In vitro and in vivo activity of ciprofloxacin/fosfomycin combination therapy against ciprofloxacin-resistant Shigella flexneri isolates

Figures & data

Table 1 MICs of ciprofloxacin and/or fosfomycin against CIP^R Shigella flexneri isolates

No.	Strain	MIC (μg/mL)				FICI
		CIP		FOS
		Alone	Combined with FOS	Alone	Combined with CIP
1	GN110052	16	4	2,048	512	0.5
2	GN110066	32	4	512	128	0.375
3	GN110123	16	8	8	2	0.75
4	GN110124	16	1	32	16	0.563
5	GN110130	16	8	256	16	0.536
6	GN110132	16	4	128	16	0.375
7	GN110157	8	2	16	8	0.75
8	GN110161	8	2	256	64	0.5
9	GN110184	8	4	8	4	1
10	GN110194	8	2	4	2	0.75
11	GN110201	8	4	2,048	256	0.625
12	GN110205	8	2	64	32	0.75
13	GN110208	32	8	64	16	0.5
14	GN110210	8	4	32	16	1
15	GN110216	16	2	16	8	0.625
16	GN110226	8	4	128	8	0.563
17	GN110227	16	8	4	2	1
18	GN110248	16	4	512	128	0.5
19	GN110250	16	8	128	16	0.625
20	GN110925	16	8	64	1	0.516
21	GN120002	8	4	128	64	1
22	GN120030	8	2	512	128	0.5
23	GN120158	8	4	32	8	0.75
24	GN120202	32	16	4	2	0.75
25	GN120207	32	4	256	128	0.625
26	GN120242	8	4	4	2	1
27	GN120252	8	4	256	32	0.625
28	GN120255	8	4	16	8	1
29	GN120267	8	2	64	16	0.5
30	GN120269	16	4	2,048	256	0.375
31	GN120278	16	8	64	2	0.531
32	GN120281	32	8	128	32	0.5
33	GN120290	16	4	8	4	0.75
34	GN120291	16	4	16	8	0.75
35	GN120301	16	4	256	32	0.375
36	GN120313	16	4	128	32	0.5
37	GN120454	32	8	64	16	0.5
38	GN120471	32	8	512	128	0.5
39	GN121842	16	8	256	32	0.625
40	GN122107	16	4	32	8	0.5
41	GN130003	8	4	16	4	0.75
42	GN130005	8	4	256	64	0.75
43	GN130014	8	1	4	2	0.625
44	GN130027	8	4	128	32	0.75
45	GN130032	16	4	64	4	0.313
46	GN130044	8	2	512	128	0.5
47	GN130047	32	4	512	128	0.375
48	GN130048	16	8	1,024	128	0.625
49	GN130050	16	2	2,048	512	0.375
50	GN130052	16	4	256	32	0.375
51	GN130057	8	4	2,048	128	0.563
52	GN130146	16	4	256	64	0.5
53	GN130151	16	8	128	16	0.625
54	GN130176	32	4	64	16	0.375
55	GN130179	32	8	128	32	0.5
56	GN130199	8	4	32	16	1
57	GN130234	8	2	64	16	0.5
58	GN130238	8	2	1,024	128	0.375
59	GN130255	8	2	8	4	0.75
60	GN131887	16	8	4	2	1
61	GN140017	8	4	16	2	0.625
62	GN140026	32	16	32	8	0.75
63	GN140028	16	4	32	16	0.75
64	GN140029	32	8	32	8	0.5
65	GN140045	32	8	32	8	0.5
66	GN140064	8	2	128	64	0.75
67	GN140066	8	1	128	64	0.625
68	GN140069	8	4	32	8	0.75
69	GN140070	32	4	64	32	0.625
70	GN140071	8	2	16	4	0.5
71	GN140082	8	2	64	32	0.75
72	GN140083	8	4	32	16	0.75
73	GN140096	16	2	256	64	0.375
74	GN140102	8	4	16	1	0.563
75	GN140110	16	4	2,048	512	0.5
76	GN140169	8	2	64	32	0.75
77	GN140197	8	2	4	2	0.75
78	GN140201	8	4	64	8	0.625
79	GN140211	8	4	128	32	0.75
80	GN140224	8	2	128	32	0.5

Abbreviations: MIC, minimal inhibitory concentration; CIP, ciprofloxacin; FOS, fosfomycin; FICI, fractional inhibitory concentration index.

Table 2 Log changes at 3, 5, 8, and 24 hours with various clinically relevant concentrations of ciprofloxacin and/or fosfomycin against CIP^R Shigella flexneri isolates

Isolate	Inoculum (CFU/mL)	Time (hours)	Log change[=log10(CFUt) – log10(CFU0)]
			CIP 0.5 μg/mL	CIP 1 μg/mL	CIP 2.5 μg/mL	FOS 30 μg/mL	FOS 150 μg/mL	FOS 300 μg/mL	CIP 0.5 μg/mL + FOS 30 μg/mL	CIP 0.5 μg/mL + FOS 150 μg/mL	CIP 0.5 μg/mL + FOS 300 μg/mL	CIP 1 μg/mL + FOS 30 μg/mL	CIP 1 μg/mL + FOS 150 μg/mL	CIP 1 μg/mL + FOS 300 μg/mL	CIP 2.5 μg/mL +FOS 30 μg/mL	CIP 2.5 μg/mL + FOS 150 μg/mL	CIP 2.5 μg/mL + FOS 300 μg/mL
GN120471	∼10^6	3	0.97	0.78	0.24	0.51	0.25	−0.64	0.49	−1.82	−2.27	0.47	−1.93	−2.38	0.13	−2.05	−2.53
		5	1.60	1.20	1.14	1.32	0.63	−1.35	1.25	−2.03	−2.69	1.15	−2.24	−2.78	0.95	−2.86	−3.06
		8	2.48	2.20	2.04	2.95	1.89	−1.47	2.20	−1.65	−2.72	2.17	−2.31	−3.08	1.84	−3.15	−3.18
		24	3.81	3.36	3.00	3.11	2.26	2.07	2.89	2.55	−0.67	2.68	1.45	−0.93	2.87	−1.92	−2.14
GN120454	∼10^6	3	0.85	0.73	0.50	−2.58	−3.04	−4.15	−2.59	−3.21	−4.56	−2.65	−3.22	−4.70	−2.84	−3.85	−5.14
		5	1.79	1.58	1.15	−2.35	−3.15	−4.10	−2.37	−3.38	−4.31	−2.58	−3.57	−4.86	−3.05	−3.91	−5.42
		8	2.57	2.37	2.08	−1.65	−2.96	−4.58	−1.75	−4.12	−5.08	−2.39	−4.62	−5.14	−3.64	−4.87	−5.66
		24	3.28	3.15	2.56	2.02	−2.53	−3.12	1.84	−4.65	−5.16	−1.23	−4.54	−5.23	−4.21	−5.09	−5.92

Note: Underlining indicates synergy (≥2 log₁₀ decrease in the CFU/mL with the combination compared with its most active monotherapy) and bold formatting indicates additivity (1 to <2 log₁₀ decrease in the CFU/mL with the combination compared with its most active monotherapy).

Abbreviations: CIP, ciprofloxacin; FOS, fosfomycin.

Figure 1 Representative time-kill curves with low, medium, and high concentrations of ciprofloxacin and fosfomycin alone and in combination against CIP^R Shigella flexneri isolates. (A) CIP (0.5 μg/mL) and FOS (30, 150, 300 μg/mL) alone and in combination against GN120471 (CIP^RFOS^R); (B) CIP (0.5 μg/mL) and FOS (30, 150, 300 μg/mL) alone and in combination against GN120454 (CIP^RFOS^S); (C) CIP (1 μg/mL) and FOS (30, 150, 300 μg/mL) alone and in combination against GN120471 (CIP^RFOS^R); (D) CIP (1 μg/mL) and FOS (30, 150, 300 μg/mL) alone and in combination against GN120454 (CIP^RFOS^S); (E) CIP (2.5 μg/mL) and FOS (30, 150, 300 μg/mL) alone and in combination against GN120471 (CIP^RFOS^R); (F) CIP (2.5 μg/mL) and FOS (30, 150, 300 μg/mL) alone and in combination against GN120454 (CIP^RFOS^S).

Abbreviations: CIP, ciprofloxacin; FOS, fosfomycin; R, resistance; S, susceptibility.

Figure 2 Survival curves of various inoculum doses of Shigella flexneri GN120471 (A) or GN120454 (B) in Galleria mellonella larvae during incubation at 37°C for 96 hours. Curves were plotted using 10 larvae for every experiment. Larvae were inoculated with 10 μL of bacterial suspensions. Mortality at 24 hours was 100% at 10⁶ CFU/larva (bacterial suspension concentration for ~10⁸ CFU/mL).

Figure 3 Survival curves for Galleria mellonella larvae by CIP (steady-state plasma concentration 2.5 μg/mL) and FOS (steady-state plasma concentration 150 μg/mL) alone and in combination against Shigella flexneriisolates GN120471 (A) or GN120454 (B). Curves were plotted using 16 larvae for every experiment during incubation at 37°C for 96 hours. Bacterial suspension concentration for 1×10⁶ CFU/mL (1×10⁴ CFU/larva) was selected as the inoculum for the treatment experiments. Univariate analysis was performed by the chi-squared test or Fisher’s exact test when appropriate. The survival rate for CIP/FOS combination against GN120471-infected larvae was 68.75% compared with 25% for CIP (χ²=6.149, P=0.013) and 43.75% for FOS (χ²=2.032, P=0.154). The survival rate for CIP/FOS combination against GN120454-infected larvae was 81.25% compared with 37.5% for CIP (χ²=6.348, P=0.012) and 62.5% for FOS (P>0.05).

Abbreviations: CIP, ciprofloxacin; FOS, fosfomycin.

Figure S1 Larvae infected with Shigella flexneri (10³, 10⁵, 10⁶, and 10⁷ CFU/larva) at 96 hours