In vitro potency of moxifloxacin, clinafloxacin and sitafloxacin against 248 genetically defined clinical isolates of Staphylococcus aureus

Franz-Josef Schmitza,b,*, Ad C. Fluitb, Dana Milatovicb, Jan Verhoefb, Hans-Peter Heinza and Sylvain Brisseb

a Institute for Medical Microbiology and Virology, Heinrich-Heine University Düsseldorf, Germany; b Eijkman-Winkler Institute for Medical Microbiology, Utrecht University, The Netherlands


    Abstract
 Top
 Abstract
 Introduction
 Material and methods
 Results and discussion
 References
 
The in vitro potency of three newer fluoroquinolones, moxifloxacin, clinafloxacin and sitafloxacin was tested against 248 genetically defined Staphylococcus aureus isolates, comprising 116 unrelated S. aureus, seven heterogeneous intermediate vancomycin-resistant S. aureus strains as well as 125 clonally related methicillin-resistant S. aureus. All strains were susceptible to clinafloxacin and sitafloxacin based on an investigational breakpoint of 1 mg/L and were less influenced by mutations within the grl and gyr gene loci. In one-quarter to one-third of the strains tested, reserpine decreased slightly the MICs of moxifloxacin, clinafloxacin and sitafloxacin. Compared with moxifloxacin, clinafloxacin and sitafloxacin showed a significantly increased anti-staphylococcal potency.


    Introduction
 Top
 Abstract
 Introduction
 Material and methods
 Results and discussion
 References
 
Fluoroquinolone resistance in Staphylococcus aureus is a widespread problem. The primary mechanism proposed for fluoroquinolone resistance comprises mutations in the grlA and grlB genes encoding DNA topoisomerase IV, and the secondary mechanism involves mutations in the gyrA and gyrB genes encoding DNA gyrase. In addition to these mutations, S. aureus strains also have multidrug efflux pumps like NorA that contribute to decreased fluoroquinolone susceptibility.1,2

Because of the inherent limitations of ciprofloxacin and other available quinolones in the treatment of staphylococcal infections, there is a need for the development of new quinolones with increased anti-staphylococcal potency. Clinafloxacin and sitafloxacin are two recently developed investigational fluoroquinolones with a broad antimicrobial spectrum.2 The promising results that have been obtained in animal protection studies support the advancement of these two compounds into clinical trials.

The purpose of the present investigation was: (i) to analyse the in vitro potencies of clinafloxacin and sitafloxacin against 116 unrelated S. aureus strains collected from eight countries with defined mutations in the grl and gyr genes;3 (ii) to determine the in vitro potencies of clinafloxacin and sitafloxacin against seven heterogeneous intermediate vancomycin-resistant S. aureus (hetero-VISA) isolates that have emerged in the Düsseldorf area;4 (iii) to investigate the range and stability of clinafloxacin and sitafloxacin MICs in three different clonal populations of methicillin-resistant S. aureus (MRSA), comprising of 125 isolates with defined mutations in the grl, gyr and norA gene loci;5 and (iv) to analyse the effect of reserpine, an inhibitor of the multidrug efflux pump NorA,6 on MICs of clinafloxacin and sitafloxacin for the 116 clonally unrelated S. aureus isolates.


    Material and methods
 Top
 Abstract
 Introduction
 Material and methods
 Results and discussion
 References
 
Bacterial isolates

One hundred and sixteen unrelated S. aureus isolates included 93 MRSA (67 ciprofloxacin resistant and 26 ciprofloxacin susceptible) and 23 methicillin-susceptible S. aureus (MSSA) (three ciprofloxacin resistant and 20 ciprofloxacin susceptible). Seventy German S. aureus isolates and 36 S. aureus isolates from Japan (8), Brazil (8), Switzerland (6), Sri Lanka (4), Spain (4), United Kingdom (3) and Hungary (3), were tested. Isolates were screened for the mecA and coa genes. All strains were isolated from clinical sources and selected on the basis of belonging to different pulse-field gel electrophoresis (PFGE) types, as described previously.3

Seven hetero-VISA, which fulfilled the criteria of Hiramatsu et al. for being hetero-VISA, were studied.7 These isolates exhibited reduced vancomycin susceptibility occurring at frequencies of 10–3–10–4 and emerged in the Düsseldorf area in 1998.4

We characterized the ranges and stability of MICs for 125 MRSA belonging to three distinct types determined by PFGE. The MRSA types were called 1, 2 and 3 (consisting of 75, 38 and 12 isolates, respectively) which are the predominant PFGE types found in western Germany. All of these 125 isolates were checked for the presence of the mecA and coa genes. The MRSA originated from diverse clinical environments and were isolated over a period of more than 3 years.5

Antimicrobial agents and MIC determinations

Reserpine was purchased from Sigma Chemical Co. (St Louis, MO, USA), whereas moxifloxacin (Bayer, Leverkusen, Germany), clinafloxacin (Parke Davis, Ann Arbor, MI, USA) and sitafloxacin (Daichi, Tokyo, Japan) were provided by the manufacturers. MICs for all 248 strains were tested by a broth microdilution method with an inoculum of approximately 105 cfu/mL and doubling dilutions of fluoroquinolones ranging from <=0.008 to 4 mg/L, according to NCCLS recommended guidelines.8

Influence of reserpine on MICs

MICs of moxifloxacin, clinafloxacin and sitafloxacin for the 116 clonally unrelated S. aureus strains with characterized mutations in the gyr and grl genes were determined as described by Neyfakh et al.9 with microdilution plates containing two-fold serial dilutions of the antibacterial drugs in 100 µL of Luria–Bertani medium (Life Technologies, Paisley, UK), an inoculum of 2 x 105 logarithmic-phase cells per mL and incubation for 12 h at 37°C. All experiments were carried out four times, in both the absence or presence of reserpine (20 mg/L). After the incubation period bacterial growth was assessed by observing turbidity of the medium.

Polymerase chain reaction, oligonucleotide primers and sequencing

Protocols for the amplification and subsequent sequencing of grlA, grlB, gyrA, gyrB and norA have been described previously.3,10 The MICs of moxifloxacin, clinafloxacin and sitafloxacin were correlated with characterized mutations in the gyrA, gyrB, grlA and grlB gene loci.


    Results and discussion
 Top
 Abstract
 Introduction
 Material and methods
 Results and discussion
 References
 
We tested the in vitro potencies of five fluoroquinolones against 70 ciprofloxacin-resistant and 46 ciprofloxacinsusceptible clinical S. aureus strains belonging to different PFGE types. The order of in vitro potency of the fluoroquinolones tested was ciprofloxacin as least active followed by ofloxacin, levofloxacin, sparfloxacin and moxifloxacin as most active. We compared the in vitro potency of moxifloxacin, the most active compound, with that of two newer fluoroquinolones, clinafloxacin and sitafloxacin, and found that sitafloxacin was the most active and moxifloxacin the least active of the three drugs (TableGo). Based on the MICs determined for ciprofloxacin-resistant isolates (n = 70), the in vitro potency of sitafloxacin was approximately two-fold greater than that of clinafloxacin and four- to eight-fold greater than that of moxifloxacin. Isolates of ciprofloxacin-resistant S. aureus were inhibited by sitafloxacin concentrations ranging from 0.06 to 0.5 mg/L. The differences between the in vitro potencies of the drugs tested against ciprofloxacin-susceptible S. aureus strains (n = 46) were smaller than those for ciprofloxacin-resistant isolates. Sitafloxacin was again the most potent with an MIC range of <=0.008–0.03 mg/L.


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Table. Mutations and consequent amino acid changes within grlA, grlB and gyrA and corresponding moxifloxacin, clinafloxacin and sitafloxacin MICs (no amino acid changes have been observed in GyrB)
 
Isolates without the GrlA alteration Ser80->Phe were ciprofloxacin susceptible and had a moxifloxacin MIC of <=0.125 mg/L, a clinafloxacin MIC of <=0.06 mg/L and a sitafloxacin MIC of <=0.03 mg/L (TableGo). An alteration in GrlA (Ser80->Phe) in combination with either a Ser84-> Leu or a Glu88->Lys alteration within GyrA was detected in all ciprofloxacin-resistant isolates. The range of MICs for isolates with these mutations was 0.5–2 mg/L for moxifloxacin, 0.12–0.5 mg/L for clinafloxacin and 0.06–0.5 mg/L for sitafloxacin. Overall, moxifloxacin was the least potent compound and clinafloxacin and sitafloxacin the most potent in response to all characterized mutations in grlA, grlB, gyrA and gyrB (TableGo).

We reported recently the emergence of heterogeneous intermediate vancomycin resistance in S. aureus in the Düsseldorf area.4 The seven hetero-VISA strains had a cell wall structure very similar, if not identical, to that of Japanese isolates.4 All hetero-VISA had the same PFGE type as the so-called ‘North German epidemic strain’, which is epidemic in several parts of Germany. The seven German hetero-VISA strains exhibited MICs of 128, 2, 0.5 and 0.25 mg/L of ciprofloxacin, moxifloxacin, clinafloxacin and sitafloxacin, respectively. Thus, clinafloxacin and sitafloxacin are also very active against the hetero-VISA emerging in the Düsseldorf area.

We analysed the range and stability of moxifloxacin, clinafloxacin and sitafloxacin MICs in three clonal populations of MRSA described previously.5 In total, MICs of the three fluoroquinolones were determined for 125 MRSA isolates, comprising three different PFGE types. Over a period of 3 years, we observed conserved mutations within the grl, gyr and norA gene loci and concomitant conserved MICs within the MRSA isolates belonging to one PFGE type and originating from quite diverse clinical environments (data not shown).5 We also tested whether clinafloxacin and sitafloxacin MICs were stable in these MRSA populations and found the MIC ranges for moxifloxacin, clinafloxacin and sitafloxacin to be 0.5–1, 0.06–0.125 and 0.015–0.06 mg/L, respectively.

In a previous study,6 efflux pump activity was inhibited by the plant alkaloid reserpine in 116 S. aureus strains. The presence of reserpine (20 mg/L), which by itself does not affect the growth of S. aureus, resulted in up to four-fold decreases in ciprofloxacin MICs.6 In contrast to its effects on ciprofloxacin MICs, the effect of reserpine on moxifloxacin, clinafloxacin and sitafloxacin MICs was negligible, with either no effect or a two-fold decrease in MICs (data not shown). Reserpine decreased slightly the MICs of moxifloxacin, clinafloxacin and sitafloxacin in 21, 26 and 29% of the isolates, respectively. Reserpine, an inhibitor of multidrug efflux pumps, seems to increase the intracellular concentration of the three fluoroquinolones to a comparable degree in these strains. This inhibition was not influenced by mutations detected within the grl, gyr or norA gene loci. Furthermore, the inhibitory effect observed was not dependent on fluoroquinolone MICs for the isolates. Thus our data suggest that in some isolates the three quinolones are excreted to some extent by efflux transporters. Differences in MICs, with or without reserpine, between various quinolones may result from differences in their chemical structure.

In summary, all strains tested (116 unrelated S. aureus, seven hetero-VISA strains and 125 clonally related MRSA) were susceptible to clinafloxacin and sitafloxacin based on an investigational breakpoint of 1 mg/L. Compared with other fluoroquinolones,3 these two new fluoroquinolones are less influenced by mutations within the gene loci that are known to be involved in fluoroquinolone resistance in S. aureus. In a quarter to a third of the strains tested, reserpine decreased slightly the MICs of moxifloxacin, clinafloxacin and sitafloxacin, suggesting the existence of multidrug efflux pumps which seem to decrease the intracellular concentration of the three fluoroquinolones. Compared with other fluoroquinolones, clinafloxacin and sitafloxacin showed a significantly increased anti-staphylococcal potency.


    Notes
 
* Correspondence address. Institute for Medical Microbiology and Virology, Heinrich-Heine-Universität Düsseldorf, Universitätsstrasse 1, Geb. 22.21, 40225 Düsseldorf, Germany. Tel: +49-2132-72040; Fax: +49-2132-72040; E-mail: schmitfj{at}uni-duesseldorf.de Back


    References
 Top
 Abstract
 Introduction
 Material and methods
 Results and discussion
 References
 
1 . Ferrero, L., Cameron, B. & Crouzet, J. (1995). Analysis of gyrA and grlA mutations in stepwise-selected ciprofloxacin-resistant mutants of Staphylococcus aureus. Antimicrobial Agents and Chemotherapy 39, 1554–8.[Abstract]

2 . Gootz, T. D. & McGuirk, P. R. (1994). New quinolones in development. Expert Opinion on Investigational Drugs 3, 93–114.

3 . Schmitz, F.-J., Hofmann, B., Hansen, B., Scheuring, S., Lückefahr, M., Klootwijk, M. et al. (1998). Relationship between ciprofloxacin, ofloxacin, levofloxacin, sparfloxacin and moxifloxacin (BAY 12-8039) MICs and mutations in grlA, grlB, gyrA and gyrB in 116 unrelated clinical isolates of Staphylococcus aureus. Journal of Antimicrobial Chemotherapy 41, 481–4.[Abstract]

4 . Geisel, R., Schmitz, F.-J., Thomas, L., Berns, G., Zetsche, O., Ulrich, B. et al. (1999). Emergence of heterogeneous intermediate vancomycin resistance in Staphylococcus aureus isolates in the Düsseldorf area. Journal of Antimicrobial Chemotherapy 43, 846–8.[Free Full Text]

5 . Schmitz, F.-J., Köhrer, K., Scheuring, S., Verhoef, J., Fluit, A., Heinz, H.-P. et al. (1999). The stability of grlA, grlB, gyrA, gyrB and norA mutations and MIC values of five fluoroquinolones in three different clonal populations of methicillin-resistant Staphylococcus aureus. Clinical Microbiology and Infection 5, 287–90.[Medline]

6 . Schmitz, F.-J., Fluit, A. C., Lückefahr, M., Engler, B., Hofmann, B., Verhoef, J. et al. (1998). The effect of reserpine, an inhibitor of multi-drug efflux pumps, on the in-vitro activities of ciprofloxacin, sparfloxacin and moxifloxacin against clinical isolates of Staphylococcus aureus. Journal of Antimicrobial Chemotherapy 42, 807–10.[Abstract]

7 . Hiramatsu, K., Aritaka, N., Hanaki, H., Kawasaki, S., Hosoda, Y., Hori, S. et al. (1997). Dissemination in Japanese hospitals of strains of Staphylococcus aureus heterogeneously resistant to vancomycin. Lancet 350, 1670–3.[ISI][Medline]

8 . National Committee for Clinical Laboratory Standards. (1997). Methods for Dilution Antimicrobial Susceptibility Tests for Bacteria that Grow Aerobically—Fourth Edition: Approved Standard M7-A4. NCCLS, Wayne, PA.

9 . Neyfakh, A. A., Borsch, C. M. & Kaatz, G. W. (1993). Fluoroquinolone resistance protein NorA of Staphylococcus aureus is a multidrug efflux transporter. Antimicrobial Agents and Chemotherapy 37, 128–9.[Abstract]

10 . Schmitz, F.-J., Hertel, B., Hofmann, B., Scheuring, S., Verhoef, J., Fluit, A. et al. (1998). Relationship between mutations in the coding and promoter regions of the norA genes in 42 unrelated clinical isolates of Staphylococcus aureus and the MICs of norfloxacin for these strains. Journal of Antimicrobial Chemotherapy 42, 561–3.[Free Full Text]

Received 14 July 1999; returned 1 November 1999; revised 15 November 1999; accepted 2 February 2000





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