Phentolamine Significantly Enhances Macrolide Antibiotic Antibacterial Activity against MDR Gram-Negative Bacteria

Ze-Hua Cui; Hui-Ling He; Zi-Jian Zheng; Zhao-Qi Yuan; Ying Chen; Xin-Yi Huang; Hao Ren; Yu-Feng Zhou; Dong-Hao Zhao; Liang-Xing Fang; Yang Yu; Ya-Hong Liu; Xiao-Ping Liao; Jian Sun

doi:10.3390/antibiotics12040760

Phentolamine Significantly Enhances Macrolide Antibiotic Antibacterial Activity against MDR Gram-Negative Bacteria

Antibiotics (Basel). 2023 Apr 14;12(4):760. doi: 10.3390/antibiotics12040760.

Authors

Ze-Hua Cui^{1

2}, Hui-Ling He^{1

2}, Zi-Jian Zheng^{1

2}, Zhao-Qi Yuan^{1

2}, Ying Chen^{1

2}, Xin-Yi Huang^{1

2}, Hao Ren^{1

2}, Yu-Feng Zhou^{1

2}, Dong-Hao Zhao^{1

2}, Liang-Xing Fang^{1

2}, Yang Yu^{1

2}, Ya-Hong Liu^{1

2

3}, Xiao-Ping Liao^{1

2}, Jian Sun^{1

2}

Affiliations

¹ Guangdong Laboratory for Lingnan Modern Agriculture, National Risk Assessment Laboratory for Antimicrobial Resistance of Animal Original Bacteria, College of Veterinary Medicine, South China Agricultural University, Guangzhou 510642, China.
² Guangdong Provincial Key Laboratory of Veterinary Pharmaceutics, Development and Safety Evaluation, South China Agricultural University, Guangzhou 510642, China.
³ Jiangsu Co-Innovation Center for the Prevention and Control of Important Animal Infectious Diseases and Zoonoses, Yangzhou University, Yangzhou 225009, China.

Abstract

Objectives: Multidrug-resistant (MDR) Gram-negative bacterial infections have limited treatment options due to the impermeability of the outer membrane. New therapeutic strategies or agents are urgently needed, and combination therapies using existing antibiotics are a potentially effective means to treat these infections. In this study, we examined whether phentolamine can enhance the antibacterial activity of macrolide antibiotics against Gram-negative bacteria and investigated its mechanism of action.

Methods: Synergistic effects between phentolamine and macrolide antibiotics were evaluated by checkerboard and time-kill assays and in vivo using a Galleria mellonella infection model. We utilized a combination of biochemical tests (outer membrane permeability, ATP synthesis, ΔpH gradient measurements, and EtBr accumulation assays) with scanning electron microscopy to clarify the mechanism of phentolamine enhancement of macrolide antibacterial activity against Escherichia coli.

Results: In vitro tests of phentolamine combined with the macrolide antibiotics erythromycin, clarithromycin, and azithromycin indicated a synergistic action against E. coli test strains. The fractional concentration inhibitory indices (FICI) of 0.375 and 0.5 indicated a synergic effect that was consistent with kinetic time-kill assays. This synergy was also seen for Salmonella typhimurium, Klebsiella pneumoniae, and Actinobacter baumannii but not Pseudomonas aeruginosa. Similarly, a phentolamine/erythromycin combination displayed significant synergistic effects in vivo in the G. mellonella model. Phentolamine added singly to bacterial cells also resulted in direct outer membrane damage and was able to dissipate and uncouple membrane proton motive force from ATP synthesis that, resulted in enhanced cytoplasmic antibiotic accumulation via reduced efflux pump activity.

Conclusions: Phentolamine potentiates macrolide antibiotic activity via reducing efflux pump activity and direct damage to the outer membrane leaflet of Gram-negative bacteria both in vitro and in vivo.

Keywords: Gram-negative pathogens; macrolide antibiotics; phentolamine; synergistic antibacterial activity.

Grants and funding

31972735/National Natural Science Foundation of China