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Updated: Jun 24, 2025
Antimicrobial Synergy Testing by the Inkjet Printer-assisted Automated Checkerboard Array and the Manual Time-kill Method
Published on: April 18, 2019
Fingolimod synergizes and reverses K. pneumoniae resistance to colistin
Xiang Geng1, Zhen-Dong Zhang1, Yu-Xi Li1
1Key Lab of New Animal Drug of Gansu Province, Key Lab of Veterinary Pharmaceutical Development of Ministry of Agriculture and Rural Affairs, Lanzhou Institute of Husbandry and Pharmaceutical Sciences of CAAS, Lanzhou, China.
Abstract:
Klebsiella pneumoniae (K. pneumoniae) infection and the rapid spread of multi-drug resistant (MDR) bacteria pose a serious threat to global healthcare. Polymyxin E (colistin), a group of cationic antimicrobial polypeptides, is currently one of the last resort treatment options against carbapenem-resistant Gram-negative pathogens. The effectiveness of colistin has been compromised due to its intensive use. This study found that fingolimod (FLD), a natural product derivative, exhibited a significant synergistic bactericidal effect on K. pneumoniae when combined with colistin, both in vitro and in vivo. The checkerboard method was employed to assess the in vitro synergistic effect of FLD with colistin. FLD enhanced the susceptibility of bacteria to colistin and lowered effectively minimum inhibitory concentrations (MIC) when compared to colistin MIC, and the fractional inhibitory concentrations (FIC) value was less than 0.3. The time-kill curve demonstrated that the combination treatment of FLD and colistin had significant bactericidal efficacy. The in vitro concurrent administration of colistin and FLD resulted in heightening membrane permeability, compromising cell integrity, diminishing membrane fluidity, and perturbing membrane homeostasis. They also induced alterations in membrane potential, levels of reactive oxygen species, and adenosine triphosphate synthesis, ultimately culminating in bacterial death. Moreover, the combination of FLD with colistin significantly influenced fatty acid metabolism. In the mouse infection model, the survival rate of mice injected with K. pneumoniae was significantly improved to 67% and pathological damage was significantly relieved with combination treatment of FLD and colistin when compared with colistin treatment. This study highlights the potential of FLD in combining with colistin for treating infections caused by MDR isolates of K. pneumoniae.
Insights
Fingolimod (FLD) combined with colistin shows significant synergistic bactericidal effects against Klebsiella pneumoniae. This combination therapy enhances colistin
Area of Science:
- Microbiology
- Pharmacology
- Infectious Diseases
Background:
- Multi-drug resistant (MDR) bacteria, particularly Klebsiella pneumoniae, pose a critical global health threat.
- Colistin is a last-resort antibiotic for carbapenem-resistant Gram-negative infections, but its efficacy is declining due to resistance.
- There is an urgent need for novel therapeutic strategies to combat MDR K. pneumoniae infections.
Purpose of the Study:
- To investigate the synergistic bactericidal effect of fingolimod (FLD) in combination with colistin against Klebsiella pneumoniae.
- To elucidate the in vitro and in vivo mechanisms underlying the synergistic action of FLD and colistin.
- To evaluate the therapeutic potential of FLD-colistin combination therapy in a mouse infection model.
Main Methods:
- Checkerboard method to determine in vitro synergy and fractional inhibitory concentration (FIC) index.
- Time-kill curve assays to assess bactericidal efficacy.
- Analysis of bacterial membrane permeability, integrity, fluidity, membrane potential, reactive oxygen species (ROS), ATP synthesis, and fatty acid metabolism.
- In vivo efficacy assessment in a K. pneumoniae mouse infection model.
Main Results:
- Fingolimod (FLD) demonstrated significant in vitro synergy with colistin against K. pneumoniae, reducing minimum inhibitory concentrations (MICs) with an FIC value < 0.3.
- The combination treatment exhibited potent bactericidal activity, as evidenced by time-kill curves.
- FLD and colistin disrupted bacterial membrane integrity, altered membrane potential, increased ROS, and perturbed ATP synthesis, leading to cell death.
- Combined therapy significantly improved survival rates to 67% and reduced pathological damage in a K. pneumoniae mouse infection model compared to colistin alone.
Conclusions:
- Fingolimod (FLD) exhibits a significant synergistic bactericidal effect with colistin against Klebsiella pneumoniae.
- The combination therapy targets multiple bacterial cellular processes, including membrane integrity and energy metabolism.
- FLD holds promise as a potential adjuvant therapy to combat infections caused by multidrug-resistant Klebsiella pneumoniae.
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