In-vitro activity of fosfomycin against vancomycin-resistant enterococci

Franz Allerbergera,* and Ingo Klareb

a Institute of Hygiene, Fritz Pregl Strasse 3, A-6020 Innsbruck, Austria; b Robert Koch Institute, Burgstrasse 37, D-38855 Wernigerode, Germany


    Abstract
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The effect of fosfomycin against 69 vancomycin-resistant isolates of Enterococcus faecium (VanA), five of E. faecium (VanB), 11 of Enterococcus faecalis (VanA), three of E. faecalis (VanB), 10 of Enterococcus gallinarum (VanC1 ) and two of Enterococcus casseliflavus (VanC2) and glycopeptide-sensitive E. faecium (n = 8) and E. faecalis (n = 10) was tested in vitro. Fosfomycin inhibited 97%, 94% and 96% of the vancomycin-resistant strains, according to results of agar dilution, broth microdilution, and a disc diffusion method (DIN 58940). The disc diffusion test by the NCCLS method does not include fosfomycin; using breakpoints suggested by Andrews et al. ( <=11 mm, resistant; >= 18 mm, susceptible), 5% of the vancomycin-resistant strains tested would have been considered fosfomycin resistant. Minimal inhibitory concentrations of most vancomycin-resistant isolates were in the intermediate sensitivity range, yielding an MIC50 of 32 mg/L and an MIC90 of 64 mg/L. Moreover the majority of inhibitory zone sizes by the disc diffusion method (DIN 58940) corresponded to intermediate susceptibility. These results suggest that fosfomycin at a high dosage and possibly used in combination with other drugs could be a potentially useful drug for the treatment of infections caused by vancomycin-resistant enterococci.


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Fosfomycin is a small molecule that inhibits the first step in peptidoglycan (cell wall) synthesis by acting as an analogue of phosphoenolpyruvate. This original antimicrobial agent, with a serum half-life of approximately 1.75 h, exhibits a broad spectrum of antimicrobial activity. 1 Fosfomycin is widely used in Europe, Japan and Latin America; in the USA (where it was developed), it never reached the market. It was the aim of this study to evaluate the in-vitro activity of fosfomycin against vancomycin-resistant (VR) enterococci. VR enterococci, first reported in Europe in 1988, 2 are emerging as a global threat to public health. Alternatives to vancomycin are few due to the multiple drug resistance typical of VR Enterococcus faecium. To our knowledge, no data on the in-vitro activity of fosfomycin against VR enterococci have been published.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Bacterial strains

One hundred and eighteen enterococcal strains (with and without different types of resistance to glycopeptides) of the following species were investigated for their in-vitro susceptibility to fosfomycin: E. faecium (VanA, n = 69; VanB, n = 5; glycopeptide-sensitive, n = 8), Enterococcus faecalis (VanA, n = 11; VanB, n = 3; glycopeptide sensitive, n = 10), Enterococcus gallinarum (VanC1, n = 10), and Enterococcus casseliflavus (VanC2n = 2). Isolates originated from the strain collection of the Robert Koch Institute, Wernigerode, Germany.

Susceptibility testing

Minimal inhibitory concentrations (MICs) of fosfomycin were determined by agar dilution test (inoculum 5 x 104 cfu/spot)3 and broth microdilution test (inoculum 5 x 105 cells/mL).4 In all susceptibility test methods, Mueller–Hinton (MH) agar or broth were used as nutrient media (Oxoid, Basingstoke, UK). As recommended by the producer of fosfomycin (Sigma Chemical Co., St Louis, MO, USA), the liquid and the solid MH media were supple?mented with {alpha}-D-glucose-6-phosphate (Sigma; final concentration 25 mg/L). An MIC of >= 128 mg/L characterizes a strain as resistant, 32– 64 mg/L as intermediate, and <=16 mg/L as susceptible. 5,6 Isolates were also tested by the disc diffusion test (according to the NCCLS method 7 with confluent bacterial growth on the agar plates and according to the DIN 58940 technique 8 with a lighter inoculum resulting in a semiconfluent growth). For preparing the inocula the same cultures of the corresponding strains in nutrient broth were used in all four susceptibility tests. Oxoid discs loaded with fosfomycin (50 µg) {alpha}-D-glucose-6-phosphate (50 µg) were used for disc susceptibility testing. Andrews et al. 6 proposed breakpoints for fosfomycin (corresponding to intravenous administration of 2 or 4 g), of 16 and 64 mg/L, corresponding to zone sizes (NCCLS method) of <=11 and >=18 mm, respectively. No official NCCLS zone sizes exist for fosfomycin. According to DIN 58940, zone diameters of <=13 mm are read as resistant, and those of >=20 mm as susceptible. 5 The recommended daily dosage of fosfomycin for severely ill patients is 8–16 g.9


    Results
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Summarized results of the agar dilution test are shown in Table I. By agar dilution, fosfomycin inhibited 97% of the VR strains and all vancomycin-susceptible (VS) isolates at concentrations <=64 mg/L. The fosfomycin MICs for the four resistant E. faecium isolates were >=256 mg/L.


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Table I. In-vitro susceptibility of enterococci to fosfomycin according to agar dilution test (>=128 mg/L, resistant; 32-64 mg/L, intermediately susceptible; <=16 mg/L, susceptible)
 
Table IIpresents summarized results of the broth microdilution test on the same 118 enterococcal isolates. By broth microdilution, fosfomycin inhibited 94% of the VR strains at concentrations <=64 mg/L. The four VR E. faecium isolates showing fosfomycin resistance by agar dilution yielded identical results from broth microdilution (fosfomycin MICs >=256 mg/L). Three further VR E. faecium isolates showed resistance against fosfomycin when tested by broth microdilution test (fosfomycin MICs 128 mg/L) but not by agar dilution (MICs for two strains 64 mg/L and one strain 32 mg/L).


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Table II. In-vitro susceptibility of enterococci to fosfomycin according to the broth microdilution test (>=128 mg/L, resistant; 32-64 mg/L, intermediately susceptible; <=16 mg/L, susceptible)
 
Summarized results from the disc diffusion test according to the DIN 58940 technique are presented in Table III. By this method, fosfomycin inhibited 96% of the VR strains (zone diameter >=14 mm). The four VR E. faecium isolates showing fosfomycin resistance by agar dilution were also resistant by disc diffusion (DIN 58940): three strains gave inhibition zones of <=6 mm diameter and one had a zone of 8 mm. One E. faecium isolate possessed an inhibition zone of 13 mm (5 resistant) despite having fosfomycin MICs of 64 mg/L by the two dilution methods.


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Table III. In-vitro susceptibility (zone diameter) of enterococci to fosfomycin according to the DIN 58940 disc diffusion test (<=13 mm, resistant; >=20 mm, susceptible)
 
These three test methods showed good correlation for recognition of fosfomycin resistance. Major discrepancies were found between the two dilution methods and the disc diffusion (DIN 58940) method in discriminating fully susceptible from intermediately susceptible strains. Twenty of 100 VR enterococci (four of 18 VS) were determined to be fully susceptible to fosfomycin by the agar dilution method; 17 of 100 VR enterococci (seven of 18 VS) were found to be fully susceptible by the broth microdilution method. Disc diffusion (DIN 58940) found 68 of 100 VR enterococci (17 of 18 VS) to be fully susceptible to fosfomycin.

Inoculating plates for disc diffusion testing according to the NCCLS method, with the breakpoints stated by Andrews et al., 6 gave rates of resistance comparable with those of the other three methods (Table IV). Of 74 VR E. faecium isolates, 6 were found to be fosfomycin resistant. Finally, full resistance to fosfomycin could only be detected in VanA and VanB type E. faecium.


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Table IV. In-vitro susceptibility (zone diameter) of enterococci to fosfomycin according to the NCCLS disc diffusion test (there are no official NCCLS zone sizes for fosfomycin)
 

    Discussion
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Treating infections caused by VR enterococci presents major problems. Many of these strains are also highly resistant to aminoglycosides and other antimicrobial agents. Fortunately, most VR E. faecalis remain relatively susceptible to penicillin and ampicillin; thus standard principles of enterococcal infection therapy can be applied to these organisms. 10

Vancomycin-resistant E. faecium are often highly resistant to ß-lactams and combinations of other antimicrobials. For infections due to strains for which MICs of ampicillin are <=64 mg/L, high-dose ampicillin may be tried, in combination with an aminoglycoside if there is no high-level resistance to the corresponding aminoglycoside. 10

A variety of other regimens have been utilized but there are no published data on the effectiveness of these regimens in humans. Teicoplanin has been used in cases of infection due to VR enterococci exhibiting the VanB phenotype. The investigational drugs quinupristin/dalfopristin, oxazolidinone, everninomycin, a new glycopeptide (LY 333328), and several fluoroquinolones including trovafloxacin and clinafloxacin with activity against VR enterococci are presently undergoing clinical evaluation. 10

Fosfomycin inhibited 97, 94 and 96% of the VR strains tested in our study by means of agar dilution, broth microdilution, and disc diffusion by DIN 58940, respectively. Using zone size diameters suggested by Andrews et al., 6 the disc diffusion test (according to the NCCLS method) gave susceptibility results concordant with those of the three other methods tested here. NCCLS criteria do not exist for disc diffusion testing of fosfomycin.

Recently Barry et al. 11 and Fuchs et al. 12 used NCCLS methods for testing susceptibility to fosfomycin tromethamine, a drug to be used for single-dose treatment of uncomplicated lower urinary tract infections, but they abstained from defining breakpoints for enterococci.

Hamilton-Miller13 found that fosfomycin could be a useful addition to the limited number of antibiotics currently available that are effective against infections caused by problematic Gram-positive cocci but no detailed information on species results or glycopeptide susceptibility was given for the 20 enterococci tested in his study, 19 of which were found to be susceptible to fosfomycin (MIC range 8–128 mg/L, MIC50 22 mg/L, MIC90 50 mg/L).

Kayser 14tested 50 VS E. faecalis and 31 VS E. faecium isolates and concluded that with mode MICs of 64 mg/L this drug exhibits only very limited activity against these enterococci (MIC range 32–512 mg/L, MIC50 64 mg/L, MIC90 128 mg/L).

Fosfomycin may be especially useful for treatment of enterococcal infection in combination. For example, Caron et al. 15 reported that in an animal endocarditis study using a VR E. faecium strain (inhibited by fosfomycin plus ceftriaxone in vitro), most vegetations were sterilized. Landman & Quale 16 found that this combination showed bactericidal synergy against some VRE.

Screening for the ß-lactam with the strongest beneficial effect in combination with fosfomycin, Pestel et al. 17 showed that cefotaxime (at 64 mg/L) combined with fosfomycin (at <=64 mg/L) was bactericidal against six of ten bloodstream isolates of VS enterococci tested. Studying the in-vivo activity of the combination of daptomycin and fosfomycin against a VS E. faecalis strain in a relapse model of rat endocarditis, Rice et al. 18 in contrast found that there was no difference between the number of valves sterilized by daptomycin alone and by daptomycin plus fosfomycin, despite synergic bactericidal activity found in in-vitro time–kill studies. In-vitro synergy does not necessarily translate into greater therapeutic efficacy against clinical infection.

In conclusion, the MICs of fosfomycin for most VR isolates were in the intermediate range, yielding an MIC50 of 32 mg/L and an MIC90 of 64 mg/L. The majority of zone sizes corresponded to intermediate susceptibility. Fosfomycin as a single agent at a low dosage therefore seems unsuitable for the treatment of infections due to VR enterococci. Fosfomycin at a high dosage could have a potential for the treatment of infections due to VR enterococci, especially if used in combination with other drugs. However, in-vitro activity does not guarantee in-vivo success. Until more effective agents are available, cure of some deep-seated infections due to VR enterococci may even require surgical intervention.16


    Acknowledgments
 
We thank Mrs D. BadstüCbner and Mrs C. Konstabel for accurate technical assistance and Mrs D. Eitze for secretarial support.


    Notes
 
* Corresponding author. Tel: +43-512-583391; Fax: +43-512-574414; E-mail: franz.allerberger{at}uibk.ac.at Back


    References
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
1 . Fernandez Lastra, F. C., Marino, E. L., Dominguez-Gil, A., Tabernero, J. M., Gonzalez Lopez, A. & Yuste Chaves, M. (1983). The influence of uremia on the accessibility of phosphomycin into interstitial tissue fluid. European Journal of Clinical Pharmacology 25, 333–8.[ISI][Medline]

2 . Uttley, A. H. C., Collins, C. H., Naidoo, J. & George, R. C. (1988).Vancomycin-resistant enterococci.Lancet, i, 57–8.

3 . Deutsches Institut füCr Normung e.V. (1992). DIN 58940, Teil 6: Methoden der EmpfindlichkeitsprüCfung von bakteriellen Krankheitserregern (außer Mykobakterien) gegen Chemotherapeutika— Bestimmung der minimalen Hemmkonzentration nach der Agar-Dilutionsmethode. In DIN-Taschenbuch Medizinische Mikrobiologie und Immunologie, Normen und weitere Unterlagen. 2. Aufl., pp. 376–80. Beuth-Verlag, Berlin and Cologne.

4 . Deutsches Institut füCr Normung e.V. (1992). DIN 58940, Teil 8: Methoden der EmpfindlichkeitsprüCfung von bakteriellen Krankheitserregern (außer Mykobakterien) gegen Chemotherapeutika— Mikrodilution. In DIN-Taschenbuch Medizinische Mikrobiologie und Immunologie, Normen und weitere Unterlagen. 2. Aufl., pp. 381–4. Beuth-Verlag, Berlin and Cologne.

5 . Grimm, H. & Haag, R. EmpfindlichkeitsprüCfung mit Fosfomycin im Agardiffusionstest auf Mueller–Hinton-agar. Immunitiät und Infektion 10, 159–63.

6 . Andrews, J. M., Baquero, F., Beltran, J. M., Canton, E., Crokaert, F., Gobernado, M. et al. (1983).International collaborative study on standardization of bacterial sensitivity to fosfomycin. Journal of Antimicrobial Chemotherapy, 12, 357–61.[Abstract]

7 . National Committee for Clinical Laboratory Standards. (1997). Performance Standards for Antimicrobial Disc Susceptibility Tests—Sixth Edition: Approved Standard M2-A6. NCCLS, Villanova, PA.

8 . Deutsches Institut füCr Normung e.V. (1992). DIN 58940, Teil 3: Methoden der EmpfindlichkeitsprüCfung von bakteriellen Krankheitserregern (außer Mykobakterien) gegen Chemotherapeutika— Agar-Diffusionstest. In DIN-Taschenbuch Medizinische Mikrobiologie und Immunologie, Normen und weitere Unterlagen. 2. Aufl., pp.354–60. Beuth-Verlag, Berlin and Cologne.

9 . Anonymous (1998). In Vidal Arzneimittelverzeichnis Österreich 1998, pp. 678–9. OVP-Vidal Verlag, Innsbruck.

10 . Moellering, R. C. (1997). VRE: what therapeutic options do we have? In Abstracts of the Thirty-Seventh Interscience Conference on Antimicrobial Agents and Chemotherapy, Toronto, Canada, 1997, Abstract S-145, p. 399. American Society for Microbiology, Washington, DC.

11 . Barry, A. L., Pfaller, M. A., Fuchs, P. C., Tenover, F. C., Reller, L. B., Allen, S. D. et al.(1993).Interpretive criteria and quality control parameters for determining bacterial susceptibility to fosfomycin tromethamine.European Journal of Clinical Microbiology and Infectious Diseases 12, 352–6.[ISI][Medline]

12 . Fuchs, P. C., Barry, A. L. & Brown, S. D. (1997).Susceptibility testing quality control studies with fosfomycin tromethamine.European Journal of Clinical Microbiology and Infectious Diseases 16, 538–40.[ISI][Medline]

13 . Hamilton-Miller, J. M. (1992).In vitro activity of fosfomycin against `problem' Gram-positive cocci.Microbios 71, 95–103.[ISI][Medline]

14 . Kayser, H. (1987). Activity of fosfomycin against grampositive bacteria. In New Aspects for Treatment with Fosfomycin, (Guggenbichler, J. P., Ed.), pp. 9–18. Springer Verlag, Vienna and New York.

15 . Caron, F., Pestel, M., Gutmann, L., Humbert, G. & Lemeland, J. F. (1995). Synergy between ceftriaxone plus fosfomycin in the treatment of an experimental endocarditis due to vancomycin-susceptible or vancomycin-resistant Enterococcus faecium. In Abstracts of the Thirty-Fifth Interscience Conference on Antimicrobial Agents and Chemotherapy, San Francisco, CA, 1995, Abstract B 47, p. 34. American Society for Microbiology, Washington, DC.

16 . Landman, D. & Quale, J. M. (1997).Management of infections due to resistant enterococci: a review of therapeutic options.Journal of Antimicrobial Chemotherapy 40, 161–70.[Abstract]

17 . Pestel, M., Martin, E., Aucouturier, C., Lemeland, J.-F. & Caron, F. (1995).In vitro interactions between different ß-lactam antibiotics and fosfomycin against bloodstream isolates of enterococci.Antimicrobial Agents and Chemotherapy 39, 2341–4.[Abstract]

18 . Rice, L. B., Eliopoulos, C. T., Yao, J. D., Eliopoulos, G. M., Moellering, R. C. (1992).In vivo activity of the combination of daptomycin and fosfomycin compared with daptomycin alone against a strain of Enterococcus faecalis with high-level gentamicin resistance in the rat endocarditis model.Diagnostic Microbiology and Infectious Disease 15, 173–6.[ISI][Medline]

Received 26 January 1998; returned 17 March 1998; revised 6 April 1998; accepted 6 May 1998