In this study, 8 colistin-resistant P. aeruginosa clinical strains were isolated from the First Affiliated Hospital of Wenzhou Medical University during 2015–2017, and the same strain from the same patient was eliminated. All species’ identification was performed by using the matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS, BioMerieux, France) according to the manufacturer’s instructions. The isolates were frozen in Luria Bertani (LB) (Thermo Fisher Scientific, America) broth medium supplemented with 30% glycerin at −80°C for further research. As a quality control strain, P. aeruginosa ATCC27853 was purchased from the National Center for Clinical Laboratories.

Mefloquine (MFQ) was purchased from Shanghai Yuanye Biotechnology Co., Ltd. (Shanghai, China). Other clinically commonly used antibiotics used in the present study included aztreonam (ATM), ceftazidime (CAZ), cefepime (FEP), imipenem (IPM), meropenem (MEM), ciprofloxacin (CIP), levofloxacin (LVX), gentamicin (GEN), tobramycin (TOB), amikacin (AMK), piperacillin/tazobactam (TZP), piperacillin (PIP), and colistin (COL) (Wenzhou Kangtai Biotechnology Co., Ltd., Zhejiang China). Mefloquine was dissolved in 2.5% dimethyl sulfoxide (DMSO) (Sigma-Aldrich, Saint Louis, United States). Cation-adjusted Mueller-Hinton broth (CAMHB) (Thermo Fisher Scientific) medium was used for antimicrobial susceptibility testing.

The minimum inhibitory concentrations (MICs) of commonly used antibiotics and mefloquine against 8 colistin-resistant P. aeruginosa were determined by the microbroth dilution method, as described elsewhere (Andrews, 2001), with slight modifications. Briefly, the bacteria cultured overnight were adjusted to 0.5 McFarland (0.5 McFarland ≈ 1.5 × 10⁸ CFU/mL) in sterile saline water and diluted at 1:100 in CAMHB. Then, 100 μL of the bacterial suspension was added to 96-well microplates to achieve a final bacterial concentration of 7.5 × 10⁵ CFU/mL. Next, the antibiotics were diluted with twofold serial dilutions in their respective solvents (0.25–256 μg/mL). The antibiotic was added to 96-well plates, and the diluted bacterial suspension was mixed. The results were observed after incubation of the plate at 37°C for 16–18 h. The results were interpreted with reference to the latest CLSI. MIC was defined as the lowest concentration of an antibiotic that could completely inhibit the bacterial growth. Each experiment was repeated independently three times.

The checkerboard method was employed to evaluate the synergistic effect of mefloquine and colistin, as described previously (Zharkova et al., 2019), albeit with some minor modifications. This assay was performed in a 96-well microplate. First, colistin was used as the drug A for twofold serial dilutions. Mefloquine in varying concentrations was prepared and used as the drug B. Then, the overnight bacterial culture was adjusted to 0.5 McFarland (0.5 McFarland ≈ 1.5 × 10⁸ CFU/mL) in sterile saline water and diluted at 1:100 in CAMHB. We then added 100 μL of the bacterial suspension to a 96-well microplate, which gave a final bacterial concentration of 7.5 × 10⁵ CFU/mL, followed by mixing with drugs A and B. Finally, the 96-well plates were incubated at 37°C for 16–20 h to observe the results. We then evaluated the synergistic effect of mefloquine combined with colistin by using a fractional inhibitory concentration index (FICI). The FICI was calculated using the formula: FICI = (MIC of drug A in the combination/MIC of drug A alone) + (MIC of drug B in the combination/MIC of drug B alone). Interactions were interpreted as follows: synergy for FICI ≤ 0.5; no interaction for 0.5 < FICI ≤ 4; antagonism for FICI > 4 (She et al., 2021). Each test was performed in triplicate.

The time-kill assay was performed to further evaluate the synergistic effect of mefloquine and colistin (Fadwa et al., 2021), with some minor modifications. Four groups were established, namely the control group (without drug), colistin monotherapy group, mefloquine monotherapy group, and combined group. Then, 200 μL of 0.5 McFarland bacterial suspension was added to a 50-mL centrifuge tube containing 20 mL of LB broth. The colistin monotherapy group with a final colistin concentration of 0.25–1 μg/mL, the mefloquine monotherapy group with a final mefloquine concentration of 32–64 μg/mL, and the monotherapy concentration of the corresponding strain was added to the combined group. The 50-mL centrifuge tube was then shaken at 200 rpm at 37°C. The bacterial counts were enumerated on LB agar plates at 0, 2, 4, 6, 12, and 24 h. According to the growth rate of bacteria, appropriate dilution concentration was prepared, and 100 μL of the bacterial suspension was taken at each time point for counting purpose. The bactericidal action was defined as a ≥ 3 log₁₀ reduction in CFU/mL at 24 h. The synergistic effect was defined as a ≥ 2 log₁₀ reduction in CFU/mL for the combination in comparison to its most active agent after 24 h (Fadwa et al., 2021). All studies were conducted in duplicate.

The effect of mefloquine combined with colistin on biofilm formation was studied after crystal violet staining (Djordjevic et al., 2002), albeit with some minor modifications. First, fresh single colonies grown on blood plates were shaken overnight in 3-mL LB broth medium at 37°C. After that, sterile normal saline was used to adjust the turbidity of the strains to 0.5 McFarland, and 1:100 diluted culture in LB broth before being added to the 96-well plate. Mefloquine and colistin were then added to the 96-well plate at a final concentration of 16–64 μg/mL and 0.5–1 μg/mL, respectively, in combination and alone. The 96-well plates were then incubated at 37°C for 24 h. We washed the 96-well plate thrice with sterile phosphate buffered saline (PBS) (Sigma–Aldrich, Milan, Italy) to remove the planktonic bacteria. After the plate dried naturally at room temperature, 1% crystal violet (Beijing Solarbio Biotechnology Co., Ltd., China) was added and the plate was incubated at 37°C for 15 min. After staining, the 96-well plates were cleaned with sterile PBS. After washing, the 96-well plates were naturally dried and 200 μL of 95% ethanol + 5% acetic acid was added to dissolve crystal violet. The dissolved crystal violet was then transferred to a new 96-well plate. Biofilm biomass was determined by measuring the absorbance at 595 nm. All tests were performed in triplicate in at least three independent experiments.

We evaluated the eradication effect of mefloquine combined with colistin on mature biofilm as suggested elsewhere (She et al., 2020), with minor modifications. First, fresh single colonies were picked from a blood plate and shaken overnight in a 3-mL LB broth medium at 37°C. The resultant bacterial suspension was adjusted to 0.5 McFarland with sterile saline water. Then, 0.5 McFarland bacterial suspension was diluted 1:100 in LB broth and incubated in a 96-well plate for 24 h. After incubation, mefloquine and colistin were added to the 96-well plates at a final concentration of 128 and 8 μg/mL alone or in combination. The 96-well plates were then incubated at 37°C for 24 h. The eradication of biofilm by mefloquine in combination with colistin was determined as described previously.

SEM was used to further study the inhibitory effect of mefloquine/colistin combination on biofilms (Zhang et al., 2020), with minor modifications. In the SEM experiment, TL2314 was selected as the experimental strain. We conducted experiments in sterile cell culture on 6-well plates (NEST Biotechnology Co., Ltd., China). The control group, colistin monotherapy group, mefloquine monotherapy group, and combination group were accordingly established. First, sterile cover slides (lot number: NO.10211818C, CITOGLAS Co., Ltd., China) were placed in a 6-well plate to provide a surface for biofilm formation, to which 100 μL of 0.5 McFarland bacterial suspension was added to the 6-well plate. Then, 1,900 μL of the LB broth was added to the control group, LB broth with a final concentration of 0.5 μg/mL colistin or 64 μg/mL mefloquine was added to the monotherapy group, and LB broth containing a final concentration of 0.5 μg/mL colistin and 64 μg/mL mefloquine was added to the combined group and incubated at 37°C for 24 h. Later, the cover glass was placed in a sterile 6-well cell culture plate containing 2.5% glutaraldehyde, fixed at 4°C for 4 h, and then dehydrated with a series of gradient concentrations (30, 50, 70, 90, and 100%) of ethanol for 10 min. The final samples were dried at room temperature and then observed by SEM (S-3000N, Japan).

We evaluated the synergistic effect of mefloquine and colistin by calculating the survival rate using a G. mellonella infection model in vivo (Tsai et al., 2016; Grygorcewicz et al., 2020), with minor modifications. Only the evenly uniformly milky white larvae of G. mellonella weighing about 250–300 mg were selected for the experiment. TL1671 and TL2314 were used as the experimental strains in this experiment. Briefly, first, a single colony was selected from the blood plate and adjusted to 0.5 McFarland. TL1671 was diluted to 1.5 × 10² CFU/mL and TL2314 to 1.5 × 10³ CFU/mL. The following four groups were established: control group, mefloquine monotherapy group, colistin monotherapy group, and combination group. Then, 10 μL of the bacteria suspension was injected into the rear left proleg of G. mellonella by using a microinjector. The negative control group was only injected with sterile saline water. The positive control group was injected with a corresponding concentration of bacterial suspension and sterile saline water. The colistin monotherapy group was injected with bacterial suspension and colistin (colistin 0.5, 1, and 2 μg/mL × 7). The mefloquine monotherapy group was injected with the bacterial suspension and mefloquine (64 μg/mL × 7). The combination group was injected with the bacterial suspension. The combined concentrations of mefloquine and colistin were 0.5 + 64 μg/mL × 7, 1 + 64 μg/mL × 7, 2 + 64 μg/mL × 7, respectively. All groups were injected with bacterial suspension 2 h before the drug injection. The larvae were then placed at 37°C to record the 7-day survival rate. The death of G. mellonella larvae was determined mainly by recording changes in their skin color and non-response to stimuli. Kaplan-Meier analysis and log-rank test were performed to analyze the survival rate of G. mellonella larvae. All experiments were conducted in triplicate.

Data were expressed as mean ± standard deviation of at least three independent trials. Significance was determined by using two-sample t-test, log-rank test and mentioned as P < 0.05 (noted with^∗), P < 0.01 (noted with^∗∗), and P < 0.001 (noted with^∗∗∗). Statistical analyses were performed using the Graph Pad Prism 8.0 statistical software.

The MICs for common antibiotics used in clinics for the treatment of eight colistin-resistant P. aeruginosa strains are listed in Table 1. Most of these strains were characterized by MDR phenotypes. In fact, most of the strains showed different degrees of resistance toward aminoglycosides, fluoroquinolones, β-lactam, and polypeptide antibiotics. Among the tested anti-bacterial agents, the resistance rate of these strains toward TZP, PIP, IPM, and MEM reached more than 75%. The sensitivity toward AMK and FEP was recorded to be the highest, with a resistance rate of 12.5%. MICs of colistin toward all strains were recorded to be in the range of 4–128 μg/mL. MIC for mefloquine was recorded to be > 256 μg/mL for all strains, which indicated that mefloquine exhibited no antibacterial activity against these eight colistin-resistant P. aeruginosa strains.

The synergistic effect of application of 0.125–2 μg/mL of colistin in combination with 4–32 μg/mL mefloquine was evaluated against colistin-resistant P. aeruginosa. As reported in Table 2, the combination of mefloquine with colistin showed synergistic activity against all strains, with FICI < 0.5. When colistin was combined with mefloquine, MIC values for colistin showed a significant reduction, by 8–64 times. In fact, combination of colistin resulted in a significant change in the sensitivity of all strains toward colistin, wherein drug-resistant phenotype changed to sensitive phenotype.

As shown in Figure 1, time killing curve was used to further verify the synergistic effect of mefloquine and colistin on eight colistin-resistant P. aeruginosa strains. The drug concentrations used for time-kill curve were derived from the results of checkerboard assay, with FICI < 0.5. The concentrations of mefloquine that were selected included 32 and 64 μg/mL, and the concentrations of colistin were 0.25, 0.5, and 1 μg/mL. When compared with control group, monotherapy group showed little or no time-dependent bactericidal activity, as bacterial load did not decrease. In the first 12 h, mefloquine in combination with colistin significantly reduced the growth of colistin-resistant P. aeruginosa, by more than 3 log₁₀ (CFU/mL), as compared to control group and monotherapy group. However, the inhibition effect weakened after 12 h, as the bacteria started to regrow at that time.

Next, the effects of mefloquine and colistin alone or in combination on the formation of biofilms of P. aeruginosa were studied. As shown in Figure 2, mefloquine combined with colistin inhibited biofilm formation in most of the strains (5/8), as compared to monotherapy group and control group (P < 0.05). Additionally, biofilm formation was found to be inhibited in other strains in case of combination group, when compared with the control group, however, there was no difference when compared with monotherapy group. The synergistic concentration for each strain was derived from checkerboard method.

The study also investigated whether mefloquine combined with colistin could eradicate preformed mature biofilms. The results of the same are shown in Figure 3. Mefloquine combined with colistin eradicated biofilms for most of the strains (7/8), when compared with control and monotherapy groups (P < 0.05).

Altogether, these results suggested that the combination of mefloquine and colistin could inhibit biofilm formation of P. aeruginosa, and also eradicated mature biofilm.

The inhibitory effect of mefloquine combination with colistin on biofilm formation for colistin-resistant P. aeruginosa was visualized using SEM. The results of the same are shown in Figure 4. SEM images showed that biofilms for control group, mefloquine monotherapy group (64 μg/mL), and colistin monotherapy group (0.5 μg/mL) were not damaged, when observed at 3,000 and 7,000 × magnification, wherein the cells were characterized by complete morphology and high density. When compared with control group and monotherapy group, the combined group exhibited more and significant changes, which were mainly manifested at 7,000 × magnifications. In particular, biofilm structure was greatly damaged, and the original, complete, and tightly adhered biofilm was found to be completely destroyed.

The efficacy of mefloquine in combination with colistin was further validated in vivo by recording survival rates in a model of G. mellonella infection. The results of the same are shown in Figure 5. TL1671 and TL2314 were randomly selected as experimental strains. For control group, mefloquine monotherapy group, and colistin monotherapy group, almost no survival was observed in TL1671 within 24 h. However, mefloquine combination with colistin maintained a survival rate of > 70%, after 168 h of the treatment. For the combination of 1 μg/mL of colistin and 64 μg/mL of mefloquine, the survival rate for G. mellonella reached 100%, after 168 h (P < 0.05). In case of TL2314, control group, monotherapy group, and combination of 0.5 μg/mL of colistin and 64 μg/mL of mefloquine showed no survival after 24 h. The survival rate for G. mellonella for two combinations of colistin (1 and 2 μg/mL) and mefloquine (64 μg/mL) reached > 50% (P < 0.05).