Journal of Antimicrobial Chemotherapy (1999) 43, 211-217
© 1999 The British Society for Antimicrobial Chemotherapy
In-vitro activity of fosfomycin against vancomycin-resistant enterococci
a Institute of Hygiene, Fritz Pregl Strasse 3, A-6020 Innsbruck, Austria; b Robert Koch Institute, Burgstrasse 37, D-38855 Wernigerode, Germany
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Abstract |
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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 |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
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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.
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Materials and methods |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
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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 
-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)
-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
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Results |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
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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 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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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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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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Discussion |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
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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
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Acknowledgments |
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We thank Mrs D. BadstüCbner and Mrs C. Konstabel for accurate technical assistance and Mrs D. Eitze for secretarial support.
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Notes |
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* Corresponding author. Tel: +43-512-583391; Fax: +43-512-574414; E-mail: franz.allerberger{at}uibk.ac.at
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Received 26 January 1998; returned 17 March 1998; revised 6 April 1998; accepted 6 May 1998
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