Comparative in vitro activity of ABT-773 and two macrolides against staphylococci

Christof von Eiff,* and Georg Peters

Institute of Medical Microbiology, University of Münster Hospital and Clinics, 48149 Münster, Germany


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
The anti-staphylococcal activity of ABT-773, a new member of the ketolides, was examined and compared with those of erythromycin and clarithromycin using the agar dilution method. A total of 336 well-defined clinical isolates of staphylococci, including a large number of clonally different methicillin-resistant strains with defined macrolide-lincosamide-streptogramin B (MLSB) resistance phenotypes were tested. The novel ketolide exhibited broad-spectrum anti-staphylococcal activity against both methicillin-susceptible and methicillin-resistant strains. Apart from strains with constitutive expression of MLSB resistance, all staphylococcal strains tested were inhibited by <=0.063 mg/L of ABT-773, irrespective of the resistance phenotype for methicillin.


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
A renewed interest in staphylococcal infections has emerged in recent years, since staphylococci resistant to multiple antibiotics have been reported with increasing frequency worldwide. Depending on local epidemiological conditions, a significant number of isolates are resistant to methicillin, lincosamides, macrolides, aminoglycosides and/or fluoroquinolones.1–3 Because glycopeptides are the major drugs with reliable activity against methicillin-resistant strains of Staphylococcus aureus (MRSA), the emergence of S. aureus strains with intermediate resistance to glycopeptides has heightened the fears of pan antibioticresistant strains.4 Thus, alternatives to the available antimicrobials are needed, not only for the treatment of infections due to multiply resistant strains, but also to reduce the increasing selection pressure on Gram-positive pathogens in hospitals.

The ketolides are a new class of macrolide antibiotics with a keto group replacing the L-cladinose moiety in position 3 and an alkyl-aryl extension at positions 11 and 12 of the lactone ring. The ketolides have activity against a broad range of pathogens, particularly against all major respiratory pathogens, including Streptococcus pneumoniae, Haemophilus influenzae and Moraxella catarrhalis.5,6 However, data on the activity of ABT-773 against staphylococci are limited.

The aims of this study were: (i) to evaluate the activity of ABT-773 against a large number of different and well-characterized staphylococcal species, particularly against clonally different methicillin-resistant strains isolated from several geographical locations in Germany, including strains expressing different macrolide-lincosamide-streptogramin B (MLSB) resistance phenotypes; and (ii) to compare the in vitro anti-staphylococcal activity of this novel ketolide with the macrolides erythromycin and clarithromycin.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
A total of 336 staphylococcal strains freshly isolated from clinical specimens were tested. The strains were isolated from blood or from other specimens, for example from foreign bodies, and were only included if they were considered aetiologically relevant. A large number of isolates were collected during the course of two multicentre studies, comprising 21 and 32 university and community hospitals in Germany.7,8 Only one isolate per patient was tested.

As MRSA may cause outbreaks, we used several other criteria to avoid including multiple isolates of the same strain: first, isolates were collected over a period of years, which would make a single clone unlikely. Secondly, isolates were collected from different geographical locations in Germany, which would also reduce the chance of obtaining a single clone, and finally, when S. aureus isolates with similar antibiograms and phenotypes were obtained, we carried out pulsed-field gel electrophoresis (PFGE) and selected only one example of each strain.

The 179 S. aureus strains included 32 penicillinsusceptible S. aureus (PSSA), 63 methicillin-susceptible S. aureus (MSSA) and 84 MRSA. The 157 coagulasenegative staphylococci (CoNS) comprised 41 methicillin-susceptible and 26 methicillin-resistant Staphylococcus epidermidis strains, 23 methicillin-susceptible and 32 methicillin-resistant Staphylococcus haemolyticus strains, and 35 other CoNS belonging to the following species: seven Staphylococcus hominis, six Staphylococcus saprophyticus, six Staphylococcus warneri, four Staphylococcus cohnii, three Staphylococcus lugdunensis, three Staphylococcus simulans, two Staphylococcus xylosus, two Staphylococcus sciuri, one Staphylococcus capitis and one Staphylococcus hyicus.

The staphylococci were identified from a variety of conventional phenotypic characteristics and by using the API-Staph system (ATB32 Staph; bioMérieux, Marcy l’Étoile, France). To test for inducible and constitutive expression of macrolide-lincosamide (ML) resistance, the disc diffusion test was carried out as defined classically using erythromycin (15 µg) and clindamycin (10 µg) discs (Oxoid Ltd, Basingstoke, UK) without delineating subdivisions.3 For this purpose, an aqueous suspension of bacterial growth from blood agar was adjusted to McFarland 0.5, and inoculated by swab on Mueller–Hinton agar (Difco, Augsburg, Germany). The arrangement of the discs allowed both the susceptibility pattern to individual compounds and interactions between both antibiotics to be observed with a minimal amount of repetition. As described previously, zones were interpreted according to their size and shape as indicating susceptible or resistant, the latter being inducible if a ‘D’-shaped zone was observed (the compound on the left being the inducer).3 Isolates were confirmed to be methicillin resistant by supplementation of the agar with 2% NaCl (using 5 µg oxacillin discs results were read after incubation for 48 h at 30°C) and by detection of the mecA gene in strains with non-definable resistance phenotype as reported previously.9

The MICs were determined on Mueller–Hinton agar, using the agar dilution technique with a final inoculum of c. 104 per spot. The following antimicrobial agents were used and obtained from their respective manufacturers: ABT-773, erythromycin, clarithromycin, quinupristin–dalfopristin. With the exception of quinupristin–dalfopristin, the compounds were tested in 13 different concentrations ranging from 0.031 to 128 mg/L. Quinupristin–dalfopristin was tested in eight different concentrations ranging from 0.06 to 8 mg/L. The results were read after 18 h incubation at 36°C. The following reference strains were included as controls: S. aureus ATCC 25923, ATCC 29213, ATCC 43300; Enterococcus faecium ATCC 51599; Escherichia coli ATCC 35218; Pseudomonas aeruginosa ATCC 27853. Additionally, sterility and growth controls were always carried out.


    Results
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
The MIC ranges, MIC50S and MIC90S of the 179 strains of S. aureus, including 84 methicillin-resistant strains are shown in Table 1Go. ABT-773 was highly active against all clindamycin-susceptible S. aureus strains, including 53 strains with MLSB inducible resistance phenotype. Except for the strains with constitutive MLSB expression, all S. aureus strains tested were inhibited by <=0.031 mg/L of ABT-773. The activity of this novel ketolide remained fully unchanged, irrespective of the resistance phenotype for methicillin. Thus, 48 clonally unrelated methicillin-resistant strains were inhibited by ABT-773 at the same concentration as the 91 penicillin- and methicillin-susceptible strains. Comparing the activity of both macrolides, clarithromycin was two- to four-fold more active than erythromycin. While quinupristin–dalfopristin MICs were slightly elevated for strains with constitutive MLSB phenotype (MIC90, 2 mg/L versus MIC90S for all other strains, <=1 mg/L), all other agents tested showed no activity against strains with constitutive MLSB phenotype, with MICs >=128 mg/L.


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Table 1. Activity of ABT-773 and two macrolides against S. aureus strains
 
The MIC ranges, MIC50S and MIC90S for the 67 S. epidermidis strains, the 55 S. haemolyticus strains and for 35 CoNS belonging to 10 other species are summarized in Table 2Go. Again, ABT-773 was the most active antimicrobial agent tested against all CoNS including 58 methicillin-resistant strains belonging to the species S. epidermidis and S. haemolyticus. All clindamycin-susceptible strains, as well as 29 CoNS with inducible MLSB phenotype, were susceptible to ABT-773 and were inhibited by a concentration of <=0.063 mg/L. As for S. aureus, the activity of ABT-773 remained fully unchanged, irrespective of the resistance phenotype for methicillin. However, all CoNS with constitutive MLSB expression were resistant with MICs > 128 mg/L. Among the macrolides, clarithromycin revealed higher in vitro activity compared with erythromycin, with clarithromycin being slightly more active against S. haemolyticus strains. All CoNS tested were inhibited by <=2 mg/L of quinupristin–dalfopristin (MIC90, 1 mg/L). The activity of ABT-773 remained unchanged irrespective of the MICs for the streptogramin tested. Quality control of all MIC determinations was carried out using the reference strains mentioned above. The MICs for these strains were within acceptable limits throughout testing. For example, for S. aureus ATCC 25923 the MICs (mg/L) were: quinupristin–dalfopristin, 1; erythromycin, 0.125; clarithromycin, <=0.031; ABT-773, <=0.031.


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Table 2. Activity of ABT-773 and two macrolides against CoNS
 

    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
The current empirical treatment of infections is challenged by the emerging resistance among the major pathogens. Over the last two decades, the incidence of infections particularly caused by multidrug-resistant Gram-positive bacteria has increased despite advances in antibacterial therapy. S. aureus as well as CoNS reveal a remarkable propensity for resistance to various antibiotics due to defined resistance mechanisms.10,11 Thus, there continues to be a need for highly active antimicrobial agents, above all for the therapy of infections due to multidrug-resistant staphylococci.

In this context, the ability of the ketolide ABT-773 to inhibit staphylococci with an inducible MLSB resistance phenotype may be of major clinical importance. All strains that were clindamycin susceptible or showed the inducible type of MLSB resistance were inhibited by <=0.063 mg/L of ABT-773, irrespective of the methicillin resistance phenotype.

In previous studies testing the activity of ABT-773 against staphylococci, multiple isolates of the same strain were not excluded by phenotypic or genomic typing. In addition, isolates were not classified previously regarding their MLSB phenotype.5,12,13 Thus, our results for methicillin-resistant strains differ from those of previous investigations in which methicillin-susceptible strains (MIC90S 0.03–0.06 mg/L) were generally found to be more susceptible to ABT-773 than methicillin-resistant strains (MIC90S >16–128 mg/L).5,13 In our study, we tested a large number of clonally unrelated strains. This is particularly important for MRSA, which often cause at least regional outbreaks with the same strain. Furthermore, a broad spectrum of different, well-defined staphylococcal species was included in the testing.

Overall, the newly developed agent ABT-773 was highly active against both methicillin-susceptible and resistant strains with inducible MLSB resistance, stimulating further evaluation of these agents for therapy of infections due to staphylococci. However, strains with constitutive type of MLSB resistance are not in the spectrum of this ketolide. Therefore, an accurate knowledge of the local susceptibility pattern will be necessary if ABT-773 is to be used as empirical therapy for infections thought to be caused by multi-resistant S. aureus.


    Acknowledgements
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
We sincerely thank S. Weber and M. Reilly for expert technical assistance. The study was supported by a grant from Abbott Laboratories, Abbott Park, IL, USA.


    Notes
 
* Corresponding author. Tel: +49-251-835-5360; Fax: +49-251-835-5350; E-mail: eiffc{at}uni-muenster.de Back


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
1 . Jones, R. N. (2001). Resistance patterns among nosocomial pathogens: trends over the past few years. Chest 119, 397S–404S. [Abstract/Free Full Text]

2 . Paradisi, F., Corti, G. & Messeri, D. (2001). Antistaphylococcal (MSSA, MRSA, MSSE, MRSE) antibiotics. Medical Clinics of North America 85, 1–17. [ISI][Medline]

3 . Hamilton-Miller, J. M. & Shah, S. (2000). Patterns of phenotypic resistance to the macrolide-lincosamide-ketolide-streptogramin group of antibiotics in staphylococci. Journal of Antimicrobial Chemotherapy 46, 941–9. [Abstract/Free Full Text]

4 . Fridkin, S. K. (2001). Vancomycin-intermediate and -resistant Staphylococcus aureus: what the infectious disease specialist needs to know. Clinical Infectious Diseases 32, 108–15. [ISI][Medline]

5 . Andrews, J. M., Weller, T. M., Ashby, J. P., Walker, R. M. & Wise, R. (2000). The in vitro activity of ABT773, a new ketolide antimicrobial agent. Journal of Antimicrobial Chemotherapy 46, 1017–22. [Abstract/Free Full Text]

6 . Barrett, J. F. & Dougherty, T. J. (2001). ABT-773: a new ketolide antibiotic. Expert Opinion on Investigational Drugs 10, 343–51. [ISI][Medline]

7 . Von Eiff, C., Reinert, R. R., Kresken, M., Brauers, J., Hafner, D. & Peters, G. for the Multicenter Study on Antibiotic Resistance in Staphylococci and other Gram-Positive Cocci Study (MARS) Group. (2000). Nationwide German multicenter study on prevalence of antibiotic resistance in staphylococcal bloodstream isolates and comparative in vitro activities of quinupristin–dalfopristin. Journal of Clinical Microbiology 38, 2819–23. [Abstract/Free Full Text]

8 . Von Eiff, C., Becker, K., Machka, K., Stammer, H. & Peters, G. for the Study Group. (2001). Nasal carriage as a source of Staphylococcus aureus bacteremia. New England Journal of Medicine 344, 11–6. [Abstract/Free Full Text]

9 . Murakami, K., Minamide, W., Wada, K., Nakamura, E., Teraoka, H. & Watanabe, S. (1991). Identification of methicillin-resistant strains of staphylococci by polymerase chain reaction. Journal of Clinical Microbiology 29, 2240–4. [ISI][Medline]

10 . Lowy, F. D. (1998). Staphylococcus aureus infections. New England Journal of Medicine 339, 520–32. [Free Full Text]

11 . Moellering, R. C., Jr (1998). Problems with antimicrobial resistance in Gram-positive cocci. Clinical Infectious Diseases 26, 1177–8. [ISI][Medline]

12 . Goldstein, E. J., Citron, D. M., Merriam, C. V., Warren, Y. & Tyrrell, K. (2000). Comparative in vitro activities of ABT-773 against aerobic and anaerobic pathogens isolated from skin and soft-tissue animal and human bite wound infections. Antimicrobial Agents and Chemotherapy 44, 2525–9. [Abstract/Free Full Text]

13 . Barry, A. L., Brown, S. D. & Fuchs, P. C. (2000). Antimicrobial activity of ABT-773. In Program and Abstracts of the Fifth International Conference on the Macrolides, Azalides, Streptogramins, Ketolides and Oxazolidinones, Seville, Spain, 2000. Abstract 2.12, p. 24. ICMAS, Inc., Atlanta, GA.

Received 28 March 2001; returned 20 July 2001; revised 28 August 2001; accepted 25 September 2001