We have previously linked efflux activity to crystalline biofilm formation and motility in P. mirabilis, and demonstrated the importance of the Bcr/CflA MFS transporter to catheter blockage by this organism20. Results obtained in this study now provide further evidence that efflux systems are important to the formation of crystalline biofilms by P. mirabilis, and confirm that suppression of efflux using chemical inhibitors is a potentially viable approach to control biofilm formation in the catheterised urinary tract. The molecular docking study, followed by molecular dynamic simulations, also suggests that both drugs evaluated here (fluoxetine and thioridazine) bind favorably within the channel region of the biofilm-associated Bcr/CflA transporter, and form stable complexes. These interactions are predicted to be strong enough to inhibit the functional mechanism of the transporter, and when considered in the context of our previous studies of biofilm formation in Bcr/CflA transposon mutants20, are in keeping with the reduction in crystalline biofilm formation observed in bladder models treated with fluoxetine or thioridazine.

The overall findings of this study are also congruent with a growing body of evidence supporting a role for efflux systems in numerous aspects of bacterial virulence, including in pathogens relevant to CAUTI such as P. aeruginosa, E. coli, Staphylococcus aureus, and Klebsiella pneumoniae. These include aspects of survival and fitness within the host, ability to initiate infection, survival and replication within host cells, roles related to the expression of virulence attributes, and also biofilm formation20,21,23,24,28,29,30,31,32,33,34,35,36. Perhaps of most relevance to this study are investigations of biofilm formation in E. coli and P. aeruginosa, which have identified genes encoding efflux systems to be up-regulated during biofilm formation (compared to planktonic cells), among the most highly expressed genes in mature biofilms, and have linked the reduced antibiotic susceptibility of biofilms to over expression of efflux in these communities23,28,37,38,39. An EPI-mediated reduction in biofilm formation, including through the use of thioridazine, has also previously been reported in E. coli 23. Collectively these previous studies fit well with the mitigation of biofilm formation we also observed in E. coli and P. aeruginosa with thioridazine treatment.

Recent investigations have also pointed to a range of potential mechanisms through which inhibition of efflux may impact on biofilm formation. For example, signalling molecules which mediate cell-cell communication, and regulate population density dependent processes are substrates for some efflux systems33,40,41. This opens the possibility for efflux inhibitors to interfere with quorum-sensing and in turn disrupt processes orchestrated by this inter-cellular communication system, including biofilm formation. In addition, studies of Pseudomonas syringae demonstrate how the accumulation of normally extruded substrates within the cell, resulting from loss of a particular efflux pump, can alter expression of unexpected gene sets leading to changes in disparate phenotypes42. Collectively these observations afford the potential for efflux pumps to contribute to regulation of a wide range of traits relevant to virulence and biofilm formation, and indicate that these transporters may perform regulatory functions beyond their basic accepted role in cellular detoxification.

Alternatively, it has been proposed that some efflux pumps act as a waste management system during maturation of the biofilm23, allowing the densely packed population of biofilm-associated cells to deal with the inevitable build-up of toxic metabolic end products as the biofilm expands. Our previous data from a P. mirabilis Bcr/ClfA MFS transporter mutant are well aligned with this theory, and disruption of the Bcr/CflA system led to defects and reductions in later stages of biofilm development and expansion, but did not appear to impede early events in this process20. While the present study did not specifically examine the effects of thioridazine or fluoxetine on distinct stages of biofilm formation, data from timed 10 h bladder model experiments are compatible with potential effects on biofilm maturation and expansion, and EPI treatments generated similar results to those obtained from the defined Bcr/CflA mutant in analogous experiments20. Nevertheless, further investigation will be required to understand how efflux inhibition leads to reduced crystalline biofilm formation in P. mirabilis, and currently available data do not presently rule out any of the mechanisms described above.

Swarming is a complex multicellular behaviour in P. mirabilis, sharing many characteristics of biofilm formation, and also likely to be regulated by quorum sensing43. Given the almost complete abolition of swarming observed in our previous Bcr/CflA efflux mutant20, the strong effects of thioridazine and fluoxetine on swarming motility are in keeping with the inhibition of efflux by these drugs, and their predicted interaction with the Bcr/CflA system. The effects of thioridazine and fluoxetine on swimming motility are also congruent with the considerable reductions in swimming ability observed in our P. mirabilis bcr/ClfA mutant20. Moreover, reductions in both swimming and swarming have been noted in other species when efflux systems are disrupted, and linked to aberrations in flagella biosynthesis, and reductions in expression of genes related to motility30,34,42. Although the impact of thioridazine or fluoxetine treatment on flagella production in P. mirabilis is yet to be determined, this is in keeping with reductions in both swimming and swarming motilities when efflux systems are inhibited, and it is a plausible hypothesis that these drugs may influence flagellar production in P. mirabilis as well.

In the context of catheter blockage, both swimming and swarming motilities have been linked with biofilm formation and virulence in several uropathogens44,45, and are proposed to contribute to P. mirabilis crystalline biofilm formation and CAUTI. However, no concrete role for either motility has yet been established in P. mirabilis crystalline biofilm formation specifically, and mutants with defects in motility did not exhibit reduced biofilm formation in bladder model experiments5. While this does not fully preclude the potential for reductions in swimming or swarming to play a part in the attenuation of blockage generated by thioridazine and fluoxetine, it seems unlikely that this is a major factor in the effect of these drugs on crystalline biofilm formation. Nevertheless, motility and particularly swarming is considered to be important in the initial colonisation of the catheterised urinary tract5,43, and interventions that can suppress motility in P. mirabilis may have applications in preventing colonisation of the catheterised urinary tract.

In contrast to swimming and swarming, the ability to grow and persist under the conditions encountered in the catheterised urinary tract, are undoubtedly critical to the pathogenesis of P. mirabilis CAUTI, and the formation of crystalline biofilms. Although thioridazine and fluoxetine treatment showed no statistically significant impact on the numbers of viable cells in bladder models at the end of time-to-blockage experiments, a trend towards reduction in viable cell counts was observed as concentrations of thioridazine increased. Moreover, in timed models where responses to treatment were evaluated after 10 h, statistically significant reductions in viable cells were evident in both fluoxetine and thioridazine treated models.

The reduced cell numbers observed at earlier stages of colonisation or infection in bladder models suggest thioridazine and fluoxetine treatment may also delay blockage by impairing P. mirabilis growth in the bladder model system. Although this would seem to conflict with use of these drugs at concentrations well below the established MICs (0.5x and below), these MIC values were defined in standard LB broth cultures, and not the specific conditions P. mirabilis must cope with in bladder models. Furthermore, only relatively small impacts on growth in bladder models were observed at the concentrations of thioridazine and fluoxetine used in these experiments, which is compatible with use at sub-MIC concentrations.

Overall the inhibition of efflux by thioridazine and fluoxetine appears to perturb a range of traits and processes relevant to P. mirabilis CAUTI and catheter blockage, and has comparable effects to the loss of the Bcr/CflA transporter on motility and ability to encrust urethral catheters. However, it should be noted that although our molecular modelling predicts these drugs interact strongly with, and can potentially inhibit, the biofilm-associated Bcr/CflA transporter, and the effect of fluoxetine and thioridazine treatment generate phenotypes comparable to disruption of the Bcr/CflA gene, the effects of chemical EPIs are not certain to be restricted to a single specific pump, and there is much potential for the results obtained in this study to originate from a broader, more generalised interference with the P. mirabilis “effluxome”. In addition, the possibility that these drugs may also have effects on P. mirabilis unrelated to efflux inhibition cannot yet be fully excluded. Nonetheless, these data provide further support for efflux systems as a viable target for control of bacterial biofilm formation, and the Bcr/CflA MFS transporter in P. mirabilis in particular.

Further research will not only need to provide greater understanding of the specific transporters and their substrates affected by thioridazine and fluoxetine, but also how such EPIs may best be utilised to develop strategies for control of CAUTI, or wider bacterial biofilm formation. The use of these drugs according to standard current clinical practice and prescribing approaches is unlikely to be feasible or useful for the control of CAUTI (for reasons relating to safety, efficacy, and pharmacokinetics), and the concentrations utilized in these laboratory studies are much higher than those normally achieved in body fluids such as urine in practice46,47,48,49. Nevertheless, there is considerable scope to explore localised delivery in the catheterized urinary tract, the use of combinations of EPIs, or the synergistic use of EPIs with antibiotics, which may yield strategies to deploy these drugs for CAUTI control. Moreover, regardless of the potential to utilize these drugs directly, they can nevertheless serve as valuable lead compounds to stimulate the development of new classes of anti-biofilm agents with novel modes of action.