood was drawn by puncturing the periorbital sinuses of the animals (one puncture per animal) at several times during and after antibiotic administration. The concentration of levofloxacin in cardiac vegetations was also determined on day 2 of therapy, in animals receiving levofloxacin 500 mg q12 h. Rats were killed 1 h after initiating a new dose, at the time of peak levofloxacin level in the serum. Drug concentrations were measured in the blood and in the vegetations by a bioassay as described previously.15 The limits of detection of the assays were 0.15 mg/L for levofloxacin, 0.12 mg/L for ciprofloxacin and 3 mg/L for ceftriaxone. The 24 h area under the serum concentration–time curve (AUC0-24h) was calculated by the trapezoidal summation method only for levofloxacin.

Evaluation of infection

Control rats were killed at the time of treatment onset, i.e. 24 h after inoculation, in order to measure both the frequency and the severity of valve infection at the start of therapy. Treated rats were killed 8–12 h after the trough level in serum of the last dose of antibiotic was achieved. At that time, no residual antibiotic was detected in the serum, except in animals treated with a total dose of levofloxacin 1 g/day. In these animals, the residual serum concentration of levofloxacin was 0.3 ± 0.1 mg/L (mean ± s.d. of 12 individual determinations). However, these low residual drug concentrations were not likely to interfere with the valve cultures. First, the residual concentrations of levofloxacin in the serum were already lower than the MICs of the drug for the test organisms. Second, processing of the organs before plating resulted in a ≥100× dilution of the original sample.

The valve vegetations were dissected under sterile conditions, weighed, homogenized in 1 mL saline, and diluted serially before being plated to determine the number of cfu. Quantitative blood and spleen cultures were performed in parallel. Few animals died before the end of treatment from either complications of the operation itself (such as possible catheter-induced arrhythmia) or the infection process, or both. Among these, only rats that had received at least two-thirds of the treatment were taken into account for vegetation bacterial counts. Blood and spleen cultures were not performed for these animals. The number of colonies growing on the plates was determined after 48 h of incubation at 35°C in a CO2 atmosphere. Bacterial densities in the vegetations were expressed as log10 cfu/g of tissue. The dilution technique permitted the detection of ≥ 2 log10 cfu/g of vegetation. For statistical comparisons of differences between the median densities of bacterial vegetations in the various treatment groups, culture-negative vegetations were considered to contain 2 log10 cfu/g.

Detection of the emergence of antibiotic resistance in vivo

To detect the emergence of quinolone-resistant streptococci during endocarditis therapy, 0.1 mL portions of each undiluted homogenate of vegetations from animals receiving either levofloxacin or ciprofloxacin treatment were plated in parallel on plain agar and on agar plates supplemented with the test drug at either 2× or 4× MIC. In addition, standard MICs were also determined for bacteria that grew from vegetations on antibiotic-free agar. In this case, one in c. 100 colonies growing from the infected vegetations were picked at random from the plates, grown in an independent liquid culture and retested for susceptibility to the drug.

Selection of quinolone-resistant variants in vitro

Additional experiments were performed to test the propensity of ciprofloxacin and levofloxacin to select for quinolone-resistant organisms in vitro. First, large inocula (109–1010 cfu) were spread on to agar plates containing either no antibiotic or the antibiotic at 2–16 × MIC of the drug for the test organism. The plates were incubated for 48 h at 35°C before colony count. The colonies growing on antibiotic-containing plates were then retested for their new, increased MIC.

Second, selection for quinolone resistance was also performed in broth cultures by exposing bacteria to stepwise increasing concentrations of antibiotic.14 Series of tubes containing two-fold increasing concentrations of drugs were inoculated with 106 cfu/mL (final concentration) as for MIC determinations. After 24 h of incubation, 0.1 mL samples from the tubes containing the highest antibiotic concentration and still showing turbidity were used to inoculate a new series of tubes containing antibiotic dilutions. The experiment was repeated several times and the increase in MICs was followed. The stability of resistance was assessed by serial passage of the resistant derivatives in antibiotic-free BHI broth.

Statistical analysis

The incidences of valve infection were compared by the Fisher's exact test. The median bacterial densities in the vegetations of various treatment groups were compared by the non-parametric Kruskal–Wallis one-way analysis of variance on ranks, with subsequent pairwise multiple comparison procedures by Dunn's method. Overall, differences were considered significant when P ≤ 0.05 by use of two-tailed significance levels. Ciprofloxacin-treated animals, which were used as negative controls, were not considered in the statistical evaluation.

Results

Antibiotic susceptibility

Table I presents the MICs and MBCs of penicillin, ceftriaxone, ciprofloxacin and levofloxacin, for the five streptococci used in animals. For all these isolates the MIC of levofloxacin was two to four times lower than that of ciprofloxacin. There was no correlation between susceptibility or resistance to quinolones and to β-lactams. However, there was a degree of cross-resistance within the two antibiotic classes. The two ciprofloxacin-resistant isolates (S. mitis 531 and S. sanguis‘ Du’, ciprofloxacin MIC = 2 and 8 mg/L, respectively) had a parallel increase in their levofloxacin MIC (1 and 2 mg/L, respectively). Likewise, the intermediate penicillin-resistant and fully penicillin-resistant isolates (S. gordonii IPR and S. mitis 531, respectively) also had a parallel increase in their ceftriaxone MIC (Table I).

MBCs indicated that all the organisms were susceptible to antibiotic-induced killing, except for penicillin-tolerant S. gordonii (Tol+). This bacterium had an MBC/MIC ratio ≥ 32 for both penicillin and ceftriaxone, thus complying with the definition of tolerance.19 Note, however, that S. gordonii Tol+ was not cross-tolerant to the quinolones.

An additional panel of 17 clinical isolates of viridans group streptococci was also tested for quinolone susceptibility. The MIC at which 90% of these bacteria were inhibited (MIC90) by levofloxacin was 1 mg/L (range 0.5–2 mg/L), compared with 4 mg/L (range 1–8 mg/L) of ciprofloxacin. All of these organisms fell within the levofloxacin-susceptible range as defined by Sutton and Jones,28 whereas almost one-third of them (five out of 17) were already beyond the therapeutic range of ciprofloxacin. This supported the fact that ciprofloxacin is not a first choice treatment against streptococcal infection. In some of the subsequent experiments, ciprofloxacin was used as a negative control, to compare this ‘older' quinolone with relatively poor activity against Gram-positive pathogens with the newer levofloxacin.

Bactericidal kinetics of the drugs

A typical bactericidal kinetic experiment is presented in Figure 1. Cultures of S. mitis 531 were exposed to drug concentrations simulating peak drug levels in the human serum produced by administration of either oral levofloxacin 500 mg (peak concentration at 1 h c. 7 mg/L), oral ciprofloxacin 750 mg (peak concentration at 1 h c. 4 mg/L), or iv ceftriaxone 2 g (peak concentration at 0.5 h c. 200 mg/L). Levofloxacin produced a decrease of 3–4 log cfu/mL in the cultures after 24 h of treatment. This bactericidal activity was observed against all the streptococcal isolates used in vivo (Table I), irrespective of their individual antibiotic susceptibilities. Moreover, this bactericidal activity was not concentration dependent. Figure 1 indicates that even low multiples of levofloxacin MIC (e.g. 2× and 4 × MIC) were highly bactericidal against S. mitis 531. The same observation was made against the other isolates presented in Table I (data not shown). In contrast, ciprofloxacin used at peak concentrations in the serum (4 mg/L) never produced a reduction of 3 log cfu/mL in viable counts after 24 h of drug treatment, whichever test organism was used (Figure 1).

With regard to ceftriaxone, peak concentrations in the serum following standard therapy (200 mg/L)27 were highly bactericidal (>5 log cfu/mL loss of viability) against the penicillin-susceptible isolate S. sanguis ‘Du’ (data not presented). On the other hand, ceftriaxone was not bactericidal against the other four organisms tested in animals (loss of <3 log cfu/mL in viability after 24 h of drug treatment), including S. mitis 531 presented in Figure 1.

Concentration of antibiotics in the serum of rats and in cardiac vegetations

The antibiotic concentrations in the serum of rats were measured on day 2 of therapy (Table II). Depending on the antibiotic regimen, drug levels were determined 0.5, 1 and 1.5 h (peak concentration) and 12 or 24 h (trough concentration) after a new drug dose was started. The values presented in Table II were close to values reported in healthy volunteers.2324252627 The kinetics of the three levofloxacin regimens are also presented graphically in Figures 2 and 3.

In certain experiments, the intravegetation penetration of levofloxacin was also determined (see Materials and methods). Intravegetation penetration was expressed as the percentage of the drug concentration measured in the vegetation compared with the concentration measured in the serum (at peak concentration) of six individual rats. Intravegetation penetration of levofloxacin was 90.5% (range 75–113%). For comparison, retrospective controls using the same techniques with other quinolones gave intravegetation penetration values that were: (i) 59% (range 54–89%) for ciprofloxacin, (ii) 92.5% (range 62–123%) for sparfloxacin and (iii) 22.5% (range 13–37%) for trovafloxacin.15 Thus, levofloxacin penetrated well into cardiac lesions and compared advantageously with other quinolones in this respect.

Treatment of experimental endocarditis with levofloxacin kinetics simulating oral administration of 500 mg q24 h to humans

Figure 2a depicts the simulation, in rats, of the serum kinetics produced by oral administration of 500 mg of levofloxacin q24 h to humans. Figure 2b presents the therapeutic results in rats infected with either fully levofloxacin-susceptible streptococci (three isolates) or borderline-susceptible organisms (one isolate) expressing various penicillin-resistance phenotypes. The figure indicates that this regimen was very active against the three levofloxacin-susceptible (MIC = 0.5 mg/L) isolates, irrespective of their penicillin-tolerance or penicillin-resistance phenotypes. In all three cases, levofloxacin was equivalent to standard treatment with ceftriaxone. In contrast, ciprofloxacin failed in all of these instances, in spite of an MIC of 1 mg/L for these bacteria. Moreover, in 13 of 24 (54%) of these treatment failures, vegetation cultures grew ciprofloxacin-resistant mutants of streptococci for which the MIC of ciprafloxacin was increased two- to 32-fold. No such resistant variants were detected in the levofloxacin treatment failures.

In contrast, neither levofloxacin nor ceftriaxone was very effective against the borderline levofloxacin-susceptible (MIC 1 mg/L) and highly penicillin-resistant isolate S. mitis 531. Although both treatments significantly reduced the vegetation bacterial densities compared with untreated controls (P < 0.05), their biological effect was rather marginal. Only very few animals had undetectable bacterial counts in the valve cultures (Figure 2). Nevertheless, none of the treatment failures contained resistant derivatives in their vegetations.

Among the control rats killed at the start of therapy, 36 of 36 (100%) had positive spleen cultures, and 31 of 36 (86%) had positive blood cultures. At the end of therapy, 21 of 21 (100%) available rats in the ciprofloxacin group had positive spleen cultures, and 17 of them (87%) also had positive blood cultures. These values were similar to those of untreated animals. In the levofloxacin-treated groups, only 11 of 34 (32%) rats had positive spleen cultures, seven of them being recovered from animals infected with the borderline-susceptible isolate S. mitis 531. Similarly, only five of 34 (14%) levofloxacin-treated rats had positive blood cultures, four of them from animals infected with S. mitis 531. The positive spleen and blood cultures in the ceftriaxone-treated groups were three of 31 (9%) and two of 31 (6%), respectively.

Treatment of experimental endocarditis with levofloxacin kinetics simulating oral administration of 500 mg of the drug q12 h, or 1 g q24 h to humans

Since 500 mg of levofloxacin q24 h failed against the borderline-susceptible isolate S. mitis 531, this raised the question as to whether merely increasing the drug dosage might restore the therapeutic efficacy. Indeed, while 500 mg of levofloxacin q24 h is a standard dosage recommended in adults,29,30 it was recently shown that 1 g daily levofloxacin was well tolerated and could be an acceptable alternative against less susceptible bacteria.25 To test this hypothesis, additional experiments were run using kinetics simulating a total daily dose of levofloxacin of 1 g, given either as 2 × 500 mg (500 mg q12 h), or as 1 × 1 g of the drug q24 h.

Figure 3a depicts these kinetics simulated in rats. As expected, the two profiles looked different. Nevertheless, they generated very similar pharmacodynamics. Their AUC0–24 values were 112.4 mg.h/L and 116.6 mg.h/L for the q12 h and the q24 h regimens, respectively. The therapeutic results of these two regimens are presented in Figure 3b. In contrast to a single daily administration of 500 mg of levofloxacin, 2 × 500 mg of the drug was effective against S. mitis 531. This also contrasted with ceftriaxone, which was still poorly effective against this isolate (P < 0.05 when compared with levofloxacin-treated rats). Moreover, both 1 g daily regimens were also effective against an additional Streptococcus isolate, S. sanguis‘ Du’, with a borderline levofloxacin MIC (2 mg/L).

In the levofloxacin-treated groups, only two of 26 (7%) available rats had positive spleen cultures and none had positive blood cultures. Again, no levofloxacin-resistant streptococcal mutants were selected among the treatment failures. In the ceftriaxone-treated group, one of four (25%) of the available rats had positive spleen and blood cultures.

Selection of quinolone resistance by ciprofloxacin and levofloxacin in vitro

To evaluate more accurately the risk of selection for quinolone resistance in viridans group streptococci, the five isolates tested in animals were submitted to two kinds of test. In this study, large inocula (c. 7 × 109 cfu) of the test organisms were spread on to agar plates containing increasing concentrations of antibiotics. Drug concentrations were determined in multiples of MIC to ensure that the antibiotic pressure applied to the organisms was comparable between the two drugs. Streptococcal colonies grew readily on ciprofloxacin plates containing 2× and 4 × MIC of this drug at a frequency varying between 4× 10–7 and 7.5 × 10–8 of the original inoculum. Upon retesting, all these variants had acquired a high level of resistance to ciprofloxacin (MIC > 32 mg/L), but had only minimal alterations in their MICs of levofloxacin: i.e. no changes were observed for S. gordoniiand S. mitis531, whereas an increase in one dilution was observed in the MIC of levofloxacin for S. sanguis‘ Du’.

When bacteria were spread on to levofloxacin-containing agar, levofloxacin-resistant variants grew only on plates containing 2 3 MIC of the drug, at a frequency similar to that observed on ciprofloxacin-containing plates. However, no bacteria grew on plates containing higher drug concentrations. Upon retesting, these variants showed a two- to four-fold increase in their MICs of levofloxacin, but were highly resistant to ciprofloxacin (MIC > 32 mg/L). Resistance to ciprofloxacin and levofloxacin was stable for up to five passages on antibiotic-free plates, as tested in 24 individual colonies randomly isolated from the plates.

In a second study, the five test isolates were exposed to two-fold stepwise increasing concentrations of either ciprofloxacin or levofloxacin, as described in Materials and methods. Figure 4 indicates that stepwise exposure to ciprofloxacin resulted in the rapid emergence of high-level resistant derivatives in all the strains tested. In contrast, levofloxacin was notably less prone than ciprofloxacin to select for resistance in these experiments. Importantly, borderline-susceptible isolates were not more likely than fully susceptible bacteria to develop high-level resistance to levofloxacin. Quinolone resistance in these derivatives was stable after daily subculture (for 5 days) in quinolone-free medium.

Thus, these in-vitro observations supported the impression provided by animal experiments that levofloxacin was a much poorer selective agent than ciprofloxacin with regard to the development of quinolone resistance in this restricted panel of viridans group streptococci.

Discussion

The present results indicate that modelled human kinetics of levofloxacin successfully treated experimental endocarditis in rats caused by viridans group streptococci that expressed different antibiotic-resistance phenotypes. The observation is relevant, because viridans group streptococci with decreased susceptibility to commonly used antibiotics exist in the clinical environment. With regard to β-lactams, penicillin-tolerant and penicillin-resistant streptococci are recovered with an increasing frequency in a number of clinical situations.31,32,33 Both penicillin tolerance and penicillin resistance can jeopardize the success of β-lactam treatment in experimental and non-experimental infections,17,19,34,35 and thus represent a genuine threat to the success of β-lactam treatment of severe streptococcal diseases.

Besides β-lactams, a dramatic surge in macrolide resistant pneumococci and group A streptococci was recently reported in France, Italy and Spain.36,37,38 Moreover, these two types of bacteria were shown to have more than one mechanism of resistance against this group of compounds, illustrating their flexibility in escaping the effect of antimicrobial drugs. In addition to the well-known mechanism of ribosome methylation, they now also have an active drug efflux system, mediated by the mefE gene in pneumococci and the mefA gene in group A streptococci.39,40 Another determinant for macrolide efflux, called mreA, was also described in Streptococcus agalactiae.41 There is no reason to think that viridans group streptococci will behave differently.

Because there is no cross-resistance between these antibiotic classes and the quinolone family, newer quinolones with improved Gram-positive activity are a potential alternative against such resistant bacteria. However, quinolones have their limitations as well. First, they have a relatively low therapeutic margin, as peak concentrations in the serum rarely reach values ≥ 10 × MIC of the target organisms.4,13 Second, prolonged exposure to low MIC multiples of quinolones tends to select for bacterial mutants with decreased drug susceptibility, thus bringing them closer to the drug's susceptibility breakpoint. Third, this borderline susceptibility further decreases the antibiotic's therapeutic margin, and thus may allow the bacterium to resist quinolone therapy in vivo.

The difficulty of treating experimental infections caused by borderline-susceptible bacteria with quinolones was observed with S. aureus. Increase in the antibiotic dosage was necessary for the effective treatment of experimental endocarditis caused by bacteria with borderline susceptibility to sparfloxacin42 and trovafloxacin (our unpublished observation). The present results confirm these observations with viridans group streptococci. While a treatment equivalent to 500 mg of oral levofloxacin once a day treated experimental infection with fully susceptible streptococci (MIC of levofloxacin ≤ 0.5 mg/L) successfully, it was less effective against borderline-susceptible bacteria (MIC of levofloxacin = 1 mg/L). Success or failure of levofloxacin therapy was independent of the susceptibility toβ-lactams. It relied purely on the MIC of levofloxacin for the test bacterium, and on the amount of drug that could be delivered to the animals. Indeed, doubling the total dose of levofloxacin, from 500 mg to 1 g per day, restored full efficacy. In the case of levofloxacin, this larger dose is still acceptable in humans.25 However, such flexibility in drug dosing might not be possible with other quinolones that are more prone to result in dose-related toxicity.43

While the relationship between the pharmacodynamics and treatment outcome of quinolones has been well described, 44,45,46 the present results are one more warning for the appropriate use of these newer molecules against Gram-positive pathogens. Using appropriate doses will be essential not only to ensure therapeutic success, but also to lower the risk of emergence of resistance to this class of drugs. Indeed, although less prone than ciprofloxacin to select for resistance, levofloxacin was not devoid of selective power in the present experiments. In severe infections, it will be essential to test the precise susceptibility of the infective organism, since the therapeutic margin of the drug is very narrow at MICs > 1 mg/L of levofloxacin.

Finally, as a consequence of their limited therapeutic margin, it will be essential to follow the possible increase in the MICs of quinolones for Streptococcus spp., as well as for other Gram-positive pathogens in the near future. This is particularly important when considering that these types of drugs are now being launched for the treatment of community-acquired respiratory tract infections. A direct corollary of this epidemiological study will also be to set up clear susceptibility breakpoints to help determine the most appropriate therapeutic regimens. The present results and previous studies provide clear evidence of the need for adequate drug dosage and resistance control. The goal is not to ban the use of newer quinolones in the community, but rather to avoid their inappropriate use. Indeed, uncontrolled use of these compounds is bound to select for quinolone resistance in the clinical environment, and the eventual loss of these very valuable antibiotics.

In conclusion, the present results highlight the success of levofloxacin against severe streptococcal infection. With regard to streptococci, levofloxacin has several advantages over other newer quinolones: (i) it can achieve relatively high serum levels in humans (up to 10 mg/L, as compared with <5 mg/L for most other quinolones), (ii) it penetrates well into cardiac vegetations and (iii) it is highly bactericidal against streptococci even at low multiples of the MIC. However, even though this represents a genuine advantage over other quinolones, levofloxacin has its own limits against borderline-susceptible streptococci. Levofloxacin dosages must be adjusted to that situation. Therefore, the susceptibility to levofloxacin, and also other newer quinolones, should be determined before treating severe streptococcal infections with these new drugs.

Table I.

MICs and MBCs of various antibiotics for the streptococci used in experimental endocarditis

 MIC/MBC (mg/L) 
Strain (phenotype)a penicillin ceftriaxone ciprofloxacin levofloxacin 
aPS, penicillin susceptible; Tol+, penicillin tolerant; IPR, intermediate penicillin resistant; PR, highly penicillin resistant. 
S. gordonii (PS) 0.004/0.008 0.008/128 1/2 0.5/1 
S. gordonii (Tol+0.004/>128 0.008/>128 1/1 0.5/1 
S. gordoni (IPR) 0.125/2 0.125/>128 1/4 0.5/1 
S. mitis 531 (PR) 2/4 2/4 2/4 1/2 
S. sanguis‘ Du’ (PS) 0.004/0.008 0.06/0.06 8/16 2/4 
 MIC/MBC (mg/L) 
Strain (phenotype)a penicillin ceftriaxone ciprofloxacin levofloxacin 
aPS, penicillin susceptible; Tol+, penicillin tolerant; IPR, intermediate penicillin resistant; PR, highly penicillin resistant. 
S. gordonii (PS) 0.004/0.008 0.008/128 1/2 0.5/1 
S. gordonii (Tol+0.004/>128 0.008/>128 1/1 0.5/1 
S. gordoni (IPR) 0.125/2 0.125/>128 1/4 0.5/1 
S. mitis 531 (PR) 2/4 2/4 2/4 1/2 
S. sanguis‘ Du’ (PS) 0.004/0.008 0.06/0.06 8/16 2/4 

Table II.

Pharmacokinetic parameters of levofloxacin, ciprofloxacin and ceftriaxone in the treatment of experimental streptococcal endocarditis

Drug (regimen simulated in rats) Peak concentration Trough concentration  AUC0–24 
 (mg/L) (time) (mg/L) (time) (mg.h/L) 
The concentration of antibiotics in the serum of rats is presented as mean ± s.d. of six to nine determinations in individual animals pooled from more than one experiment. 
ND, not determined. 
Levofloxacin (500 mg q24 h) 6.92 ± 0.39 (1 h) 0.45 ± 0.16 (24 h) 49.8 
Levofloxacin (500 mg q12 h) 9.53 ± 1.86 (1 h) 1.81 ± 0.33 (12 h) 112.4 
Levofloxacin (1 g q24 h) 13.30 ± 1.28 (1.5 h) 1.90 ± 0.67 (24 h) 116.6 
Ciprofloxacin (750 mg q12 h) 3.44 ± 0.53 (1 h) 0.31 ± 0.10 (12 h) ND 
Ceftriaxone (2 g q24 h) 193.50 ± 79.8 (0.5 h) 13.80 ± 8.10 (24 h) ND 
Drug (regimen simulated in rats) Peak concentration Trough concentration  AUC0–24 
 (mg/L) (time) (mg/L) (time) (mg.h/L) 
The concentration of antibiotics in the serum of rats is presented as mean ± s.d. of six to nine determinations in individual animals pooled from more than one experiment. 
ND, not determined. 
Levofloxacin (500 mg q24 h) 6.92 ± 0.39 (1 h) 0.45 ± 0.16 (24 h) 49.8 
Levofloxacin (500 mg q12 h) 9.53 ± 1.86 (1 h) 1.81 ± 0.33 (12 h) 112.4 
Levofloxacin (1 g q24 h) 13.30 ± 1.28 (1.5 h) 1.90 ± 0.67 (24 h) 116.6 
Ciprofloxacin (750 mg q12 h) 3.44 ± 0.53 (1 h) 0.31 ± 0.10 (12 h) ND 
Ceftriaxone (2 g q24 h) 193.50 ± 79.8 (0.5 h) 13.80 ± 8.10 (24 h) ND 

Figure 1.

(Bactericidal kinetics of levofloxacin, ciprofloxacin and ceftriaxone against the borderline levofloxacin-susceptible isolate S. mitis 531 (MIC 1 mg/L). Antibiotics were added to the cultures at final concentrations simulating peak drug levels in human and rat serum. Concentrations were: 8 mg/L for levofloxacin (8 × MIC of the drug for the test bacterium) (⋄), 4mg/L for ciprofloxacin (2 × MIC) (□) and 200 mg/L for ceftriaxone (100 × MIC) (▴). In addition, levofloxacin was also tested at final concentrations of 2 (▴) and 4 mg/L (◯) (2× and 4 ×MIC); (•), control. The results indicate the mean of at least three separate experiments. )

Figure 1.

(Bactericidal kinetics of levofloxacin, ciprofloxacin and ceftriaxone against the borderline levofloxacin-susceptible isolate S. mitis 531 (MIC 1 mg/L). Antibiotics were added to the cultures at final concentrations simulating peak drug levels in human and rat serum. Concentrations were: 8 mg/L for levofloxacin (8 × MIC of the drug for the test bacterium) (⋄), 4mg/L for ciprofloxacin (2 × MIC) (□) and 200 mg/L for ceftriaxone (100 × MIC) (▴). In addition, levofloxacin was also tested at final concentrations of 2 (▴) and 4 mg/L (◯) (2× and 4 ×MIC); (•), control. The results indicate the mean of at least three separate experiments. )

Figure 2.

(Therapeutic results of human-like kinetics of levofloxacin 500 mg q24 h against experimental endocarditis caused by viridans group streptococci with various antibiotic-resistance phenotypes. (a) The simulation, in the serum of rats, of the kinetics of levofloxacin following oral administration of 500 mg of the drug to humans. Mean and standard deviations of peak and trough drug concentrations in the serum are specified in the text. (b) The therapeutic results of 5 days' treatment with this regimen in rats with experimental endocarditis. The MICs of levofloxacin for these test organisms are indicated in the figure. PS stands for penicillin susceptible, Tol+ for penicillin tolerant, IPR for intermediate penicillin resistant (MIC 0.125 mg/L) and PR for penicillin resistant (MIC 2 mg/L). Each dot in the figure represents the bacterial density in the vegetations of a single animal. The open dot in the ciprofloxacin treatment groups represents animals in which bacteria isolated from the valve had a decreased sensitivity to the drug. The median bacterial densities recovered from vegetations (expressed as log cfu/g of tissue) are indicated at the bottom of the column for each group. P < 0.05 when the results were compared by both the Fisher's exact test and the Kruskall–Wallis test with Dunn's correction for pairwise multiple comparisons. Ciprofloxacin-treated animals were used as negative controls and were not considered in this statistical evaluation. Abbreviations: Contr, controls; LVFX, levofloxacin; Cipro, ciprofloxacin; CRO, ceftriaxone.)

Figure 2.

(Therapeutic results of human-like kinetics of levofloxacin 500 mg q24 h against experimental endocarditis caused by viridans group streptococci with various antibiotic-resistance phenotypes. (a) The simulation, in the serum of rats, of the kinetics of levofloxacin following oral administration of 500 mg of the drug to humans. Mean and standard deviations of peak and trough drug concentrations in the serum are specified in the text. (b) The therapeutic results of 5 days' treatment with this regimen in rats with experimental endocarditis. The MICs of levofloxacin for these test organisms are indicated in the figure. PS stands for penicillin susceptible, Tol+ for penicillin tolerant, IPR for intermediate penicillin resistant (MIC 0.125 mg/L) and PR for penicillin resistant (MIC 2 mg/L). Each dot in the figure represents the bacterial density in the vegetations of a single animal. The open dot in the ciprofloxacin treatment groups represents animals in which bacteria isolated from the valve had a decreased sensitivity to the drug. The median bacterial densities recovered from vegetations (expressed as log cfu/g of tissue) are indicated at the bottom of the column for each group. P < 0.05 when the results were compared by both the Fisher's exact test and the Kruskall–Wallis test with Dunn's correction for pairwise multiple comparisons. Ciprofloxacin-treated animals were used as negative controls and were not considered in this statistical evaluation. Abbreviations: Contr, controls; LVFX, levofloxacin; Cipro, ciprofloxacin; CRO, ceftriaxone.)

Figure 3.

(Therapeutic results of human-like kinetics of levofloxacin 500 mg q12 h or 1 g q24 h against experimental endocarditis caused by viridans group streptococci with various antibiotic-resistance phenotypes. (a) The simulation, in the serum of rats, of the kinetics of levofloxacin following oral administration of 500 mg q12 h (♦) or 1 g q24 h (□)of the drug to humans. Mean and standard deviations of peak and trough drug concentrations in serum are specified in the text. (b) The therapeutic results of 5 days' treatment with this regimen in rats with experimental endocarditis. The MICs of levofloxacin for these test organisms are indicated in the figure. PS stands for penicillin susceptible and PR for penicillin resistant (MIC 2 mg/L). Each dot in the figure represents the bacterial density in the vegetations of a single animal. The median bacterial densities recovered from vegetations (expressed as log cfu/g of tissue) are indicated at the bottom of the columns for each group. P < 0.05 when the results were compared by both the Fisher's exact test and the Kruskall–Wallis test with Dunn's correction for pairwise multiple comparisons. Abbreviations: Contr, controls; LVFX, levofloxacin; CRO, ceftriaxone.)

Figure 3.

(Therapeutic results of human-like kinetics of levofloxacin 500 mg q12 h or 1 g q24 h against experimental endocarditis caused by viridans group streptococci with various antibiotic-resistance phenotypes. (a) The simulation, in the serum of rats, of the kinetics of levofloxacin following oral administration of 500 mg q12 h (♦) or 1 g q24 h (□)of the drug to humans. Mean and standard deviations of peak and trough drug concentrations in serum are specified in the text. (b) The therapeutic results of 5 days' treatment with this regimen in rats with experimental endocarditis. The MICs of levofloxacin for these test organisms are indicated in the figure. PS stands for penicillin susceptible and PR for penicillin resistant (MIC 2 mg/L). Each dot in the figure represents the bacterial density in the vegetations of a single animal. The median bacterial densities recovered from vegetations (expressed as log cfu/g of tissue) are indicated at the bottom of the columns for each group. P < 0.05 when the results were compared by both the Fisher's exact test and the Kruskall–Wallis test with Dunn's correction for pairwise multiple comparisons. Abbreviations: Contr, controls; LVFX, levofloxacin; CRO, ceftriaxone.)

Figure 4.

(Selection of levofloxacin (LVFX)-resistant and ciprofloxacin (CIPRO)-resistant derivatives by exposure of the test streptococci to stepwise increasing concentrations of antibiotics. Series of tubes containing two-fold serial dilutions of either test drug were inoculated with a final concentration of 106 cfu/mL of the test organisms and were further incubated for 24 h at 35°C, as described for the MIC tests. On the next day, the tube with the highest antibiotic concentration still showing turbidity was used to inoculate a new series of antibiotic-containing tubes. The procedure was repeated, and the increase in the MIC was followed over time. Key: ▪, ciprofloxacin; ▴, levofloxacin. (a) S. gordonii (PS); (b) S. gordonii (Tol+); (c) S. gordonii (IPR); (d) S. mitis 531 (PR); (e) S. sanguis‘ Du’ (PS). PS stands for penicillin susceptible, Tol+ for penicillin tolerant, IPR for intermediate penicillin resistant (MIC 0.125 mg/L) and PR for penicillin resistant (MIC 2 mg/L). )

Figure 4.

(Selection of levofloxacin (LVFX)-resistant and ciprofloxacin (CIPRO)-resistant derivatives by exposure of the test streptococci to stepwise increasing concentrations of antibiotics. Series of tubes containing two-fold serial dilutions of either test drug were inoculated with a final concentration of 106 cfu/mL of the test organisms and were further incubated for 24 h at 35°C, as described for the MIC tests. On the next day, the tube with the highest antibiotic concentration still showing turbidity was used to inoculate a new series of antibiotic-containing tubes. The procedure was repeated, and the increase in the MIC was followed over time. Key: ▪, ciprofloxacin; ▴, levofloxacin. (a) S. gordonii (PS); (b) S. gordonii (Tol+); (c) S. gordonii (IPR); (d) S. mitis 531 (PR); (e) S. sanguis‘ Du’ (PS). PS stands for penicillin susceptible, Tol+ for penicillin tolerant, IPR for intermediate penicillin resistant (MIC 0.125 mg/L) and PR for penicillin resistant (MIC 2 mg/L). )

*
Corresponding author. Tel. +41-21-314-10-26; Fax: +41-21-314-10-36 E-mail: pmoreill@chuv.hospvd.ch

We thank Marlyse Giddey and Jacques Vouillamoz for outstanding technical assistance. Part of this work was presented at the Thirty-Seventh Interscience Conference on Antimicrobial Agents and Chemotherapy, Toronto, Ontario, Canada, September 28–October 1, 1997 and at the Thirty-Eighth Interscience Conference on Antimicrobial Agents and Chemotherapy, San Diego, CA, USA, September 24–27, 1998.

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