Sitafloxacin (DU-6859a) and trovafloxacin: postantibiotic effect and in vitro interactions with rifampin on methicillin-resistant Staphylococcus aureus

ANTIMICROBIAL SUSCEPTIBILITY STUDIES

Sitafloxacin (DU-6859a) and Trovafloxacin: Postantibiotic Effect and in Vitro Interactions with Rifampin on Methicillin-resistant Staphylococcus aureus Evangelos J. Giamarellou-Bourboulis, Helen Sambatakou, Paraskevi Grecka, Zoi Chryssouli, and Helen Giamarellou

Sitafloxacin (DU-6859a) and trovafloxacin are novel quinolones potent on methicillin-resistant Staphylococcus aureus (MRSA) that are designed for once daily administration. In order to define the adequacy of the above regimen for the therapy of infections by multiple drug–resistant MRSA, their postantibiotic effect (PAE), their bactericidal activity, and their interactions with rifampin were determined on 14 MRSA isolates resistant to both ciprofloxacin and rifampin. PAE was defined after 1-h exposure to 1⫻, 4⫻, and 10⫻ MIC and the killing effect after exposure to 1⫻ and 4⫻ MIC. Rifampin was applied for interactive studies at a concentration of 2 ␮g/mL, which is equal to its mean serum level. Median PAEs produced by 1⫻, 4⫻, and 10⫻ MIC of sitafloxacin were 1.39, 3.75, and 6.61 h respectively, and by 1⫻, 4⫻, and 10⫻ MIC of trovafloxacin 0.87, 2.07, and 2.23 h respectively. PAEs achieved by sitafloxacin were statistically shown to be longer

than those achieved by trovafloxacin; PAEs achieved by a concentration of 10⫻ MIC of each quinolone did not differ significantly from those achieved by a concentration of 4⫻ MIC. Both the 4⫻ and 10⫻ MIC concentrations produced a more prolonged PAE than the 1⫻ MIC concentration. A rapid bactericidal activity was expressed over the first 6 h of growth by each quinolone involving 80% of isolates enhanced in some isolates by their interaction with rifampin. The above findings revealed an extended PAE and a rapid killing effect of both sitafloxacin and trovafloxacin on MRSA resistant to ciprofloxacin and to rifampin, thus supporting their once daily administration in the therapy of infections by multiple drug– resistant MRSA. However little in vitro benefit is derived by their interaction with rifampin. © 1999 Elsevier Science Inc.

INTRODUCTION

methicillin-resistant Staphylococcus aureus (MRSA) (Giamarellos-Bourboulis et al. 1997). Both these agents are designed for once daily oral administration at a dose of 200 mg for seven to ten days (Brighty and Gootz 1997). This dosage results in a mean Cmax of trovafloxacin in serum equal to 2.9 ␮g/mL (Wise et al. 1996) and of sitafloxacin equal to 1.86 ␮g/mL

Sitafloxacin (DU-6859a) and trovafloxacin are newer fluoroquinolones that are very potent in vitro against Gram-positive cocci and especially against From the First Department of Propaedeutic Medicine (EJGB), Athens Medical School; the Department of Special Infections (HS), Athens General Hospital “G. Gennimatas,”and the Fourth Department of Internal Medicine (PG, ZC, HG), Athens Medical School, Athens, Greece. Address reprint requests to H. Giamarellou, M.D., Ph.D., 4th Department of Internal Medicine, Sismanoglion General Hospital, 151 26 Maroussi Attikis, Greece.

DIAGN MICROBIOL INFECT DIS 1999;34:301–307 © 1999 Elsevier Science Inc. All rights reserved. 655 Avenue of the Americas, New York, NY 10010

Presented in part at The 37th Interscience Conference on Antimicrobial Agents and Chemotherapy, September–October 1997, Toronto, Canada, abstract no. A-81. Received 2 November 1998; accepted 29 March 1999.

0732-8893/99/$–see front matter PII S0732-8893(99)00044-9

302 (Nakashima et al. 1995). The above concentrations are well above the MIC90s of sitafloxacin and of trovafloxacin, which were found to be 0.025 (Nakane et al. 1995) and 0.5 ␮g/mL (Brighty and Gootz 1997) respectively for staphylococci. The above pharmacodynamic data along with the fact that the t1/2 of the above compounds in plasma are found to be 4.6 and 7.8 h respectively support the adequacy of the once daily regimen for infections by staphylococci with the above MICs. However it has been reported by our study group that sitafloxacin and trovafloxacin inhibit MRSA isolates resistant to ciprofloxacin at much higher MICs close to their Cmax concentrations in serum (Giamarellos-Bourboulis et al. 1997). As a consequence their dosing at more frequent intervals than once daily might be necessary to eradicate an infection caused by MRSA resistant to ciprofloxacin and to rifampin. The postantibiotic effect (PAE) of both quinolones is a pharmacodynamic parameter that could be applied to resolve that point. PAE is considered as the period of time needed by a bacterial population to overcome the suppression imposed to its growth after a short exposure to an antimicrobial agent (Craig and Gudmundsson 1991). A relative prolonged duration of the PAE produced by the tested quinolones might reveal the adequacy or not of the proposed once daily regimen for infections caused by multiple drug–resistant MRSA. The perspective that newer quinolones may eradicate an infection by such isolates derives from studies revealing that they possess a different mechanism of bacterial killing than ciprofloxacin (Morrisey 1996). On that basis their bactericidal activity and their interactions with rifampin might be applied as an indicator of their potential on MRSA resistant to both ciprofloxacin and to rifampin.

MATERIALS AND METHODS Selection of Strains and Determination of MICs Fourteen MRSA strains were included in the study derived from pus cultures of different patients suffering from nosocomial infections. Resistance to methicillin was determined by the growth of single colonies after an 18-h incubation at 30°C on Mueller– Hinton agar (Oxoid Ltd, London, UK) with incorporated oxacillin (Sigma Co., St. Louis, MO, USA) at a concentration of 6 ␮g/mL and 4% NaCl (Hindler 1992a). The criteria for the inclusion of the strains in the study were their susceptibility to both sitafloxacin and to trovafloxacin, and their resistance to both ciprofloxacin and to rifampin as defined by the determination of their MIC. The latter was performed by a microdilution technique of the antimicrobial

E.J. Giamarellos-Bourboulis et al. agent in Mueller–Hinton broth (Oxoid Ltd.) at final volumes of 0.1 mL where an inoculum of 5 ⫻ 105 CFU/mL was applied. To prepare the needed inoculum fresh colonies were incubated in Mueller–Hinton broth for 1 h in a shaking water bath of 37°C until they adapted a visible turbidity, and they were then diluted by applying 0.5 of the McFarland climax (Woods and Washington 1995). MIC was defined as the lowest concentration limiting visible growth after 18 h of incubation at 35°C. A 2-␮g/mL concentration of all quinolones and a 4-␮g/mL one of rifampin were considered as their susceptibility breakpoints (Giamarellos-Bourboulis et al. 1997; Woods and Washington 1995).

Determination of PAE Sitafloxacin (Daichi Pharmaceutical, Tokyo, Japan) and trovafloxacin (Pfizer, Groton, CT, USA) were provided as white amorphous powders and reconstituted with sterile and pyrogen-free water prior to experimentation according to the manufacturer’s instructions. They were then added to tubes with a final volume of 10 mL at concentrations of 1⫻, 4⫻, and 10⫻ MIC. To assure that the added inoculum of each isolate was of a logarithmic phase of growth, 12 h before the experiment single colonies were incubated for 5 h in Mueller–Hinton broth at 37°C and the resulting inoculum was serially diluted five times 1:10. All dilutions were incubated overnight at 37°C and their absorbance was then read at 580 nm (Hitachi Spectophotometer, Tokyo, Japan). The dilution with an absorbance closest to 0.3 was selected as the most appropriate to yield a log-phase inoculum (Craig and Gudmundsson 1991). A 1-mL volume of each dilution was added along with Mueller–Hinton broth in the tubes containing the quinolone so that the applied inoculum was 5 ⫻ 106 CFU/mL. One tube served as growth control. All tubes were incubated for 1 h at 37°C in a shaking water bath after which a 2-mL volume of each tube was centrifuged at 1200 g for 2 min. The supernatant was discarded and the bacterial pellet was washed twice with Mueller–Hinton broth to assure removal of any remnants of the quinolones. The bacterial pellet was then diluted with 2 mL of Mueller–Hinton broth and it was left to incubate in the same conditions for 24 h. The viable cell counts were determined at standard time intervals (0 time and just prior to centrifugation, 0 time, 1, 2, 3, 4, 5, and 24 h postcentrifugation) by removing a 0.1-mL aliquot that was five times serially diluted 1:10 in sterile, cold 0.9% NaCl and by plating another 0.1-mL aliquot of each dilution onto blood agar (Columbia agar base, BBL Becton Dickinson, Cockeysville, MD, USA). The log10 of the number of viable cell counts at each time interval was plotted over

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time and the PAE (in hours) was estimated by the following equation: PAE: T ⫺ C, where T is the time required for the count in the test tube to increase by 1log10 above the count observed immediately after drug removal and C is the same period of time in the untreated growth control (Craig and Gudmundsson 1991).

Fisher’s exact test (Zelterman and Louis 1992). The change of log10 cell counts by both quinolones and by their interactions with rifampin was expressed by their median values and compared by the Mann– Whitney U test (Moses et al. 1992). Any value of p below 0.05 was considered as significant.

Time Kill and Interactive Studies

RESULTS

A log-phase inoculum of 5 ⫻ 106 CFU/mL of each MRSA isolate was exposed over time to 1⫻ and to 4⫻ MIC concentrations of sitafloxacin and of trovafloxacin and to their interaction with rifampin. All quinolones were added into tubes of a final volume of 10 mL containing both the starting inoculum and Mueller–Hinton broth. Rifampin was provided as a yellowish amorphous powder (Marion Merrel Dow, Richmond, Ontario, Canada) and it was dissolved in methanol 99% (Merck, Darmstadt, Germany) to become water soluble. Since all isolates were resistant to rifampin (MIC ⬎4 ␮g/mL) its applied concentration was 2 ␮g/mL to be equal to its mean serum level (Sambatakou et al. 1998) so that results might reflect to the in vivo conditions. Rifampin was added so that the final concentration of methanol 99% in each tube was 0.001 v/v. One tube served as growth control and another one with added rifampin at a concentration of 2 ␮g/mL as an internal control. All tubes were left to incubate at 37°C in a shaking water bath and bacterial growth was determined as stated above at standard time intervals (0 time, 2, 4, 6, and 24 h of incubation). That manner of viable cell count determination permitted avoidance of any antimicrobial carry-over effect. At each time interval the log10 change of viable cells compared with the starting inoculum was determined in each tube. Any change equal to or more than a 3log10 decrease was considered as a bactericidal effect (Hindler 1992b). Whenever the interaction of a quinolone and of rifampin resulted in a more than or equal to 2log10 decrease compared with the most active single agent, synergy was defined, and whenever it resulted in a respective 1–2log10 decrease, an additive effect between both agents was established (Hindler 1992b). One-hundred and forty killing curves were performed in total.

The postantibiotic effect of sitafloxacin and of trovafloxacin on all tested MRSA isolates in correlation to their MICc is shown in Table 1. Their bactericidal activity is shown in Table 2 and the effect of both single agents and of their interaction with rifampin in Table 3. Synergy between 1⫻ MIC of sitafloxacin and rifampicin was found at 2, 4, 6, and 24 h of growth in one (7.1%), two (14.3%), two (14.3%), and three (21.4%) isolates respectively, and between 4⫻ MIC of sitafloxacin and rifampin in nil (0%), one (7.1%), two (14.3%), and three (21.4%) isolates respectively. Synergy between 4⫻ MIC of trovafloxacin and rifampin was found at 24 h in two (14.3%) isolates. Concerning the additive effect between the tested quinolones and rifampin, it was found in one isolate (7.1%) in the presence of 1⫻ MIC of sitafloxacin at 24 h and in two (14.3%) and two (14.3%) isolates at 24 h in the presence of 1⫻ MIC and of 4⫻ MIC of trovafloxacin respectively. The killing curves for one MRSA isolate are shown in Figure 1.

Statistical Analysis Median PAE produced by the concentration of each quinolone was determined. Differences between the concentrations of each quinolone and between both quinolones were evaluated by the Mann–Whitney U test (Moses et al. 1992). Comparisons between both quinolones and between both concentrations regarding their bactericidal activity was performed by the

DISCUSSION The present study focused on the determination of the PAE and of the bactericidal activity of two novel quinolones, sitafloxacin (DU-6859a) and trovafloxacin, on MRSA isolates resistant to both ciprofloxacin and rifampin. The objective of the present study was to define the adequacy of the proposed once daily dose regimen of both compounds to eradicate infections caused by multiple drug–resistant MRSA. PAE is a pharmacodynamic parameter that permits clarification of whether the exposure of a bacterial population for a short period of time to a new agent might suppress the continuation of its growth (Craig and Gudmundsson 1991). If PAE produced by antimicrobial concentrations close to their levels achieved in body fluids is long enough, then the time intervals between doses are adequate. Concerning these two quinolones the only existing information in the literature comes from studies of trovafloxacin on only six susceptible Pseudomonas aeruginosa and two MRSA strains (Boswell et al. 1997; Pankuch et al. 1998). As a consequence the present study is the first dealing with their PAE on a large number of multiple drug–resistant MRSA.

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TABLE 1 Postantibiotic Effect of Sitafloxacin and of Trovafloxacin on 14 MRSA Isolates Sitafloxacin

Trovafloxacin

Strain

MIC (␮g/mL)

1⫻ MIC

4⫻ MIC

10⫻ MIC

MIC (␮g/mL)

1⫻ MIC

4⫻ MIC

10⫻ MIC

1 4 5 14 16 27 31 40 45 47 56 161 163 164 Median

0.03 0.03 0.03 0.03 0.12 0.25 1 0.25 0.25 0.03 0.03 2 1 2

1.50 1.66 2.28 0 1.38 1.88 3.6 0 2.84 0 0 0.28 1.4 0 1.39a

1.50 1.46 2.35 0 3.51 5 4 5 5 1.3 1.75 5 4 5 3.75b,c

1.50 7.21 4.18 0.5 1.51 ⬎24 3.8 6 ⬎24 0.7 ⬎24 ⬎24 ⬎24 ⬎24 6.61d,e,f

0.12 0.03 0.03 0.5 0.5 0.06 0.12 0.12 0.03 0.03 0.03 0.12 0.25 1

0.25 0.86 0 0.88 0 1.98 2.06 2.8 0 1.21 0 1.19 2.22 0 0.87

0.25 1.02 2.23 1.94 0 2.25 3.21 5 3.21 1.26 2.20 0.11 2.51 0 2.07g

0.25 2.20 2.25 2.00 4.25 1.82 3.28 5 4.48 1.48 2.65 1.25 4.16 0 2.23g,h

a

p Between 1⫻ MIC of sitafloxacin and 1⫻ MIC of trovafloxacin NS. p Between 4⫻ MIC and 1⫻ MIC of sitafloxacin 0.006. c p Between 4⫻ MIC of sitafloxacin and 4⫻ MIC of trovafloxacin 0.047. d p Between 10⫻ MIC and 4⫻ MIC of sitafloxacin NS (nonsignificant). e p Between 10⫻ MIC and 1⫻ MIC of sitafloxacin 0.001. f p Between 10⫻ MIC of sitafloxacin and 10⫻ MIC of trovafloxacin 0.032. g p Between 10⫻ MIC and 4⫻ MIC of trovafloxacin NS, p between 4⫻ MIC and 1⫻ MIC of trovafloxacin NS. h p Between 10⫻ MIC and 1⫻ MIC of trovafloxacin 0.006. b

TABLE 2 Bactericidal Activity of Sitafloxacin and of Trovafloxacin on 14 MRSA Isolates Number (%) of Isolates Sitafloxacin Time (hours) 2 4 6 24 a b

1⫻ MIC 2 (14.3) 5 (35.7) 8 (72.7) 8 (72.7)

Trovafloxacin

4⫻ MIC a

4 (28.6) 8 (72.7)a 11 (78.6)a 12 (85.7)b

1⫻ MIC

4⫻ MIC

— 1 (7.1) 6 (42.8) 4 (28.6)

2 (14.3)a 5 (35.7)a 9 (64.3)a 11 (78.6)a

p Between 4⫻ MIC and 1⫻ MIC nonsignificant. p Between 4⫻ MIC and 1⫻ MIC 0.021.

Our results revealed that the median PAEs produced by 1⫻, 4⫻, and 10⫻ MIC of sitafloxacin on MRSA were 1.39, 3.75, and 6.61 h respectively, whereas by the same concentrations of trovafloxacin they were 0.87, 2.07, and 2.23 h respectively (Table 1). However, a large range of values was presented with the PAEs of 1⫻, 4⫻, and 10⫻ MIC of sitafloxacin being within the ranges of 0 to 2.84, 0 to 5, and 0.5 to ⬎24 h respectively, and with those of 1⫻, 4⫻, and 10⫻ MIC of trovafloxacin of 0 to 2.22, 0 to 3.21, and 0 to 4.48 h respectively. Despite the above ranges it was clear that higher MICs produced higher PAE, with the exception of isolate 164 with MIC of trovafloxacin above 0.5 ␮g/mL failing to give any PAE. However, our study should include a greater number of isolates with such MICs to draw safe conclu-

sions whether trovafloxacin produces any PAE at concentrations above 1 ␮g/mL or not. The PAEs produced by the 4⫻ MIC and the 10⫻ MIC concentrations of either agent were statistically the same whereas both the 4⫻ MIC and the 10⫻ MIC concentrations were superior to the 1⫻ MIC one. When comparing the two quinolones it was found that with the exception of the 1⫻ MIC concentration PAEs produced by sitafloxacin were longer than those of trovafloxacin. Considering the PAEs of both sitafloxacin and trovafloxacin on multiple drug–resistant MRSA it should be kept in mind that the majority of the tested isolates possessed such MICs that at multiples of 1⫻, 4⫻, and 10⫻ MIC produce concentrations of both quinolones below than or equal to the Cmax of sita-

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305

TABLE 3 Median log10 Changes of Viable Cell Counts from the Baseline by the Two Tested Quinolones and After Their Interaction with Rifampicin Sitafloxacin

Trovafloxacin

Sitafloxacin ⫹ Rifampina

Trovafloxacin ⫹ Rifampina

Time (hours)

1⫻ MIC

4⫻ MIC

1⫻ MIC

4⫻ MIC

Rifampin 2 ␮g/mLa

1⫻ MIC

4⫻ MIC

1⫻ MIC

4⫻ MIC

2 4 6 24

⫺1.30 ⫺1.22 ⫺1.85 ⫺2.35

⫺2.00 ⫺3.00b ⫺4.00b ⫺4.39b

⫺0.09 ⫺1.81 ⫺2.21 ⫹0.11

⫺1.15 ⫺2.49b ⫺2.91b ⫺3.52c

⫺0.69 ⫺0.16 ⫺1.04 ⫹0.69

⫺0.61 ⫺1.39d ⫺2.00d ⫺2.22d

⫺1.00 ⫺1.69d ⫺2.22d ⫺3.00d

⫺1.09 ⫺1.69d ⫺2.00d ⫺3.39

⫺1.69d ⫺2.69d ⫺3.69d ⫺4.00d

b

b

d

d

d

Rifampin was applied at a concentration of 2 ␮g/mL, which is equal to its mean serum level. p Not significant compared with the change of viable cells by 1⫻ MIC. c p ⫽ 0.029 compared with the change of viable cells by 1⫻ MIC. d p Not significant compared with the change of viable cells by single agents. a

b

FIGURE 1 Killing curves of MRSA isolate no. 14 exposed to (a) 1⫻ MIC of sitafloxacin, (b) 4⫻ MIC of sitafloxacin, (c) 1⫻ MIC of trovafloxacin, and (d) 4⫻ MIC of trovafloxacin (Œ, growth control; ■, growth in the presence either of sitafloxacin or of trovafloxacin; }, growth in the presence of rifampin (RIF) at a concentration of 2 ␮g/mL; F, interaction of either sitafloxacin or of trovafloxacin with rifampin).

floxacin (Nakashima et al. 1995) and of trovafloxacin (Wise et al. 1996) in serum achieved after a single 200 mg dose. It should be noted however that analogous levels of trovafloxacin are found in the bronchial

mucosa, the epithelial lining fluid, and the alveolar macrophages after a single dose where they remain elevated for more than 12 h (Andrews et al. 1997). These pharmacokinetic data on trovafloxacin along

306 with the extended period of the PAEs of both quinolones even surpassing 7 h support the adequacy of their once daily administration for the eradication of infections by multiple drug–resistant MRSA. The extended PAEs of both sitafloxacin and trovafloxacin led to the study of their killing effect on such isolates. As found (Table 2, Figure 1) both agents provoked a rapid bactericidal effect expressed over the first 6 h of growth, reaching its peak at 24 h. The latter effect involved 72.7 and 85.7% of isolates exposed to 1⫻ and 4⫻ MIC of sitafloxacin and 42.8 and 78.6% of those exposed to 1⫻ and 4⫻ MIC of trovafloxacin respectively. Most important to state is that no statistical difference was found between 1⫻ and 4⫻ MIC of sitafloxacin as opposed to trovafloxacin where the concentration of 4⫻ MIC was more effective than the one of 1⫻ MIC at 24 h of growth (Tables 2 and 3). The rapid and efficacious killing effect of both novel compounds might be attributed to the existence of a B mechanism of bactericidal activity on S. aureus (Morrisey 1996). Interactive studies between either sitafloxacin or trovafloxacin and rifampin revealed that synergism of either novel quinolone with rifampin was limited to simple isolates not exceeding 21.4% of the their total number. A minor decrease of viable cells was found with rifampicin alone after 6 h of growth without ever being above the criteria of bactericidal effect and which might be explained by the fact that the MIC of rifampin was for some isolates equal to 8 ␮g/mL; i.e., close to the applied concentration of 2

E.J. Giamarellos-Bourboulis et al.

␮g/mL for the interactive studies. Because the bactericidal activity of both quinolones surpassed 80% of the tested isolates and the observed synergy involved only 21.4% of them, that interaction simply leads to an attenuation of the already observed killing effect of the novel compounds on multiple drug– resistant MRSA. The present findings of the extended PAE of sitafloxacin (DU-6859a) and of trovafloxacin even at low concentrations equal to 1⫻ MIC on MRSA resistant to both ciprofloxacin and rifampin signify the adequacy of their single daily dose regimen (Brighty and Gootz 1997; Nakashima et al. 1995) for the eradication of infections caused by multiple drug–resistant MRSA. However, it should be borne in mind that the principal criteria for dosing are the t1/2 of the antimicrobial agent and the achievement of continuous drug levels above the MIC of the infective microorganism whereas dosing based solely on PAE might result for some period of time in levels below the MIC (Craig and Gudmundsson 1991). As already mentioned both sitafloxacin and trovafloxacin are characterized by relatively long t1/2s of 4.6 and 7.8 h respectively (Nakashima et al. 1995; Wise et al. 1996), which in accordance with their extended PAEs permit their once daily dose regimen. These results are further supported by the rapid bactericidal effect of these newer quinolones on the above isolates, which might be enhanced by their interaction with rifampin.

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