Assessing the Emergence of Resistance in vitro and Invivo: Linezolid Combined with Fosfomycin Against Fosfomycin-Sensitive and Resistant Enterococcus

Figures & data

Table 1 MICs of Antimicrobial Agents Against Nineteen Strains of Enterococcus

Isolates	MIC (mg/L)		MIC in Combination
	LZD	FOS	LZD+FOS	FICI
22	2	64	1+8	0.625
23	2	64	1+8	0.625
24	2	64	1+8	0.625
116	4	64	1+32	0.75
118	2	64	1+32	1.0
226	2	64	1+16	0.75
231	2	128	1+128	1.5
300	2	64	1+8	0.625
593	8	64	4+8	0.625
605	2	128	2+16	1.125
633	2	128	1+16	0.625
643	1	64	0.25+32	0.75
662	2	256	0.25+128	0.625
673	2	128	1+32	0.75
674	2	64	2+16	1.25
694	2	64	1+32	1.0
680	2	128	2+16	1.125
681	2	128	1+32	0.75
29,212	2	128	1+64	1.0

Abbreviations: FOS, fosfomycin; LZD, linezolid; FICI, fractional inhibitory concentration index; MIC, minimal inhibitory concentration of antibiotics.

Table 2 ∆logCFU_0–24h Values of Linezolid and Fosfomycin as Monotherapy and in Combination

Strains (MICLZD/ MICFOS)	∆logCFU0–24h by Antibiotic Therapy (Mean ± SD) (n = 3)
	No drug	Linezolid		Fosfomycin		Combination
Concentration (mg/L)	–	2	4	64	128	2+64	4+128
NO.22 (2/64)	2.07±0.10	−0.64±0.14	−0.81±0.11	0.53±0.48	1.14±0.51	−2.99±0.3	−3.22±0.07
NO.23 (2/64)	2.5±0.09	−1.38±0.10	−1.27±0.06	1.37±0.17	0.57±0.11	−2.73±0.07	−3.11±0.07
NO.24 (2/64)	2.27±0.07	–1.39±0.13	−1.61±0.14	0.86±0.07	1.49±0.04	−2.77±0.06	−3.02±0.02

Figure 1 Static time-kill assays displaying the activity of linezolid, fosfomycin and their combination against Enterococcus. NO.22 (A), NO.23 (B), and NO.24 (C); Control: no drug.

Abbreviations: FOS, fosfomycin; LZD, linezolid.

Figure 2 In vivo assays using G.mellonella model. (A) exploration of the concentration of 50% lethal bacteria (B) survival curves of infected G.mellonella larvae treated with with linezolid and fosfomycin at different concentrations alone or in combination.(C) Haemolymph CFU burden of G. mellonella after infection with NO.22 followed by antibiotic treatment. CFU/mL values are shown for the drug treatment groups in relation to the mean CFU/mL value for the control group, indicating the reduction in log10 CFU/mL due to treatment. Data expressed as mean ± standard deviation of three independent experiments (*P<0.05, **P<0.01).

Table 3 MICs of Antimicrobial Agents Against Four Strains

Stains	MIC (LZD) mg/L	MIC (FOS) mg/L
NO.22R	2	2048
NO.23R	2	1024
NO.24R	2	1024
29,212	2	128

Figure 3 Virulence analysis of fosfomycin-resistant mutants (A–C). The Galleria mellonella infection model was used to explore the difference in pathogenicity between baseline isolates (NO.22, NO.23, NO.24) and fosfomycin-resistant mutants (NO.22, NO.23, NO.24). (D–F) The relative mRNA expression levels of virulence genes (D) asaL, (E) esp, (F) cylA were compared between baseline isolates and fosfomycin-resistant isolates mutants. (*P < 0.05, **P < 0.01 and ***P < 0.001).NO.22R, isolate resistant to NO.22; NO.23R, isolate resistant to NO.23; NO.24R, isolate resistant to NO.24.

Figure 4 Fitness cost analysis of fosfomycin-resistant mutant NO.22. (A and B) Growth curve of drug-resistant mutant NO.22 and parental strain NO.22 cultured separately in vitro and in vivo. (C and D) Relative fitness of fosfomycin-resistant mutant NO.22R and parental strain NO.22 in co-culture in vitro and vivo. Relative fitness value less than 1 indicates fitness defect, which incurs fitness cost and a value greater than 1 indicates fitness benefit.

Figure 5 Static time-kill assays displaying the activity of linezolid, fosfomycin and their combination against Enterococcus NO.22R; Control: no drug.

Abbreviations: FOS, fosfomycin; LZD, linezolid.