Abstract
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 (RIFR) 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.
Highlights
During the development of new antimicrobial approaches, it is important to assess the risk of tolerance and/or resistance development
When the bactericidal efficacy of Rose Bengal (RB)-dependent Antimicrobial photodynamic inactivation (aPDI) was investigated against both S. aureus control samples and S. aureus treated with 15 cycles of RB-aPDI, antimicrobial blue light (aBL) or CIP, significant tolerance development was observed exclusively in the S. aureus that underwent the RB-aPDI treatment (Fig. 3)
We demonstrated that sub-lethal aBL and aPDI (employing various photosensitizing compounds, i.e., RB, new methylene blue (NMB), toluidine blue O (TBO), a cationic porphyrin derivative (TMPyP) and zinc phthalocyanine (ZnPc)) drive substantial DNA damage in S. aureus, leading to the activation of RecA and an increased SOS response[2]
Summary
During the development of new antimicrobial approaches, it is important to assess the risk of tolerance and/or resistance development. Antimicrobial photodynamic inactivation (aPDI) and antimicrobial blue light (aBL) are defined as treatments involving exogenously- or endogenously produced photosensitizing compounds (photosensitizers, PS) that can be activated with light of the appropriate wavelength This photoactivation leads to the formation of reactive oxygen species (ROS) and free radicals that result in the oxidation of membrane lipids and damage to proteins and nucleic acids[1]. Tolerance development has been extensively studied separately for both aPDI and aBL and reviewed by Kashef and Hamblin in depth in 20175 All of these studies follow the existing dogma that aPDI and aBL, due to the nonselective, multifactorial and ROS-dependent mechanisms of action of these phototreatments, are unlikely to induce bacterial tolerance and/or resistance. There is no standard protocol to predict bacterial resistance development; when introducing new or reviving old antimicrobials, the studies need to meet the following requirements[6,7,8,9,10]:
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