Synergistic activity of fosfomycin and flucloxacillin against methicillin-susceptible and methicillin-resistant Staphylococcus aureus: in vitro and in vivo assessment

Abstract

Fosfomycin (FOF) exhibits broad-spectrum antimicrobial activity, and is mainly used in combination therapy. Previous in vitro studies have shown synergistic effects of FOF in combination with flucloxacillin (FLX) against Staphylococcus aureus isolates. This study aims to validate these findings in vitro and investigate the synergistic effect in an in vivo Galleria mellonella model. Five methicillin- and FOF-susceptible isolates (ATCC-29213 & 4 clinical isolates); one methicillin- and FOF-resistant strain (DSMZ-23622) and four methicillin-resistant and FOF-susceptible strains (ATCC-33592 & 3 clinical isolates) were tested with checkerboard assays to assess synergism. Time-kill curves were generated for two MSSA (ATCC 29213 and 231/20) and two MRSA strains (ATCC 33592 and DSMZ 23622). The in vivo efficacy of FOF and/or FLX was evaluated by a G. mellonella survival assay and by determining the total bacterial count (TBC) in hemolymph. Checkerboard assays revealed additive or indifferent effects, with some indicating synergism. Time-kill curves demonstrated higher reduction in TBC with combination therapy compared to monotherapy. In vivo, the combination therapy showed the greatest reduction of TBC in larval haemolymph compared to monotherapy, and the survival assay showed highly synergistic activity of FLX plus FOF against MRSA (ATCC-33592) and MSSA (ATCC 6538), resulting in an average reduction in mortality of 48 and 40%, respectively, compared to monotherapies. Therefore, FOF plus FLX could be an alternative for the calculated or definitive treatment of S. aureus infections without antimicrobial susceptibility results or even for salvage therapy of MRSA infections after treatment failure or necessary discontinuation of classical MRSA drugs.

Keywords: Antimicrobial resistance; Combination therapy; Experimental model; In vivo synergy; MRSA.

Fig. 1

CFU/mL with standard deviation (partly overlaid by symbols) of the Time Kill Curves with fosfomycin (FOF) and flucloxacillin (FLX) of two MSSA strains a ATCC 29213 and b MSSA 231/20 and two MRSA strains c ATCC 33592 (FOF susceptible) and d DSMZ 23622 (FOF resistant) are shown over 24 h. The individual MICs of the strains are a FOF 2 mg/L and FLX 0.5 mg/L, b FOF 4 mg/L and FLX 0.125 mg/L, c FOF 8 mg/L, FLX 8 mg/L and d FOF 128 mg/L, FLX 1024 mg/L. Concentrations evaluated by the checkerboard assay showing synergy or additive behavior were tested in single and in combination. Circles represent the growth control, red and blue symbols depict individual concentrations of FOF and FLX respectively and green symbols show the combination of both antibiotics

Fig. 2

a Survival curves of G. mellonella larvae infected with methicillin-susceptible S. aureus (ATCC 29213) followed by treatment with flucloxacillin (FLX) 85 mg/kg (n = 16), fosfomycin (FOF) 0.2 mg/kg (n = 16) or the combination of both, FLX 85 mg/kg plus FOF 0.2 mg/kg (n = 17). Larvae in the control group were infected but received only sterile PBS (n = 21). b Survival curves of G. mellonella larvae infected with methicillin-susceptible S. aureus (ATCC 6538) followed by treatment with FLX 85 mg/kg (n = 20), FOF 0.2 mg/kg (n = 20) or the combination of both, FLX 85 mg/kg plus FOF 0.2 mg/kg (n = 20). Larvae in the control group were infected but received only sterile PBS (n = 20). c Survival curves of G. mellonella larvae infected with methicillin-resistant S. aureus (ATCC 33592) followed by treatment with FLX 85 mg/kg (n = 33), FOF 0.8 mg/kg (n = 34) or the combination of both, FLX 85 mg/kg plus FOF 0.8 mg/kg (n = 33). Larvae in the control group were infected but received only sterile PBS (n = 34)

Fig. 3

a-f Relative gene expression of selected virulence factors/gene regulators (accessory gene regulator A (agrA), alpha-hemolysin (hla), leukotoxin ED (lukED)) of methicillin-susceptible S. aureus ATCC 29213 determined by RT-PCR with glyceraldehyde 3-phosphate dehydrogenase (gap) as housekeeping gene and an untreated control as reference. Bacteria were exposed to either fosfomycin or flucloxacillin at concentrations equivalent to 1/8, ¼, ½ or 5 times the respective minimum inhibitory concentration (MIC) for a period of 4 or 8 h, respectively. Data are expressed as mean (± SD) relative quantification values