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. 2023 Nov 18:16:7271-7288.
doi: 10.2147/IDR.S427232. eCollection 2023.

Combination of AS101 and Mefloquine Inhibits Carbapenem-Resistant Pseudomonas aeruginosa in vitro and in vivo

Rongrong Li  1   2 , Xuhang Shen  3 , Zhengyuan Li  4 , Jilong Shen  2 , Hao Tang  5 , Huaming Xu  6 , Jilu Shen  1   7 , Yuanhong Xu  1
Affiliations

Combination of AS101 and Mefloquine Inhibits Carbapenem-Resistant Pseudomonas aeruginosa in vitro and in vivo

Rongrong Li et al. Infect Drug Resist. .

Abstract

Background: In recent years, carbapenem-resistant Pseudomonas aeruginosa (CRPA) has spread around the world, leading to a high mortality and close attention of medical community. In this study, we aim to find a new strategy of treatment for CRPA infections.

Methods: Eight strains of CRPA were collected, and PCR detected the multi-locus sequence typing (MLST). The antimicrobial susceptibility test was conducted using the VITEK@2 compact system. The minimum inhibitory concentration (MIC) for AS101 and mefloquine was determined using the broth dilution method. Antibacterial activity was tested in vitro and in vivo through the chessboard assay, time killing assay, and a mouse model. The mechanism of AS101 combined with mefloquine against CRPA was assessed through the biofilm formation inhibition assay, electron microscopy, and detection of reactive oxygen species (ROS).

Results: The results demonstrated that all tested CRPA strains exhibited multidrug resistance. Moreover, our investigation revealed a substantial synergistic antibacterial effect of AS101-mefloquine in vitro. The assay for inhibiting biofilm formation indicated that AS101-mefloquine effectively suppressed the biofilm formation of CRPA-5 and CRPA-6. Furthermore, AS101-mefloquine were observed to disrupt the bacterial cell wall and enhance the permeability of the cell membrane. This effect was achieved by stimulating the production of ROS, which in turn hindered the growth of CRPA-3. To evaluate the therapeutic potential, a murine model of CRPA-3 peritoneal infection was established. Notably, AS101-mefloquine administration resulted in a significant reduction in bacterial load within the liver, kidney, and spleen of mice after 72 hours of treatment.

Conclusion: The present study showed that the combination of AS101 and mefloquine yielded a notable synergistic bacteriostatic effect both in vitro and in vivo, suggesting a potential clinical application of this combination in the treatment of CRPA.

Keywords: AS101; Pseudomonas aeruginosa; biofilm; carbapenem resistance; mefloquine; synergy.

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Conflict of interest statement

The authors have no competing interests to declare for this work.

Figures

Figure 1
Figure 1
Time killing curves of AS101 and mefloquine acting alone or jointly on eight CRPA strains.
Figure 2
Figure 2
AS101 and mefloquine used separately or jointly on the inhibition of biofilm formation of CRPA. Bars of different colors represented different treatment groups. The data were analyzed by Student’s t test; ns, no statistical significance; *P < 0.05; **P < 0.01; ***P < 0.001. The experiment was conducted three times. Data was expressed in mean±standard deviation; OD595, optical density at 595 nm.
Figure 3
Figure 3
SEM images of CRPA-3 processed by AS101 and mefloquine. (A) BHI blank control at 15 000x magnetization (B) Mefloquine at 15 000x magnetization (C) AS101 at 15 000x magnetization (D) Mefloquine–AS101 combination at 15 000x magnetization. The red arrows indicate the holes in the bacterial cell wall.
Figure 4
Figure 4
TEM images of CRPA-3 processed with AS101 and mefloquine. (A) BHI blank control at 22 000x magnetization (B) Mefloquine at 22 000x magnetization (C) AS101 at 22 000x magnetization (D) Mefloquine–AS101 combination at 22 000x magnetization. The red arrows indicate cell wall ruptured.
Figure 5
Figure 5
The flow cytometry picture of cell membrane permeability of CRPA-3 treated with AS101 and mefloquine. Compared with the control group (A) and the monotherapy group (B and C), the bacterial particles generally moved to the right after the combination of AS101 and mefloquine (D), that was, the fluorescence intensity increased and the permeability of cell membrane increased.
Figure 6
Figure 6
The statistical results of cell membrane permeability of the CRPA-3 under the action of AS101 and mefloquine by flow cytometry. Compared with the control group and the monotherapy group, the fluorescence intensity after the combination of AS101 and mefloquine was significantly increased. The data were analyzed by Student’s t test; ns, no statistical significance; **P < 0.01; ***P < 0.001. The experiment was conducted three times. Data was expressed in mean ± standard deviation.
Figure 7
Figure 7
ROS production after the CRPA-3 treated with AS101 and mefloquine. Compared with the control group and the monotherapy group, the combined use of AS101 and mefloquine significantly increased ROS. The data were analyzed by Student’s t test; ***P < 0.001. The experiment was conducted three times. Data was expressed in mean ± standard deviation.
Figure 8
Figure 8
Log10 changes of liver, kidney and spleen of CRPA-3 infected mice after 72 h of treatment with placebo (1 x PBS), single drug (mefloquine 10 mg/kg/day, AS101 3.33 mg/kg/day) or combination (mefloquine 10 mg/kg/day + AS101 3.33 mg/kg/day). The data were analyzed by Student’s t-test; *P < 0.05; **P < 0.01; ***P < 0.001. Three mice in each group. Data was expressed in mean ± standard deviation.
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Grants and funding

This work was supported by the Anhui Provincial Natural Fund (Project’s number: 9021138203) and the Subject Construction Fund of the First Affiliated Hospital of Anhui Medical University (Project’s number: 9001001855).