Increased in vitro activity of the novel des-fluoro(6) quinolone BMS-284756 against genetically defined clinical isolates of Staphylococcus aureus

Franz-Josef Schmitza,b,*, Mechthild Boosa, Susanne Mayera, Harold Jaguscha and Ad C. Fluitb

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


    Abstract
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 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
The in vitro activities of the novel des-fluoro(6) quinolone BMS-284756, ciprofloxacin, gatifloxacin and moxifloxacin were tested against 248 genetically defined Staphylococcus aureus isolates, comprised of 116 unrelated S. aureus, seven heterogeneous vancomycin-intermediate S. aureus (hetero-VISA) strains and 125 clonally related MRSA. All strains were susceptible to BMS-284756 at an investigational breakpoint of 1 mg/L. Reserpine did not decrease the MIC of BMS-284756 in any of the strains tested. The novel des-fluoro(6) quinolone BMS-284756 showed a significantly increased anti-staphylococcal activity.


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
Fluoroquinolone resistance in Staphylococcus aureus is a widespread problem. Because of the inherent limitations of ciprofloxacin and other older quinolones in the treatment of staphylococcal infections, there is a need for the development of new quinolones with increased anti-staphylococcal activity. BMS-284756 (T-3811) is a novel quinolone that lacks a fluorine at the C-6 position and possesses a difluoromethoxy group at the C-8 position. This des-fluoro(6) quinolone has a broad spectrum of antibacterial activity.1,2

The purpose of the present investigation was:

(i) to analyse the in vitro activities of the novel quinolone BMS-284756 against 116 unrelated S. aureus strains with defined mutations in the grl and gyr genes, collected from eight countries;3

(ii) to determine the in vitro activities of BMS-284756 against seven heterogeneous vancomycin-intermediate resistant S. aureus (hetero-VISA) isolates that have emerged in the Düsseldorf area;4

(iii) to investigate the range and stability of BMS-284756 MICs in three different clonal populations of methicillin-resistant S. aureus (MRSA), comprised of 125 isolates with defined mutations in the grl, gyr and norA gene loci;5

(iv) to analyse the effect of reserpine, an inhibitor of multidrug efflux pumps,6 on MICs of the novel des-fluoro(6) quinolone BMS-284756 in those 116 unrelated S. aureus.


    Materials and methods
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 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
Bacterial isolates

One hundred and sixteen S. aureus isolates, 93 MRSA (67 ciprofloxacin resistant and 26 ciprofloxacin susceptible) and 23 methicillin-susceptible S. aureus (MSSA) (three ciprofloxacin resistant and 20 ciprofloxacin susceptible) were studied. Seventy S. aureus German isolates and 36 S. aureus isolates from Japan (eight), Brazil (eight), Switzerland (six), Sri Lanka (four), Spain (four), the UK (three) and Hungary (three) were tested. Isolates were screened for the mecA and coa genes. All 116 strains were isolated from clinical sources and selected on the basis of exhibiting different pulsed-field gel electrophoresis (PFGE) types, as described previously.3 Seven hetero-VISA strains were also studied. These isolates fulfilled the criteria of Hiramatsu et al.7 for being hetero-VISA and had emerged in the Düsseldorf area in 1998.4

In addition, we characterized the ranges and stability of MIC values for 125 MRSA belonging to three distinct PFGE types. The MRSA types were designated types 1, 2 and 3 (consisting of 75, 38 and 12 isolates, respectively), which comprise the predominant PFGE types found in western Germany. All of these 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 determination

Reserpine was purchased from Sigma-Aldrich (St Louis, MO, USA); BMS-284756 (Bristol-Myers Squibb, Wallingford, CT, USA), ciprofloxacin (Bayer, Wuppertal, Germany), gatifloxacin (Grünenthal, Aachen, Germany) and moxifloxacin (Bayer) were provided by the manufacturers.

MIC values were determined using a broth microdilution method according to NCCLS guidelines.8

Influence of reserpine on MICs

MICs of BMS-284756 were determined as described previously.6 All experiments were carried out three times, both in the absence and presence of reserpine (20 mg/L).6

PCR, oligonucleotide primers and sequencing

Protocols for the amplification and subsequent sequencing of grl and gyr genes were as described previously.3

The MIC values of BMS-284756, ciprofloxacin, gatifloxacin and moxifloxacin were correlated with characterized mutations in the quinolone resistance-determining regions (QRDRs) of the grl and gyr genes.


    Results and discussion
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 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
We tested the in vitro activities of five fluoroquinolones against 70 ciprofloxacin-resistant and 46 ciprofloxacin-susceptible clinical S. aureus strains belonging to different PFGE types. In response to all characterized mutations in the gene loci grl, gyr and norA,3,9 ciprofloxacin was least active in vitro, followed by ofloxacin, levofloxacin, sparfloxacin and moxifloxacin, with moxifloxacin as most active.

Since moxifloxacin was the most active compound, we compared the in vitro activities of the novel des-fluoro(6) quinolone BMS-284756 with that of moxifloxacin and the other C-8 methoxy quinolone, gatifloxacin. BMS-284756 was the most active compound followed by the two C-8 methoxy quinolones, moxifloxacin and gatifloxacin. Ciprofloxacin was the least active of the four drugs (TableGo). Based on the MIC values for ciprofloxacin-resistant isolates (n = 70), the in vitro activity of BMS-284756 was approximately two- to four-fold greater than that of moxifloxacin and four- to eight-fold greater than that of gatifloxacin. Isolates of ciprofloxacin-resistant S. aureus were inhibited by concentrations of BMS-284756 ranging from 0.06 to 0.5 mg/L. The differences between the in vitro activities of the drugs tested against ciprofloxacin-susceptible S. aureus strains (n = 46) were smaller than those against ciprofloxacin-resistant isolates. BMS-284756 was again the most potent compound, with an MIC range of <=0.015– 0.06 mg/L.


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Table. Amino acid changes within GrlA, GrlB and GyrA and corresponding MICs in 116 genetically characterized S. aureus isolates
 
Isolates without the Ser-80->Phe alteration in GrlAwere ciprofloxacin susceptible and had a BMS-284756 MIC of <=0.06 mg/L, a moxifloxacin MIC of <=0.12 mg/L and a gatifloxacin MIC of <=0.5 mg/L (TableGo). This alteration in GrlA (Ser-80->Phe), in combination with either a Ser-84->Leu or a Glu-88->Lys alteration within GyrA, was detected in all ciprofloxacin-resistant isolates. The range of MIC values for isolates with these mutations was 0.06–0.5 mg/L of BMS-284756, 0.5–2 mg/L of moxifloxacin and 1–8 mg/L of gatifloxacin. In summary, ciprofloxacin, followed by gatifloxacin and moxifloxacin, were the least potent compounds and BMS-284756 the most potent in response to the characterized mutations in grlA, grlB and gyrA. This included the key mutations within the QRDR at codons 80 and 84 in grlA, as well as 84 and 88 in gyrA (TableGo). However, the comparative activities of the 8-methoxy quinolones versus BMS-284756 remained similar with regard to mutations in gyrA and grlA. In this respect, BMS-284756 seemed to be affected by the same mutations that conferred higher MICs to the C-8 methoxy fluoroquinolones moxifloxacin and gatifloxacin. In contrast, ciprofloxacin was more affected by specific mutations within the QRDR (TableGo).

We recently reported the emergence of hetero-VISA 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, 4, 2 and 0.5 mg/L of ciprofloxacin, gatifloxacin, moxifloxacin and BMS-284756, respectively. Thus, BMS-284756 is also very active against the hetero-VISA emerging in the Düsseldorf area.

We also analysed the range and stability in three clonal populations of MRSA described previously of the MIC values of the four quinolones tested.5 In total, MIC values of the four quinolones were determined for 125 MRSA isolates, comprised of three different PFGE types. Over a 3 year period we observed conserved mutations within the grl, gyr and norA gene loci, and concomitant conserved MIC values within MRSA isolates belonging to one PFGE type and originating from quite diverse clinical environments.5 We also investigated the stability of BMS-284756 MIC values in these MRSA populations. The ranges of MIC values of gatifloxacin, moxifloxacin and BMS-284756 were 1–4, 0.5–1 and 0.125–0.25 mg/L, respectively, in the three ciprofloxacin-resistant MRSA populations comprised of three different PFGE types.

In previous studies,6,9 efflux pump activity in S. aureus was inhibited by the plant alkaloid reserpine. The presence of reserpine 20 mg/L, which does not affect the growth of the isolates, did not result in a decrease in BMS-284756 MICs for any of the isolates tested. There is clearly considerable variation in the susceptibility of S. aureus to BMS-284756 as shown by the up to eight-fold difference in MICs recorded for isolates with a homologous QRDR. This range remains the same in the presence of reserpine. Thus, BMS-284756 does not seem to be a substrate for efflux pump activity via NorA.

In summary, all strains tested (116 unrelated S. aureus, seven hetero-VISA strains and 125 clonally related MRSA) were susceptible to BMS-284756 based on an investigational breakpoint of <=1 mg/L. BMS-284756 is not a substrate for efflux pump activity via NorA. Compared with the other fluoroquinolones, BMS-284756 showed significantly increased in vitro activity against S. aureus isolates and might be a useful adjunct in anti-staphylococcal therapy.


    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
 Materials and methods
 Results and discussion
 References
 
1 . Fung-Tomc, J. C., Minassian, B., Kolek, B., Huczko, E., Aleksunes, L., Stickle, T. et al. (2000). Antibacterial spectrum of a novel des-fluoro(6) quinolone, BMS-284756. Antimicrobial Agents and Chemotherapy 44, 3351–6.[Abstract/Free Full Text]

2 . Takahata, M., Mitsuyama, J, Yamashiro, Y., Yonezawa, M., Araki, H., Todo, Y. et al. (1999). In vitro and in vivo antimicrobial activities of T-3811ME, a novel des-F(6)-quinolone. Antimicrobial Agents and Chemotherapy 43, 1077–83.[Abstract/Free Full Text]

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, U. 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. C., 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 and Microbiological Infection 5, 287–90.

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. Methods for Dilution Antimicrobial Susceptibility Tests for Bacteria That Grow Aerobically—Fifth Edition: Approved Standard M7-A5. NCCLS, Wayne, PA.

9 . Schmitz, F.-J., Hertel, B., Hofmann, B., Scheuring, S., Verhoef, J., Fluit, A. et al. (1998). Correlation of norfloxacin MIC values with mutations within the coding and promotor region of the norA gene in 42 unrelated clinical isolates of Staphylococcus aureus. Journal of Antimicrobial Chemotherapy 42, 561–3.[Free Full Text]

Received 22 June 2001; returned 14 September 2001; revised 5 October 2001; accepted 11 October 2001