In vitro activity of fosfomycin alone and in combination against Staphylococcus aureus with reduced susceptibility or resistance to methicillin, vancomycin, daptomycin or linezolid

Abstract

Objectives

To evaluate the activity of fosfomycin against a group of MRSA strains, including isolates with reduced susceptibility or resistance to vancomycin, daptomycin, linezolid and ceftaroline and to determine the effect of combining various combinations of antimicrobial agents used in the therapy of serious Gram-positive infections.

Methods

Broth microdilution testing was used to determine the MICs of fosfomycin, vancomycin, daptomycin, linezolid, ceftaroline and cefazolin. Isolates were selected for further evaluations to determine in vitro synergy between fosfomycin and select antimicrobial agents using chequerboard broth microdilution testing. Fosfomycin was tested in combination with vancomycin, linezolid, daptomycin, ceftaroline and cefazolin.

Results

Fosfomycin maintained activity against 100% of strains of vancomycin-resistant Staphylococcus aureus (VRSA) and linezolid-resistant S. aureus (LRSA), 86% of VISA and 95% of daptomycin-resistant S. aureus (DRSA) strains. The combination of fosfomycin with ceftaroline consistently demonstrated synergy among all 18 isolates against the strains tested. The next most potent combination regimen was linezolid with fosfomycin, which demonstrated synergy in 16 of the 18 strains. Daptomycin demonstrated synergy in only 7 of the 18 strains tested when combined with fosfomycin. Cefazolin demonstrated synergy in 6 of 6 strains and vancomycin demonstrated no interaction in 6 of 6 strains tested.

Conclusions

Fosfomycin demonstrated excellent activity against MRSA as well as isolates with resistance or reduced activity to other anti-MRSA drugs including vancomycin, daptomycin and linezolid. When combined with linezolid or daptomycin, fosfomycin demonstrated synergy for all or most strains tested. Thus, these combinations may have potential clinical utility when treating patients with serious infections caused by MRSA.

Introduction

Fosfomycin is an antibacterial agent with bactericidal activity against Gram-negative and Gram-positive pathogens. It is used primarily for the treatment of urinary tract infections including those patients with resistant Gram-negative infections of the urinary tract including ESBL-producing and carbapenemase-producing enterobacteria. Fosfomycin has also been considered for the treatment of serious Gram-positive infections including MRSA. The IDSA guidelines for treatment of Staphylococcus aureus infections have suggested there is limited efficacy, along with concern for the emergence of resistance to current antibiotic therapy with either vancomycin or daptomycin.¹ Thus, the need for additional antimicrobial agents by themselves or in combination with other agents may provide helpful background in determining therapy for serious S. aureus infections.

The purpose of this study was to evaluate the activity of fosfomycin against a group of MRSA strains, including blood isolates and isolates with reduced susceptibility or resistance to vancomycin, daptomycin, linezolid and ceftaroline, and to determine the effect of combining various combinations of antimicrobial agents that may be considered in therapy for Gram-positive infections.

Materials and methods

A collection of S. aureus isolates was selected for evaluation. VISA (n = 31), vancomycin-resistant S. aureus (VRSA) (n = 15) and linezolid-resistant S. aureus (LRSA) (n = 4) isolates were obtained from the Network on Antimicrobial Resistance in Staphylococcus aureus (NARSA) programme, supported under NIAID/NIH contract HHSN272200700055C (ClinicalTrials.gov identifier: NCT00341913). NARSA is now supported by and known as BEI Resources. Two additional LRSA isolates were obtained from Robinson Memorial Hospital in Ravenna, OH, USA. Also included in the study were 110 MRSA blood isolates, 4 VISA isolates and 14 daptomycin-resistant S. aureus (DRSA) isolates obtained from patients admitted to Ascension St. John Hospital Detroit, Michigan, USA. Some isolates met criteria for both VISA and DRSA and thus were included in both categories in Table 1.

Broth microdilution testing using CAMHB was used to determine the MICs of fosfomycin, vancomycin, daptomycin, linezolid, ceftaroline and cefazolin. For all testing with fosfomycin, the CAMHB was supplemented with α-D-glucose-6-phosphate (G6P) to a final concentration of 25 mg/L.² When testing daptomycin, CAMHB was supplemented with 50 mg/L calcium.³ All antibiotic powders were purchased from Sigma–Aldrich (St Louis, MO, USA). The plates were prepared in house and inoculated with approximately 5 × 10⁵ cfu/mL of each organism. The plates were incubated at 35°C in ambient air for 16–24 h. MICs were read visually as the lowest drug concentration well with no visible bacterial growth.⁴S. aureus ATCC 29213 was used daily to monitor quality control (QC) for the agents tested. All QC results were within published specified ranges. MBCs were also determined according to CLSI guidelines.⁵

CLSI does not publish susceptibility breakpoints for fosfomycin susceptibility with Staphylococcus spp. EUCAST has published guidelines for staphylococcus isolates with an MIC of ≤32 mg/L as susceptible.⁶ We used the EUCAST breakpoints to determine fosfomycin susceptibility and the breakpoints of ceftaroline, daptomycin and linezolid. We used CLSI breakpoints for vancomycin to maintain our VISA classification consistent with our previous work with these agents.³

Eighteen isolates were selected for further evaluations to determine in vitro synergy between fosfomycin and select antimicrobial agents. These isolates were selected to give us a sampling of isolates in each of our resistant groups. CLSI and EUCAST recommend agar dilution as the reference method for MIC testing of fosfomycin. Whenever there is testing of fosfomycin, the media used must be supplemented with G6P to a final concentration of 25 mg/L.^3,6 We used the chequerboard broth microdilution method to test for synergy, supplementing the media with 25 mg/L G6P. Broth microdilution is the most practical way to perform chequerboard testing and this method of testing has been used by other investigators when testing fosfomycin for synergy.^7–9

Fosfomycin was tested in combination with vancomycin, linezolid, daptomycin, ceftaroline and cefazolin. Fosfomycin was tested at a range of 4× MIC to ¹/₂₅₆× MIC; serial 2-fold dilutions were made vertically across the microtitre plate columns. The other antibiotics were tested at the range of 4× MIC to ¹/₁₆× MIC with serial 2-fold dilutions horizontally down the rows of the microtitre plate. Each well of the microtitre plate contained a final inoculum of 5 × 10⁵ cfu/mL in a 100 µL total well volume. The plates were incubated at 35°C for 18–20 h in ambient air. The MICs of the agents alone and in combination were determined by the lowest dilution that completely inhibited growth of the organism.¹⁰

The interactions between the two tested antimicrobial agents were evaluated by the FIC index (FICI). The FICI was calculated by the following formula: FICI = (MIC of agent A in combination/MIC of agent A alone) + (MIC of agent B in combination/MIC of agent B alone). The FICI results were interpreted as follows: ≤0.5 equals synergy, >0.5 to ≤4.0 equals no interaction (or indifference) and >4.0 equals antagonism.¹¹

Chequerboard results were determined by the smallest FICI value calculated. It has been reported that reproducibility errors are considerable when using the MIC chequerboard method.¹⁰ We performed all experiments at least three separate times. A minimum of three experiments had to have results in agreement of synergy or no interaction to be finalized.

Results

According to the determined MIC values, all MRSA blood strains were susceptible to fosfomycin and linezolid (Table 1). In addition, fosfomycin maintained activity against 100% of strains of VRSA and LRSA, 86% of VISA and 95% of DRSA. The MBC values were generally within one 2-fold dilution of the MIC values, supporting that fosfomycin maintained bactericidal activity against all subgroups tested within the study.

The summarized results of chequerboard testing are listed in Table 2 for fosfomycin when combined with linezolid, daptomycin, ceftaroline, vancomycin and cefazolin. The combination of fosfomycin with ceftaroline consistently demonstrated synergy among all 18 isolates against the various strains that were tested. The next most potent combination regimen was linezolid with fosfomycin, which demonstrated synergy in 16 of the 18 strains. Daptomycin demonstrated synergy in only 7 of the 18 strains tested when combined with fosfomycin. Cefazolin demonstrated synergy in 6 of 6 strains and vancomycin demonstrated no interaction in 6 of 6 strains tested. There was a smaller number of strains tested with vancomycin and cefazolin since high MICs for these agents precluded adequate synergy testing. We did not observe any antagonism in our testing.

Discussion

The increasing prevalence of resistance among Gram-positive cocci, especially MRSA to new and established antimicrobial agents, demands additional agents to treat MRSA infections including consideration of combination therapy. Newer anti-MRSA drugs such as daptomycin have demonstrated a reduction of in vitro activity since approval. This is illustrated by the identification of 40 DRSA isolates in this study.

Fosfomycin has shown excellent in vitro activity by other investigators against both MRSA and MSSA.¹² Also, Aktas et al.¹³ have reported that daptomycin combined with fosfomycin demonstrated synergy in 25/25 strains tested. However, their study did not include any DRSA, VISA or VRSA isolates, which may account for our lower rate of synergy when daptomycin was combined with fosfomycin. Our study evaluated challenging MRSA isolates that included DRSA, VISA, VRSA and LRSA. In vitro activity of fosfomycin against these isolates with reduced susceptibility to commonly used anti-MRSA drugs has not been reported and may be of assistance in identifying options for the clinician in dealing with infections caused by these isolates.

Fosfomycin and daptomycin is the only combination in our study that has been evaluated in a prospective randomized clinical trial versus daptomycin alone in the treatment of MRSA bacteraemia and endocarditis.¹⁴ The clinical trial demonstrated treatment success in 40/74 (54.1%) of patients receiving the combination of fosfomycin plus daptomycin versus 34/81 (42.0%) for daptomycin alone (P = 0.135). In addition, there was a lower microbiological failure rate in the combination, with 0 failures versus 9 failures in the daptomycin-alone arm. These observations, though not conclusive, support that there may be candidate patients who can benefit from fosfomycin combination therapy. Thus, future trial considerations with combination therapy against MRSA may be justified, understanding the limitation that demonstration of in vitro synergy has not always been associated with enhanced clinical outcomes.

Fosfomycin demonstrated excellent activity against MRSA as well as isolates with resistance or reduced activity to other anti-MRSA drugs including vancomycin, daptomycin and linezolid. When combined with linezolid or daptomycin, fosfomycin demonstrated synergy for all or most strains. Thus, these combinations may have potential clinical utility when treating patients with serious infections caused by MRSA.

Funding

This work was supported by internal funding from the Thomas Mackey Center for Infectious Disease Research and the Ascension St. John Hospital Department of Medicine.

Transparency declarations

None to declare.

References