Fluoxetine and thioridazine inhibit efflux and attenuate crystalline biofilm formation by Proteus mirabilis

Abstract

Proteus mirabilis forms extensive crystalline biofilms on indwelling urethral catheters that block urine flow and lead to serious clinical complications. The Bcr/CflA efflux system has previously been identified as important for development of P. mirabilis crystalline biofilms, highlighting the potential for efflux pump inhibitors (EPIs) to control catheter blockage. Here we evaluate the potential for drugs already used in human medicine (fluoxetine and thioridazine) to act as EPIs in P. mirabilis, and control crystalline biofilm formation. Both fluoxetine and thioridazine inhibited efflux in P. mirabilis, and molecular modelling predicted both drugs interact strongly with the biofilm-associated Bcr/CflA efflux system. Both EPIs were also found to significantly reduce the rate of P. mirabilis crystalline biofilm formation on catheters, and increase the time taken for catheters to block. Swimming and swarming motilies in P. mirabilis were also significantly reduced by both EPIs. The impact of these drugs on catheter biofilm formation by other uropathogens (Escherichia coli, Pseudomonas aeruginosa) was also explored, and thioridazine was shown to also inhibit biofilm formation in these species. Therefore, repurposing of existing drugs with EPI activity could be a promising approach to control catheter blockage, or biofilm formation on other medical devices.

Figure 1

Effect of putative EPIs on accumulation of EtBr in P. mirabilis. The EPI activity of thioridazine and fluoxetine was assessed using the EtBr accumulation assay in artificial urine supplemented with 20 mM glucose, against the P. mirabilis catheter isolates. Fluorescence intensity relative to cell free controls was measured after 2 h exposure to EPIs at concentrations (µg/mL) reflecting 0.125x and 0.25x the MIC for each drug against P. mirabilis strain B4. Readings from treated cells were compared with untreated controls (No EPI but with EthBr substrate), and cells treated with the proton gradient decoupling agent carbonyl cyanide m-chlorophenyl hydrazine (CCCP), as a+ve control for the accumulation assay. (a) Results of accumulation assays for treatment with CCCP, thioridazine, or fluoxetine. (b) Results of accumulation assays for treatment with CCCP, thioridazine, or fluoxetine against a broader panel of P. mirabilis clinical isolates **P < 0.001, ****P < 0.00001 vs untreated controls. All data represent the mean of 3 replicates. Error bars show standard error of the mean.

Figure 2

Interaction of fluoxetine and thioridazine with the biofilm-associated Bcr/CflA transporter. (a) Average 2D and 3D structures of bcr/CflA:thioridazine and bcr/CflA:fluoxetine; Binding sites and key interactions between the Bcr/CflA transporter and (b) thioridazine; (c) fluoxetine.

Figure 3

Effect of EPI administration on P. mirabilis crystalline biofilm formation. To investigate the impact of EPI treatment on crystalline biofilm formation, timed bladder model experiments were conducted and levels of encrustation quantified and visualised at the end of experiments. Models were supplied with standardised artificial urine with initial pH of 6.1, and containing EPIs at 0.5x MIC of each drug tested (thioridazine 400 µg/mL, or fluoxetine 128 µg/mL). Models were inoculated with 10⁹ CFU/mL of P. mirabilis strain B4 (simulating established infection), and viable planktonic cells and pH measured in residual bladder after 10 h when models were deactivated. Catheters were removed and sectioned for analysis at the 10 h time point. (a) Schematic showing catheter sections analysed for levels of encrustation. (b) Quantification of calcium on catheter sections by flame photometry as a measure of encrustation. (c) Scanning Electron Microscopy of cross sections adjacent to catheter section 1 showing levels of crystalline biofilm formed in control and treated catheters. (d) pH of residual bladder urine after 10 h. (e) Number of viable cells present in residual urine after 10 h. Data represent a minimum of three replicate experiments. Error bars show standard error of the mean. *P < 0.05 vs control. The diagram of catheter section used in part a of this Figure is taken from Holling et al. 2014 Infect Immun 80: 1616–1626.

Figure 4

Effect of EPI administration on ability of P. mirabilis to block indwelling urethral catheters. Models were supplied with standardised artificial urine with initial pH of 6.1, and EPIs thioridazine and fluoxetine were supplied in artificial urine media throughout experiment. Models simulating both established infection (inoculated with 10⁹ CFU/mL of P. mirabilis strain B4), or early stages of infection (inoculated with 10³ CFU/mL of P. mirabilis strain B4) were run. Upon blockage viable planktonic cells in residual urine in “bladders” were enumerated and pH measured. (a) Impact of EPI treatment on time taken for catheters to block in models simulating established infection. Models were treated with concentrations reflecting 0.125x, 0.25x, or 0.5x the MIC of each drug (100, 200, 400 µg/mL thioridazine; 32, 64, 128 µg/mL fluoxetine). (b) Impact of EPI treatment on time taken for catheters to block in models simulating early stages of infection. Models were treated with concentrations reflecting 0.5x MIC values for each drug (400 µg/mL thioridazine, 128 µg/mL fluoxetine). Data represent a minimum of three replicate experiments. Error bars show standard error of the mean. ***P < 0.0001 vs control; ****P < 0.00001 vs control.

Figure 5

Effect of EPI treatment on motility. The impact of EPI treatment on swimming and swarming motility in strain B4 was evaluated by supplementation of LB agar (1.5% for swarming and 0.15% for swimming) with either (a) thioridazine (100, 200 or 400 µg/mL) or (b) fluoxetine (32, 64 or 128 µg/mL). Significant differences in motility upon EPI exposure are denoted by changes in distances migrated (mm) compared to untreated controls. Data represent the mean of at least 3 replicates, and error bars show standard error of the mean. *P < 0.05 vs untreated control; **P < 0.001 vs untreated control; ***P < 0.0001 vs untreated control; ****P < 0.00001 vs untreated control.

Figure 6

Effect of EPI administration on catheter biofilm formation by other uropathogens. To investigate the impact of EPI treatment on biofilm formation by Escherichia coli and Pseudomonas aeruginosa, timed bladder model experiments were conducted and levels of biofilm formation quantified at the end of experiments. Models were supplied with standardised artificial urine with initial pH of 6.1, and containing EPIs at 0.5x MIC of each drug tested (thioridazine 400 µg/mL, or fluoxetine 128 µg/mL). Models were inoculated with 10⁹ CFU/mL of E. coli or P. aeruginosa (simulating established infection), and viable planktonic cells and pH measured in residual bladder after 10 h when models were deactivated. Catheters were removed and sectioned for analysis at the 10 h time point as shown in Fig. 6. (a,b) Quantification of biomass on catheter sections using modified crystal violet biofilm quantification assay. (c,d) Number of viable cells present in residual urine after 10 h. (e,d) pH of residual bladder urine after 10 h. Data represent a minimum of three replicate experiments. Error bars show standard error of the mean. *P < 0.05, ** < 0.01 vs control.