Development of Staphylococcus aureus tolerance to antimicrobial photodynamic inactivation and antimicrobial blue light upon sub-lethal treatment

Antimicrobial photodynamic inactivation (aPDI) and antimicrobial blue light (aBL) are considered low-risk treatments for the development of bacterial resistance and/or tolerance due to their multitargeted modes of action. In this study, we assessed the development of Staphylococcus aureus tolerance to these phototreatments. Reference S . aureus USA300 JE2 was subjected to 15 cycles of both sub-lethal aPDI (employing an exogenously administered photosensitizer (PS), i.e., rose Bengal (RB)) and sub-lethal aBL (employing endogenously produced photosensitizing compounds, i.e., porphyrins). We demonstrate substantial aPDI/aBL tolerance development and tolerance stability after 5 cycles of subculturing without aPDI/aBL exposure (the development of aPDI/aBL tolerance was also confirmed with the employment of clinical MRSA and MSSA strain as well as other representatives of Gram-positive microbes, i.e. Enterococcus faecium and Streptococcus agalactiae ). In addition, a rifampicin-resistant (RIF R ) mutant selection assay showed an increased mutation rate in S . aureus upon sub-lethal phototreatments, indicating that the increased aPDI/aBL tolerance may result from accumulated mutations. Moreover, qRT-PCR analysis following sub-lethal phototreatments demonstrated increased expression of umuC , which encodes stress-responsive error-prone DNA polymerase V, an enzyme that increases the rate of mutation. Employment of recA and umuC transposon S . aureus mutants confirmed SOS-induction dependence of the tolerance development. Interestingly, aPDI/aBL-tolerant S . aureus exhibited increased susceptibility to gentamicin (GEN) and doxycycline (DOX), supporting the hypothesis of genetic alterations induced by sub-lethal phototreatments. The obtained results indicate that S . aureus may develop stable tolerance to studied phototreatments upon sub-lethal aPDI/aBL exposure; thus, the risk of tolerance development should be considered significant when designing aPDI/aBL protocols for infection treatments in vitro and in clinical settings.