Inhibitory effects of fluoroquinolone antibiotics on Babesia divergens and Babesia microti, blood parasites of veterinary and zoonotic importance

Figures & data

Figure 1 Chemical structures of fluoroquinolone antibiotics used in this study: (A) enrofloxacin, (B) enoxacin, (C) trovafloxacin, (D) norfloxacin, and (E) ofloxacin.

Figure 2 Correlation between relative fluorescence units (RFUs) and the log concentrations of fluoroquinolone antibiotics (nM) in Babesia divergens on the fourth day of treatment. Each value represents the mean of triplicate wells after subtraction of the background fluorescence for non-parasitized red blood cells.

Table 1 IC₅₀ values of fluoroquinolone antibiotic drugs evaluated for Babesia divergens parasite

Drugs	IC50 values (μM)^a
Enrofloxacin	9.54 ± 0.64
Enoxacin	45.23 ± 1.35
Trovafloxacin	13.80 ± 2.69
Norfloxacin	59.52 ± 3.36
Ofloxacin	212.14 ± 3.02
Diminazene aceturate	0.14 ± 0.05
Chloroquine diphosphate	0.40 ± 0.07
Tetracycline hydrochloride	35.27 ± 2.84

Notes: ^aIC₅₀ values for each drug were calculated on the fourth day of the in vitro culture using a Babesia fluorescence assay (BFA) in three separate experiments. Each drug concentration was made in triplicate in each experiment, and the final obtained IC₅₀ values were the mean ± SD of values obtained from three separate experiments.

Table 2 Viability test results of fluoroquinolone antibiotic drugs evaluated for Babesia divergens parasite

Drugs	Drug concentrations(μM)^a
	0.5	10	50	100	400
Enrofloxacin	+	+	-	-	-
Enoxacin	+	+	+	-	-
Trovafloxacin	+	+	-	-	-
Norfloxacin	+	+	+	+	-
Ofloxacin	+	+	+	+	-
Tetracycline hydrochloride	+	+	+	-	-
Chloroquine diphosphate	-	-	-	-	-
	Drug concentrations(μM)^a
	0.25	0.5	1	5	10
Diminazene aceturate	-	-	-	-	-

Note: ^aEach value was calculated using a Babesia fluorescence assay (BFA) in three separate experiments. Each concentration of the drug was made in triplicate in each experiment. +, viable; −, dead.

Table 3 Synergetic and additive interactions of diminazene aceturate (DA), luteolin, and pyronaridine tetraphosphate (PYR) in combination with enrofloxacin, enoxacin, or trovafloxacin on Babesia divergens parasite on the fourth day of treatment

Drug combination	M^a	FICD1	FICD2	ΣFIC	Degree of interaction^b
DAD1+enrofloxacinD2	M1	0.33	0.24	0.57	Additive
	M2	0.37	0.27	0.64	Additive
	M3	0.42	0.31	0.73	Additive
	M4	0.46	0.34	0.80	Additive
DAD1+ enoxacinD2	M1	0.33	0.26	0.59	Additive
	M2	0.35	0.27	0.62	Additive
	M3	0.37	0.29	0.66	Additive
	M4	0.47	0.36	0.83	Additive
DAD1+ trovafloxacinD2	M1	0.09	0.18	0.27	Synergetic
	M2	0.20	0.40	0.60	Additive
	M3	0.25	0.50	0.75	Additive
	M4	0.28	0.57	0.85	Additive
LuteolinD1+ enoxacinD2	M1	0.39	0.55	0.94	Additive
PYRD1+ trovafloxacinD2	M1	0.04	0.07	0.11	synergetic
	M2	0.09	0.13	0.22	synergetic
	M3	0.26	0.39	0.65	Additive

Notes: ^aM1–M4 refer to the combinations of fluoroquinolone antibiotic drugs combined with different antibabesial drugs;

b the degree of drug interaction was determined based on the following fractional inhibitory concentration (FIC) index: ≤0.5 (synergetic), >0.5–1 (additive), >1 to <2 (indifferent), and ≥2 (antagonistic). FIC_D1 refers to the fractional inhibitory concentration of DA, luteolin, or PYR. FIC_D2 refers to thefractional inhibitory concentration of fluoroquinolone.

Figure 3 Inhibitory effect of fluoroquinolone antibiotics, diminazene aceturate (DA), and the combination of enoxacin and DA on the growth of Babesia microti. Each value represents the mean ± SD of five mice per experimental group. Asterisks indicate significant differences (*P < 0.05) from day 4 to day 16 post-inoculation between the fluoroquinolone antibiotic-treated and control groups.

Figure 4 Anemia monitoring in mice treated with fluoroquinolone antibiotics: (A) RBCs, (B) hemoglobin (HGB), and (C) hematocrit (HCT). Each value represents the mean ± SD of five mice per experimental group. Asterisks indicate a significant difference (*P < 0.05) between the treated or infected mice and the uninfected mice.

Abbreviation: DA, diminazene aceturate.

Figure 5 Changes in mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), and red blood cell distribution width (RDW) in mice treated with fluoroquinolone antibiotics: (A) MCV, (B) MCH, and (C) RDW. Each value represents the mean ± SD of five mice per experimental group. Asterisks indicate a significant difference (*P < 0.05) between the treated or infected mice and the uninfected mice.

Abbreviation: DA, diminazene aceturate.

Figure 6 PCR of the ss-rRNA gene in different organs of Babesia microti-infected mice treated with DDW (positive control), 25 mg⋅kg^–1 diminazene aceturate (DA), and DA (10 mg⋅kg^–1) combined with enoxacin (50 mg⋅kg^–1).

Note: M indicates a 100 bp DNA ladder.
Abbreviations: NC, negative control; PC, positive control; DDW, double-distilled water; S, spleen; L, lung; H, heart; K, kidney; DA, diminazene aceturate; PCR, polymerase chain reaction.

Table S1 Concentrations of fluoroquinolone antibiotic drugs combined with diminazene aceturate, luteolin, and pyronaridine tetraphosphate applied to the cultures of Babesia divergens parasite

M^a	IC 50^b
	Fluoroquinolone antibiotic drugs	Diminazene aceturate	Luteolin	Pyronaridine tetraphosphate
MI	0.75	0.75	0.75	0.75
M2	0.75	0.5	0.5	0.5
M3	0.5	0.75	0.75	0.75
M4	0.5	0.5	0.5	0.5

Note: ^aM1–M4 refer to the combinations of fluoroquinolone antibiotic drugs combined with different antibabesial drugs;

b combinations were based on the calculated IC₅₀ values obtained from the in vitro fluorescence-based assay.

Table S2 Indifferent interaction of diminazene aceturate (DA), luteolin, and pyronaridine tetraphosphate (PYR) in combination with enrofloxacin, enoxacin, or trovafloxacin on Babesia divergens parasite on the fourth day of treatment

Drug combination	M^a	FICD1	FICD2	ΣFIC	Degree of interaction^b
LuteolinD1+ enrofloxacinD2	M1	0.89	0.82	1.71	Indifferent
	M2	0.92	0.85	1.77	Indifferent
	M3	0.94	0.87	1.81	Indifferent
	M4	0.96	0.88	1.84	Indifferent
LuteolinDI+ enoxacinD2	M2	0.48	0.68	1.16	Indifferent
	M3	0.50	0.71	1.21	Indifferent
	M4	0.55	0.78	1.33	Indifferent
LuteolinD1+ trovafloxacinD2	M1	0.79	0.98	1.77	Indifferent
	M2	0.78	0.96	1.74	Indifferent
	M3	0.78	0.96	1.74	Indifferent
	M4	0.58	0.71	1.29	Indifferent
PYRD1+ enrofloxacinD2	M1	0.89	0.85	1.74	Indifferent
	M2	0.90	0.86	1.76	Indifferent
	M3	0.94	0.90	1.84	Indifferent
	M4	0.95	0.91	1.86	Indifferent
PYRDI+ enoxacinD2	M1	0.63	0.69	1.32	Indifferent
	M2	0.68	0.74	1.42	Indifferent
	M3	0.80	0.86	1.66	Indifferent
	M4	0.89	0.97	1.86	Indifferent
PYRD1+ trovafloxacinD2	M4	0.67	0.98	1.65	Indifferent

Notes: ^aM1–M4 refer to the combinations of fluoroquinolone antibiotic drugs combined with different antibabesial drugs;

b the degree of drug interaction was determined based on the following fractional inhibitory concentration (FIC) index: ≤0.5 (synergism), >0.5–1 (additive), >1 to <2 (indifferent), and ≥2 (antagonism). FIC_D1 refers to the fractional inhibitory concentration of DA, luteolin, or PYR. FIC_D2 refers to thefractional inhibitory concentration of fluoroquinolone drug.