Naringenin restores colistin activation against colistin-resistant gram-negative bacteria in vitro and in vivo

Abstract

Colistin is used as the "last line of defense" against multidrug-resistant (MDR) Gram-negative bacteria (GNB). However, improper use of colistin may further lead to an increasing number of colistin-resistant (Col-R) strains worldwide, which greatly limits antibiotic treatment options. In this study, we investigated the antibacterial and antibiofilm activities of naringenin (NG) combined with colistin against Col-R GNB in vitro and in vivo. The checkerboard method and time-kill test showed that NG combined with colistin has better antibacterial activity (FICI < 0.5) compared with NG and colistin alone. Biofilm formation inhibition tests demonstrated that combining the two drugs could inhibit biofilm formation; scanning electron microscopy (SEM) confirmed that the combination of the two significantly reduces the number of cells in the biofilm compared with the drug alone. The in vivo experiment showed that the combination of NG and colistin can improve the survival rate of the Galleria mellonella (G. mellonella) and reduce the microbial load in the mouse thigh infection model. Mechanistically, the combination of NG and colistin synergistically enhances the antibacterial activity and changes the permeability of the bacterial outer membrane. More importantly, cytotoxicity tests showed no cell cytotoxicity of NG in combination with colistin. In conclusion, our data revealed that NG combined with colistin exhibited good synergistic effects in vivo and in vitro, thus providing a new therapeutic option for clinical Col-R GNB infections.

Keywords: biofilm; colistin resistance; gram-negative bacteria; naringenin; synergistic effect.

FIGURE 1

Colistin and naringenin time-killing curves against colistin-resistant Klebsiella pneumonia, colistin-resistant Escherichia coli, and colistin-resistant Acinetobacter baumannii when used alone or in combination. Naringenin (NG) and colistin (COL).

FIGURE 2

Fluorescence microscopy imaging of exponential-phase Klebsiella pneumonia FK1913, which were treated with naringenin and colistin alone or in combination, and incubated with 50 μg/ml PI for 10 min before imaging. (A) LB broth control; (B,C) cells treated with naringenin at 64, 128 μg/ml; (D) cells treated with colistin with 2 μg/ml; (E,F) cells exposed to a combination of naringenin and colistin. Naringenin (NG) and colistin (COL).

FIGURE 3

Biofilm inhibitory effects of colistin combined with naringenin on colistin-resistant Klebsiella pneumonia, colistin-resistant Escherichia coli, and colistin-resistant Acinetobacter baumannii. Drug concentration was chosen from the checkerboard method with FICI < 0.5. *P < 0.05, **P < 0.01, and ***P < 0.001 analyzed by Student’s t-test. Naringenin (NG) and colistin (COL).

FIGURE 4

Scanning electron microscopy image. Klebsiella pneumonia FK1913 treated with 64 μg/ml naringenin (C,D), 2 μg/ml colistin (E,F), or combined treatment (G,H) for 2 h. (A,B) The control condition. Naringenin (NG) and colistin (COL).

FIGURE 5

Survival rate of Galleria mellonella after 7 days of monotherapy or combination therapy against colistin-resistant Klebsiella pneumoniae FK1913, Escherichia coli DC90, and Acinetobacter baumannii BM2349. Naringenin (NG) and colistin (COL).

FIGURE 6

Changes in Log₁₀ of thigh muscle (Δlog₁₀ CFU/thigh) 24 h after monotherapy (naringenin 50 mg/kg, colistin 7.5 mg/kg) or combination therapy (50 mg/kg NG + 7.5 mg/kg colistin) for colistin-resistant Klebsiella pneumoniae FK1913. Naringenin (NG) and colistin (COL). ***P < 0.001.

FIGURE 7

Cytotoxic effect of naringenin alone and in combination with different concentrations against the RAW 264.7 murine macrophage cell line (absorbance values at 450 nm). Data were analyzed by Student’s t-test; ns, not statistically significant; ***P < 0.001. Naringenin (NG) and colistin (COL).