In vitro activity and post-antibiotic effects of linezolid in combination with fosfomycin against clinical isolates of Staphylococcus aureus

Figures & data

Table 1 MIC and MBC of antimicrobial agents against eleven strains of S. aureus

Strains	MIC/MBC (MBC/MIC) (mg/L)		MICcombination	FICI
	LIN	FOS	LIN/FOS	LIN + FOS
No. 64	4/16 (4)	2/8 (4)	1.00/0.50	0.50
No. 36	2/8 (4)	2/8 (4)	0.50/0.25	0.38
No. 24	2/8 (4)	2/16 (8)	0.50/1.00	0.75
No. 54	2/8 (4)	2/8 (4)	0.50/0.50	0.50
No. 78	4/16 (4)	2/16 (8)	1.00/0.50	0.50
No. 8	2/8 (4)	2/8 (4)	0.50/0.50	0.50
No. 27	2/8 (4)	2/4 (2)	0.50/0.50	0.50
No. 14	1/2 (2)	2/4 (2)	0.25/1.00	0.75
No. 35	2/8 (4)	2/16 (8)	0.50/0.50	0.50
No. 55	2/8 (4)	2/8 (4)	0.50/1.00	0.75
ATCC 25923	2/8 (4)	2/8 (4)	0.50/0.50	0.50

Note: FICI was defined as follows: FICI ≤0.5, synergy; 0.5< FICI ≤4.0, indifference; FICI >4.0, antagonism.

Abbreviations: FICI, Fractional Inhibitory Concentration Index; FOS, fosfomycin; LIN, linezolid; MBC, minimum bactericidal concentration; MIC, minimum inhibitory concentration; S. aureus, Staphylococcus aureus.

Figure 1 Time-kill curves of linezolid plus fosfomycin against MRSA (No.36).

Abbreviations: CFU, colony-forming units; FOS, fosfomycin; LIN, linezolid; MRSA, methicillin-resistant Staphylococcus aureus; MIC, minimum inhibitory concentration.

Table 2 PAE (h) of linezolid plus fosfomycin exposure for 1 hour against three S. aureus strains

Strains	LIN		FOS		LIN + FOS
	1× MIC	4× MIC	1× MIC	4× MIC	1+1× MIC	4+4× MIC
No. 36	0.83±0.16	1.12±0.19	1.01 ±0.18	2.80±0.24	1.76±0.32	3.44±0.41
No. 8	1.01 ±0.22	2.09±0.21	1.15±0.26	2.22±0.36	1.77±0.33	2.93±0.51
ATCC 25923	1.31 ±0.19	1.86±0.24	1.63±0.20	2.24±0.23	2.32±0.21	3.34±0.36
Mean ± SD	1.05±0.24	1.69±0.51	1.26±0.33	2.42±0.33	1.95±0.32	3.24±0.27

Note: Mean ± SD represents the mean PAE and SDs of three S. aureus strains at each drug concentration.

Abbreviations: FOS, fosfomycin; LIN, linezolid; MIC, minimum inhibitory concentration; PAE, post-antibiotic effect; S. aureus, Staphylococcus aureus.

Table 3 PAE (h) of linezolid plus fosfomycin exposure for 2 hours against three S. aureus strains

Strains	LIN		FOS		LIN + FOS
	1× MIC	4× MIC	1× MIC	4× MIC	1+1× MIC	4+4× MIC
No. 36	1.39±0.17	2.18±0.21	1.66±0.34	3.02±0.27	2.20±0.35	3.78±0.45
No. 8	2.02±0.28	2.23±0.25	2.12±0.24	2.86±0.33	2.82±0.23	3.74±0.38
ATCC 25923	1.93±0.12	2.18±0.16	2.53±0.15	2.93±0.36	2.96±0.23	3.84±0.18
Mean ± sD	1.78±0.34	2.20±0.03	2.10±0.44	2.94±0.08	2.66±0.40	3.79±0.05

Abbreviations: FOS, fosfomycin; LIN, linezolid; MIC, minimum inhibitory concentration; PAE, post-antibiotic effect; S. aureus, Staphylococcus aureus.

Table 4 PAE (h) of linezolid plus fosfomycin exposure for 3 hours against three S. aureus strains

Strains	LIN		FOS		LIN + FOS
	1× MIC	4× MIC	1× MIC	4× MIC	1+1× MIC	4+4× MIC
No. 36	1.90±0.30	2.46±0.31	1.85±0.23	3.20±0.42	3.05±0.38	4.18±0.63
No. 8	2.08±0.34	2.42±0.20	2.57±0.26	3.16±0.52	3.45±0.24	4.09±0.45
ATCC 25923	2.33±0.21	2.31 ±0.15	2.80±0.29	3.42±0.56	3.30±0.43	4.12+0.34
Mean ± sD	2.10±0.22	2.40±0.08	2.41 ±0.50	3.26±0.14	3.27±0.20	4.13±0.05

Abbreviations: FOS, fosfomycin; LIN, linezolid; MIC, minimum inhibitory concentration; PAE, post-antibiotic effect; S. aureus, Staphylococcus aureus.

Figure 2 Regrowth curves of MRSA (No.36) strains exposed to linezolid plus fosfomycin for 1, 2, and 3 hours.

Notes: Timings of antibiotics addition, removal, and the duration of exposure are indicated by a vertical black arrow, a horizontal blue arrow, and a horizontal black arrow, respectively.
Abbreviations: CFU, colony-forming units; FOS, fosfomycin; LIN, linezolid; MRSA, methicillin-resistant Staphylococcus aureus; MIC, minimum inhibitory concentration.

Table 5 PA-SME (h) of linezolid plus fosfomycin at 4x MIC exposure for 2 hours against three S. aureus strains

Strains	PA-SME (LIN)			PA-SME (FOS)			PA-SME (LIN + FOS)
	0.1× MIC	0.2x MIC	0.4× MIC	0.1× MIC	0.2x MIC	0.4× MIC	0.1× MIC	0.2x MIC	0.4× MIC
No.36	2.37±0.22	2.44±0.06	3.04±0.16	2.96±0.41	3.92±0.28	4.73±0.34	4.35±0.24	4.85±0.51	5.62±0.76
No.8	2.45±0.33	2.77±0.24	3.36±0.14	3.19±0.18	3.93±0.27	4.90±0.58	4.09±0.37	5.01±0.24	5.51±0.05
ATCC 25923	2.46±0.20	2.67±0.12	3.87±0.32	3.68±0.08	4.13±0.25	4.48±0.47	4.13±0.17	4.73±0.62	5.46±0.18
Mean ± SD	2.43±0.05	2.63±0.17	3.42±0.42	3.28±0.37	3.99±0.12	4.70±0.21	4.19±0.14	4.86±0.14	5.26±0.48

Notes: Mean ± SD represents the mean PAE and SDs of three S. aureus strains at each drug concentration.

Abbreviations: FOS, fosfomycin; LIN, linezolid; MIC, minimum inhibitory concentration; PA-SME, post-antibiotic sub-MIC effect; S. aureus, Staphylococcus aureus.

Table 6 Bacterial count changes of the three strains after 24 hours (time-kill experiments) and 12 hours (PAE and PA-SME experiments) of exposure to linezolid alone or combination fosfomycin compared to the viable bacterial counts with control

Drugs	Bacterial counts (CFU/mL)
	No. 36	No. 8	ATCC 25923
Time-kill experiments
Control	17.53±0.60	16.63±0.08	16.17±0.09
LIN 1× MIC	7.88±0.11	8.83±0.05	8.04±0.04
FOs 1× MIC	8.11 ±0.09	7.87±0.08	7.22±0.04
LIN 1/2×+FOs 1/2× MIC	3.37±0.28	3.65±0.05	4.12±0.07
LIN 1/2×+FOs 1× MIC	3.45±0.03	3.69±0.01	3.67±0.28
LIN 1×+FOs 1/2× MIC	5.03±0.06	4.52±0.17	3.55±0.05
LIN 1×+FOs 1× MIC	2.51 ±0.43	2.73±0.03	3.01 ±0.06
LIN 2×+FOs 2× MIC	1.88±0.15	1.55±0.04	2.99±0.09
LIN 4×+FOs 4× MIC	0.00±0.00	0.00±0.00	0.00±0.00
PAE experiments
Control	13.39±0.04	13.65±0.02	14.12±0.03
LIN 1× MIC	10.72±0.04	9.85±0.02	8.33±0.05
LIN 4× MIC	7.43±0.06	9.71 ±0.04	6.27±0.12
FOs 1× MIC	9.66±0.02	10.51 ±0.04	8.94±0.15
FOs 4× MIC	8.29±0.05	8.49±0.02	7.07±0.15
LIN 1×+FOs 1× MIC	8.72±0.02	8.83±0.06	8.82±0.27
LIN 4×+FOs 4× MIC	7.37±0.13	6.79±0.04	5.31 ±0.05
PA-SME experiments
Control (LIN 0.1× MIC)	13.56±0.05	13.88±0.03	13.35±0.04
Control (LIN 0.2× MIC)	12.29±0.03	13.71 ±0.11	12.38±0.14
Control (LIN 0.4 × MIC)	8.05±0.08	11.26±0.15	9.26±0.14
LIN 0.1× MIC	6.43±0.07	7.27±0.09	7.17±0.15
LIN 0.2× MIC	5.25±0.07	6.29±0.01	5.26±0.16
LIN 0.4× MIC	4.30±0.00	4.82±0.17	4.22±0.17
Control (FOs 0.1× MIC)	12.54±0.04	13.65±0.05	14.00+0.03
Control (FOs 0.2× MIC)	11.31 ±0.10	13.43±0.04	13.50+0.10
Control (FOs 0.4× MIC)	10.99±0.22	12.85±0.19	11.43±0.11
FOs 0.1× MIC	7.33±0.08	8.27±0.10	7.26±0.01
FOs 0.2× MIC	5.61 ±0.05	7.23±0.11	6.69±0.05
FOs 0.4× MIC	5.32±0.09	6.19±0.10	5.49±0.15
Control (LIN 0.1×+FOs 0.1× MIC)	13.74±0.05	13.43±0.04	13.88±0.15
Control (LIN 0.2×+FOs 0.2× MIC)	11.24±0.11	11.09±0.11	12.37±0.30
Control (LIN 0.4× MIC+ FOs 0.4× MIC)	9.73±0.07	9.12±0.17	10.36±0.16
LIN 0.1×+FOs 0.1× MIC	6.77±0.04	6.01 ±0.11	7.54±0.12
LIN 0.2×+FOs 0.2× MIC	5.68±0.03	4.33±0.07	4.72±0.05
LIN 0.4×+FOs 0.4× MIC	4.52±0.15	3.78±0.07	3.18±0.16

Note: All data are presented as mean ± SD.

Abbreviations: CFU, colony-forming units; FOS, fosfomycin; LIN, linezolid; MIC, minimum inhibitory concentration; PAE, post-antibiotic effect; PA-SME, post-antibiotic sub-MIC effect.

Figure S1 Time-kill curves of linezolid plus fosfomycin against MSSA (No.8) and ATCC 25923.

Abbreviations: CFU, colony-forming units; FOS, fosfomycin; LIN, linezolid; MSSA, methicillin-resistant Staphylococcus aureus; MIC, minimum inhibitory concentration.

Figure S2 Regrowth curves of MSSA (No.8) (A–C) and ATCC 25923 (D–F) exposed to linezolid plus fosfomycin for 1, 2, and 3 hours.

Note: Timings of antibiotic addition, removal, and the duration of exposure are indicated by a vertical black arrow, a horizontal blue arrow, and a horizontal black arrow, respectively.
Abbreviations: CFU, colony-forming units; FOS, fosfomycin; LIN, linezolid; MSSA, methicillin-susceptible Staphylococcus aureus; MIC, minimum inhibitory concentration