Polymicrobial biofilms, in which mixed microbial species are present, play a significant role in persistent infections. Furthermore, polymicrobial biofilms promote antibiotic resistance by allowing interspecies transfer of antibiotic resistance genes. In the present study, we investigated the effectiveness of antimicrobial blue light (aBL; 405 nm), an innovative non-antibiotic approach, for the inactivation of polymicrobial biofilms. Dual-species biofilms with Pseudomonas aeruginosa and methicillin-resistant Staphylococcus aureus (MRSA) as well as with P. aeruginosa and Candida albicans were reproducibly grown in 96-well microtiter plates or in the CDC biofilm reactor for 24 or 48 h. The effectiveness of aBL inactivation of polymicrobial biofilms was determined through colony forming assay and compared with that of monomicrobial biofilms of each species. aBL-induced morphological changes of biofilms were analyzed with confocal laser scanning microscopy (CLSM) and scanning electron microscopy (SEM). For 24-h old monomicrobial biofilms formed in 96-well microtiter plates, 6.30-log 10 CFU inactivation of P. aeruginosa , 2.33-log 10 CFU inactivation of C. albicans and 3.48-log 10 CFU inactivation of MRSA were observed after an aBL exposure of 500 J/cm 2 . Under the same aBL exposure, 6.34-log 10 CFU inactivation of P. aeruginosa and 3.11-log 10 CFU inactivation of C. albicans were observed, respectively, in dual-species biofilms. In addition, 2.37- and 3.40-log 10 CFU inactivation were obtained in MRSA and P. aeruginosa , dual-species biofilms. The same aBL treatment of the biofilms developed in the CDC-biofilm reactor for 48 h significantly decreased the viability of P. aeruginosa monomicrobial and polymicrobial biofilm when cocultured with MRSA (3.70- and 3.56-log 10 CFU inactivation, respectively). 2.58-log 10 CFU inactivation and 0.86-log 10 CFU inactivation was detected in MRSA monomicrobial and polymicrobial biofilm when cocultured with P. aeruginosa . These findings were further supported by the CLSM and SEM experiments. Phototoxicity studies revealed a no statistically significant loss of viability in human keratinocytes after an exposure to 216 J/cm 2 and a statistically significant loss of viability after 500 J/cm 2 . aBL is potentially an alternative treatment against polymicrobial biofilm-related infections. Future studies will aim to improve the efficacy of aBL and to investigate aBL treatment of polymicrobial biofilm-related infections in vivo .

Background and Objectives Biofilms cause more than 80% of infections in humans, including more than 90% of all chronic wound infections and are extremely resistant to antimicrobials and the immune system. The situation is exacerbated by the fast spreading of antimicrobial resistance, which has become one of the biggest threats to current public health. There is consequently a critical need for the development of alternative therapeutics. Antimicrobial blue light (aBL) is a light‐based approach that exhibits intrinsic antimicrobial effect without the involvement of exogenous photosensitizers. In this study, we investigated the antimicrobial effect of this non‐antibiotic approach against biofilms formed by microbial isolates of multidrug‐resistant bacteria. Study Design/Materials and Methods Microbial isolates of Acinetobacter baumannii, Candida albicans, Escherichia coli, Enterococcus faecalis, MRSA, Neisseria gonorrhoeae, Pseudomonas aeruginosa, and Proteus mirabilis were studied. Biofilms were grown in microtiter plates for 24 or 48 hours or in the CDC biofilm reactor for 48 hours and exposed to aBL at 405 nm (60 mW/cm2, 60 or 30 minutes). The anti‐biofilm activity of aBL was measured by viable counts. Results The biofilms of A. baumannii, N. gonorrhoeae, and P. aeruginosa were the most susceptible to aBL with between 4 and 8 log10 inactivation after 108 J/cm2 (60 mW/cm2, 30 minutes) or 216 J/cm2 (60 mW/cm2, 60 minutes) aBL were delivered in the microplates. On the contrary, the biofilms of C. albicans, E. coli, E. faecalis, and P. mirabilis were the least susceptible to aBL inactivation (−0.30, −0.24, −0.84, and −0.68 log10 inactivation, respectively). The same aBL treatment in biofilms developed in the CDC biofilm reactor, caused −1.68 log10 inactivation in A. baumannii and −1.74 and −1.65 log10 inactivation in two different strains of P. aeruginosa. Conclusions aBL exhibits potential against pathogenic microorganisms and could help with the significant need for new antimicrobials in clinical practice to manage multidrug‐resistant infections. Lasers Surg. Med. © 2019 Wiley Periodicals, Inc.

Antimicrobial resistance in Neisseria gonorrhoeae is a major issue of public health, and there is a critical need for the development of new antigonococcal strategies. In this study, we investigated the effectiveness of antimicrobial blue light (aBL; wavelength, 405 nm), an innovative nonpharmacological approach, for the inactivation of N. gonorrhoeae. Our findings indicated that aBL preferentially inactivated N. gonorrhoeae, including antibiotic-resistant strains, over human vaginal epithelial cells in vitro. Furthermore, no aBL-induced genotoxicity to the vaginal epithelial cells was observed at the radiant exposure used to inactivate N. gonorrhoeae. aBL also effectively inactivated N. gonorrhoeae that had attached to and invaded into the vaginal epithelial cells in their cocultures. No gonococcal resistance to aBL developed after 15 successive cycles of inactivation induced by subtherapeutic exposure to aBL. Endogenous aBL-activatable photosensitizing porphyrins in N. gonorrhoeae were identified and quantified using ultraperformance liquid chromatography, with coproporphyrin being the most abundant species in all N. gonorrhoeae strains studied. Singlet oxygen was involved in aBL inactivation of N. gonorrhoeae. Together, these findings show that aBL represents a potential potent treatment for antibiotic-resistant gonococcal infection.