Fosfomycin Synergy In Vitro with Amoxicillin, Daptomycin, and

Linezolid against Vancomycin-Resistant Enterococcus faecium from

Renal Transplant Patients with Infected Urinary Stents
Jillian L. Descourouez,b Margaret R. Jorgenson,b Justine E. Wergin,a Warren E. Rosea,b
Pharmacy Practice Division, University of Wisconsin School of Pharmacy, Madison, Wisconsin, USAa; Department of Pharmacy, University of Wisconsin Hospital and Clinics,
Madison, Wisconsin, USAb

Fosfomycin is a potential option for vancomycin-resistant enterococcus (VRE) infections despite limited in vitro and clinical
data. In this study, 32 VRE isolates from renal transplant patients with urinary stent infections were susceptible to fosfomycin,
daptomycin, and linezolid and resistant to amoxicillin, minocycline, and nitrofurantoin based on their MIC50s and MIC90s. Fos-
fomycin was bacteriostatic at 0.5 to 16ⴛ the MIC (32 to 2,048 ␮g/ml); synergy occurred when fosfomycin was combined with
daptomycin (2.8 to 3.9 log10 CFU/ml kill; P < 0.001) or amoxicillin (2.6 to 3.4; P < 0.05). These combinations may be potent op-
tions to treat VRE urinary infections pending investigation of clinical efficacy.

S olid-organ transplant recipients are at increased risk for colo-

nization with vancomycin-resistant enterococcus (VRE) and
vulnerable to active infections with this organism (1). At our in-
Antibiotic activities were determined in calcium-adjusted Mu-
eller-Hinton broth (MHB) appropriate for the antibiotics tested
(5). For assays involving fosfomycin, MHB was also supplemented
stitution, a 7-French, 16-cm double-J stent is inserted through the with 25 ␮g/ml glucose-6 phosphate. All biofilm assays were eval-
ureter into the renal pelvis and then into the bladder at the time of uated in tryptic soy broth plus 1% dextrose and supplemented
kidney transplantation. Biofilm development often precipitates with the appropriate requirements for selected antibiotics (6).
colonization of the stents and results in sequestered, stationary- MICs and minimum bactericidal concentrations (MBCs)
phase bacteria that further resist the effects of antibiotics. against planktonic cultures were determined by broth microdilu-
Fosfomycin, a phosphonic acid derivative initially isolated in tion (5). The MIC and minimum biofilm eradication concentra-
1969 from cultures of Streptomyces species, is an oral therapy for tion (MBEC) were determined using a transferable solid-phase

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uncomplicated urinary tract infections (UTIs) (2). It is often bac- pin lid (Nunc) as described by Ceri et al. (7).
tericidal against multidrug-resistant Gram-positive and Gram- Antibiotic activity against four of the clinical isolates as well as
negative pathogens, although it has not been well studied against ATCC 29212 was analyzed in a 24-h time-kill curve in duplicate
VRE. The antibacterial activity of fosfomycin is achieved by inhib- with an inoculum of 5 ⫻ 105 CFU/ml. The standard kill curve
iting the enzyme N-acetylglucosamine (UDP-GlcNAc) enolpy- method for synergy (8) utilized variable concentrations of the pri-
ruvyl transferase (MurA), which synthesizes UDP-N-acetylenol- mary antibiotic (fosfomycin, 0.5 to 16⫻ the MIC) in combination
pyruvylglucosamine, an essential component in the biosynthesis with a static subinhibitory concentration (0.5⫻ the MIC) of the
of peptidoglycan (2). It is highly concentrated in the urine, with secondary antibiotic. Synergy, additive effect, antagonism, and
peak values of 1,053 to 4,415 ␮g/ml within 4 h after a single oral indifference were defined as ⱖ2 log kill, ⬍2 but ⬎1 log kill, ⬎1 log
3-g dose (2). Fosfomycin has gained recent interest as a potential growth, and ⫾1 log kill, respectively (8). Bactericidal activity was
therapeutic option for treating infections caused by VRE despite defined as 99.9% kill from the initial inoculum.
limited efficacy data (3). Clinical VRE isolates were susceptible to fosfomycin, dapto-
The aim of this study was to investigate fosfomycin activity in mycin, and linezolid and resistant to amoxicillin, minocycline,
vitro alone and in combination with other antibiotic treatment and nitrofurantoin based on MIC50 and MIC90 results (Table 1).
options against VRE urine isolates collected from renal transplant Linezolid and daptomycin were the most active antibiotics, with
patients with urinary stent infections. We present synergistic com- MIC90s of 2 and 4 ␮g/ml, respectively. Only linezolid maintained
binations with fosfomycin that may be useful treatment options the same MIC profile when tested against biofilm cultures, while
for these situations. fosfomycin and daptomycin had at least a 2-fold increase in the
(Portions of this work were presented at the 51st Interscience MIC90 in biofilm (Table 1).
Conference on Antimicrobial Agents and Chemotherapy, Chi- Fosfomycin was bacteriostatic against VRE in the kill curve at
cago, IL, 2011 [4].)
Thirty-two VRE (Enterococcus faecium) isolates were collected
from renal transplant patients with urinary stent infections from Received 12 October 2012 Returned for modification 1 December 2012
2007 to 2010 at a tertiary medical center. Enterococcus faecalis Accepted 16 December 2012
ATCC 29212 was used as a control strain. The antibiotics evalu- Published ahead of print 21 December 2012
ated were amoxicillin, fosfomycin, nitrofurantoin, and minocy- Address correspondence to Warren E. Rose, [email protected].
cline, purchased from Sigma-Aldrich (St. Louis, MO), as well as Copyright © 2013, American Society for Microbiology. All Rights Reserved.
linezolid (Pfizer, NY) and daptomycin (Cubist, Lexington, MA), doi:10.1128/AAC.02099-12
which were commercially purchased.

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 Antibiotic Synergy with Fosfomycin against VRE

TABLE 1 Susceptibility results of Enterococcus faecium isolatesa was the most active combination against VRE and resulted in bac-
Susceptibility (␮g/ml)b terial kill at or close to the detection limit. Based on the ability of
fosfomycin to inhibit essential enzymes in the biosynthesis of pep-
Planktonic cultures Biofilm cultures
tidoglycan (11), we hypothesize that this synergistic effect is
MBC MBEC largely due to fosfomycin increasing the sensitivity of the bacterial
Antibiotic MIC50 MIC90 range MIC50 MIC90 range cell envelope to daptomycin.
Amoxicillin 64 128 16–1,024 64 128 64–1,024 Aminopenicillins are key agents used in the treatment of En-
Daptomycin 2 4 1–32 2 8 1–32 terococcus sp. infections, although the activities of ampicillin and
Fosfomycin 64 64 128 64 128 256–512 amoxicillin alone against VRE are poor (9). Fosfomycin modifies
Linezolid 2 2 16–32 2 2 16–32 the production of penicillin binding proteins (PBPs) in Staphylo-
Minocycline 16 32 4–32 8 32 4–256 coccus aureus and Streptococcus pneumoniae (12). This suggests
Nitrofurantoin 64 128 32–1,024 64 128 512–1,024 that the combination of fosfomycin and amoxicillin has potential
a
Susceptibility results in planktonic and biofilm cultures of 32 vancomycin-resistant to overcome beta-lactam resistance caused by PBPs (12). In our
Enterococcus faecium isolates collected from renal transplant patients with urinary stent
infections.
study, synergy was observed for fosfomycin in combination with
b
MBC, minimum bactericidal concentration; MBEC, minimum biofilm eradication amoxicillin despite amoxicillin resistance in VRE. This combina-
concentration. tion may be a potent and sought-after oral option to treat VRE
urinary infections, since both agents achieve high urine concen-
trations.
concentrations of 0.5 to 16⫻ the MIC. Concentrations of 0.5 to Linezolid has in vitro activity against VRE, with susceptibility
4⫻ the MIC resulted in 0.2 to 1.6 log10 CFU/ml kill at 8 h followed rates over 99% (13). The isolates in our study displayed similar
by regrowth. Fosfomycin concentrations of 16⫻ the MIC were the susceptibility rates (97%), and importantly linezolid susceptibility
most active, with 0.9 to 2.9 log10 CFU/ml maximum 24-h kill. in biofilm was maintained (MIC, ⱕ2 ␮g/ml). Fosfomycin com-
Fosfomycin was bactericidal and considerably more active against bined with linezolid was either synergistic or additive in the kill
vancomycin-susceptible E. faecalis ATCC 29212 (maximum kill, curve. One previous study with fosfomycin and linezolid suggests
3.4 to 3.5 log10 CFU/ml; P ⬍ 0.001). potential for this combination therapy when used against Staph-
The addition of daptomycin, amoxicillin, or linezolid to fosfo- ylococcus epidermis and Staphylococcus aureus (11). This combina-
mycin in the kill curve increased bacterial killing (Fig. 1, each tion could be an effective oral option for the treatment of VRE
antibiotic at 0.5⫻ the MIC), while combination with nitrofuran- UTIs, including those with biofilm development on urinary
toin or minocycline had no further antimicrobial effects, with ⫾1 stents.
log difference in kill compared to any agent alone (data not The treatment of UTIs caused by VRE in renal transplant pa-
shown). The most potent and highly synergistic combination was tients is often difficult to manage due to host immunosuppres-

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fosfomycin plus daptomycin, which often achieved bactericidal sion, hardware placement, and lack of optimally studied antibiotic
activity against clinical strains (maximum kill, 2.8 to 3.9 log10 options (14). A recent case series of fosfomycin outcomes in com-
CFU/ml; P ⬍ 0.001 versus either agent alone). Fosfomycin plus plex urinary tract infections due to multidrug-resistant organ-
amoxicillin was also synergistic (2.6 to 3.4 log10 CFU/ml kill; P ⬍ isms, including VRE, reported that urinary stents were associated
0.05) but was bactericidal in only half of the clinical strains tested. with microbiologic failure in fosfomycin monotherapy (3). In or-
Fosfomycin in combination with linezolid in the kill curve was der to effectively treat such infections, early hardware removal and
bacteriostatic and produced a synergistic or additive effect. No complex antimicrobial therapy are often required (9). Based on
antagonism was detected.
The use of fosfomycin in the treatment of VRE has recently
gained renewed interest although few in vitro or clinical data sup-
port its use. Of the VRE isolates in our study, 91% were susceptible
(MIC, ⱕ64 ␮g/ml) to fosfomycin (5), consistent with the limited
prior reports of fosfomycin susceptibility in VRE (3, 9). Fosfomy-
cin exhibited a concentration-dependent effect in the standard kill
curve. The antibacterial activity of fosfomycin increased from low
to high concentrations but still remained bacteriostatic up to 16⫻
the MIC. Although fosfomycin concentrations are high at this
exposure (1,024 to 2,048 ␮g/ml), they represent concentrations
comparable to those achieved in the urine of patients after a single
3-g oral dose (9). Interestingly, low exposures of fosfomycin (0.5⫻
the MIC) in combination with daptomycin, amoxicillin, or lin-
ezolid produced greater bacterial killing than high concentrations
of fosfomycin alone (1 to 16⫻ the MIC) and bacterial killing sim-
ilar to that induced by high fosfomycin exposures in combination
with 0.5⫻ the MICs of secondary antibiotics. FIG 1 Change in log10 CFU/ml after 24-h antibiotic exposures in the kill
Synergy studies with fosfomycin and daptomycin are limited, curve. Data represent antibiotics evaluated alone or in combination at 0.5⫻
the MIC. Fosfomycin (FOS) combined with either daptomycin (DAP) or
but one case report describes successful treatment with this com- amoxicillin (AMO) was synergistic and often bactericidal (ⱖ3 log kill), while
bination for S. aureus endocarditis caused by a daptomycin-non- FOS combined with linezolid (LIN) was either synergistic or additive yet bac-
susceptible strain (10). In our study, fosfomycin plus daptomycin teriostatic.

March 2013 Volume 57 Number 3 aac.asm.org 1519

Descourouez et al.

our findings, combination therapy with fosfomycin and either vancomycin alone and in combination with tigecycline and rifampicin
daptomycin or amoxicillin is highly synergistic and results in en- against Staphylococcus aureus. J. Antimicrob. Chemother. 63:485– 488.
7. Ceri H, Olson ME, Stremick C, Read RR, Morck D, Buret A. 1999. The
hanced bactericidal activity compared to fosfomycin alone against
Calgary Biofilm Device: new technology for rapid determination of anti-
VRE. Additional modeling of these two combinations using phar- biotic susceptibilities of bacterial biofilms. J. Clin. Microbiol. 37:1771–
macokinetic concentrations achieved in urine will help to define 1776.
their potential utilities. These combinations should be further ex- 8. Chin JN, Jones RN, Sader HS, Savage PB, Rybak MJ. 2008. Potential
plored for clinical efficacy. synergy activity of the novel ceragenin, CSA-13, against clinical isolates of
Pseudomonas aeruginosa, including multidrug-resistant P. aeruginosa. J.
ACKNOWLEDGMENT Antimicrob. Chemother. 61:365–370.
9. Heintz BH, Halilovic J, Christensen CL. 2010. Vancomycin-resistant
This work was supported by the University of Wisconsin Hospital and enterococcal urinary tract infections. Pharmacotherapy 30:1136 –1149.
Clinics. 10. Chen LY, Huang CH, Kuo SC, Hsiao CY, Lin ML, Wang FD, Fung CP.
2011. High-dose daptomycin and fosfomycin treatment of a patient with
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