Inhibitors of Bacterial Multidrug Efflux Pumps Potentiate Antimicrobial Photoinactivation

Abstract

Antimicrobial photodynamic inactivation (APDI) combines a nontoxic photoactivatable dye or photosensitizer (PS) with harmless visible light to generate singlet oxygen and reactive oxygen species that kill microbial cells. Cationic phenothiazinium dyes, such as toluidine blue O (TBO), are the only PS used clinically for APDI, and we recently reported that this class of PS are substrates of multidrug efflux pumps in both gram-positive and gram-negative bacteria. We now report that APDI can be significantly potentiated by combining the PS with an efflux pump inhibitor (EPI). Killing of Staphylococcus aureus mediated by TBO and red light is greatly increased by coincubation with known inhibitors of the major facilitator pump (NorA): the diphenyl urea INF271, reserpine, 5-methoxyhydnocarpin, and the polyacylated neohesperidoside, ADH7. The potentiation effect is greatest in the case of S. aureus mutants that overexpress NorA and least in NorA null cells. Addition of the EPI before TBO has a bigger effect than addition of the EPI after TBO. Cellular uptake of TBO is increased by EPI. EPI increased photodynamic inactivation killing mediated by other phenothiazinium dyes, such as methylene blue and dimethylmethylene blue, but not that mediated by nonphenothiazinium PS, such as Rose Bengal and benzoporphyrin derivative. Killing of Pseudomonas aeruginosa mediated by TBO and light was also potentiated by the resistance nodulation division pump (MexAB-OprM) inhibitor phenylalanine-arginine beta-naphthylamide but to a lesser extent than for S. aureus. These data suggest that EPI could be used in combination with phenothiazinium salts and light to enhance their antimicrobial effect against localized infections.

Antibacterial photodynamic therapy: overview of a promising approach to fight antibiotic-resistant bacterial infections

“…Several PSs, such as the amphiphilic cationic phenothiazinium, are substrates of multidrug resistance efflux pumps [25] and may therefore be removed from the intracellular environment before APDT. Hence, inhibition of efflux pump function before photosensitization may improve APDT outcome, as has been demonstrated in several studies [27,28]. Specific export pump inhibitors were combined with several PSs, both cationic (methylene blue) and anionic (Rose Bengal), and evaluated in APDT of biofilm-derived Enterococcus faecalis [28].…”

“…Multidrug resistance pump inhibitors may therefore be employed when phenothiazinium-APDT yields suboptimal results. For example, Tegos et al have shown that co-incubation of toluidine blue O with different efflux pump inhibitors (EPIs) such as NorA and Mex-AB increased the bactericidal effect of toluidine blue O by at least 2 logs in both S. aureus and P. aeruginosa [27]. It is noteworthy that this effect was more prominent when the EPI was administered before adding toluidine blue O than after, indicating that toluidine blue O competitively binds the pump binding site of EPI and therefore blocks its access when the EPI inhibitor is administered after incubation with toluidine blue O.…”

“…APDT with methylene blue was considerably more cytotoxic to E. faecalis compared to Rose Bengal, and the combination with EP inhibitor verapamil hydrochloride significantly enhanced APDT-induced cytotoxicity by 3 Log at a relatively low light dose (5 J/cm 2 ). Another study [27] showed that the order in which EP inhibitors (the diphenyl urea INF271, reserpine, 5'-methoxyhydnocarpin, and the polyacylated neohesperidoside, ADH7; all inhibit S. aureus NorA) and PS (toluidine blue O) were applied governs the bacterial killing efficacy. When the inhibitor was applied before the PS, a superior outcome was achieved.…”

Bioactive Chitosan Nanoparticles and Photodynamic Therapy Inhibit Collagen Degradation In Vitro

“…Phenothaizinium, a commonly used PSs, which includes methylene blue and its analogues, are classified as amphipathic cations and have been reported to be substrates of bacterial multi-drug efflux pumps (85). Recently, agents known as efflux pump inhibitors (EPI) have been studied as a means of overcoming this mode of bacterial resistance (86). One such study was reported by Kishen et al (87) who demonstrated an increased disinfection potential against E. faecalis biofilm when an EPI was combined with phenothiazinium PSs for PDT disinfection.…”

All you need is light

“…86 This synergistic discovery platform has been exploited in PDT for the potentiation of the phototoxic action of PS that are designated substrates of efflux systems. 87 It has been shown that near-infrared light can cause selective photodamage of multidrug-resistant pathogens. 88 In a recent study, it has been demonstrated that photodamage of multidrug-resistant Gram-positive and Gram-negative bacteria by near-infrared (870 nm/930 nm) light potentiates erythromycin, tetracycline and ciprofloxacin.…”