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.

The emergence of antimicrobial drug resistance requires development of alternative therapeutic options. Multidrug-resistant strains of Enterococcus spp., Staphylococcus aureus, Klebsiella pneumonia...

Enterococcus faecium and Enterococcus faecalis are opportunistic pathogens that can cause a vast variety of nosocomial infections. Moreover, E. faecium belongs to the group of ESKAPE microbes, which are the main cause of hospital-acquired infections and are especially difficult to treat because of their resistance to many antibiotics. Antimicrobial photodynamic inactivation (aPDI) represents an alternative to overcome multidrug resistance problems. This process requires the simultaneous presence of oxygen, visible light, and photosensitizing compounds. In this work, aPDI was used to resensitize Enterococcus spp. isolates to antibiotics. Antibiotic susceptibility testing according to European Committee on Antimicrobial Susceptibility Testing (EUCAST) recommendations was combined with synergy testing methods recommended by the American Society for Microbiology. Two clinical isolates, E. faecalis and E. faecium, were treated with a combination of aPDI utilizing rose bengal (RB) or fullerene (FL) derivative as photosensitizers, antimicrobial blue light (aBL), and 10 recommended antibiotics. aPDI appeared to significantly impact the survival rate of both isolates, while aBL had no significant effect. The synergy testing results differed between strains and utilized methods. Synergy was observed for RB aPDI in combination with gentamycin, ciprofloxacin and daptomycin against E. faecalis. For E. faecium, synergy was observed between RB aPDI and gentamycin or ciprofloxacin, while for RB aPDI with vancomycin or daptomycin, antagonism was observed. A combination of FL aPDI gives a synergistic effect against E. faecalis only with imipenem. Postantibiotic effect tests for E. faecium demonstrated that this isolate exposed to aPDI in combination with gentamycin, streptomycin, tigecycline, doxycycline, or daptomycin exhibits delayed growth in comparison to untreated bacteria. The results of synergy testing confirmed the effectiveness of aPDI in resensitization of the bacteria to antibiotics, which presents great potential in the treatment of infections caused by multidrug-resistant strains.

Streptococcus agalactiae (Group B Streptococcus, GBS) is a common commensal bacterium in adults but remains a leading source of invasive infections in newborns, pregnant women, and the elderly, and more recently, causes an increased incidence of invasive disease in nonpregnant adults. Reduced penicillin susceptibility and emerging resistance to non-β-lactams pose challenges for the development and implementation of novel, nonantimicrobial strategies to reduce the burden of GBS infections. Antimicrobial photodynamic inactivation (aPDI) via the production of singlet oxygen or other reactive oxygen species leads to the successful eradication of pathogenic bacteria, affecting numerous cellular targets of microbial pathogens and indicating a low risk of resistance development. Nevertheless, we have previously reported possible aPDI tolerance development upon repeated sublethal aPDI applications; thus, the current work was aimed at investigating whether aPDI tolerance could be observed for GBS and what mechanisms could cause it. To address this problem, 10 cycles of sublethal aPDI treatments employing rose bengal as a photosensitizer, were applied to the S. agalactiae ATCC 27956 reference strain and two clinical isolates (2306/02 and 2974/07, serotypes III and V, respectively). We demonstrated aPDI tolerance development and stability after 5 cycles of subculturing with no aPDI exposure. Though the treatment resulted in a stable phenotype, no increases in mutation rate or accumulated genetic alterations were observed (employing a RIF-, CIP-, STR-resistant mutant selection assay and cyl sequencing, respectively). qRT-PCR analysis demonstrated that 10 sublethal aPDI exposures led to increased expression of all tested major oxidative stress response elements; changes in sodA, ahpC, npx, cylE, tpx and recA expression indicate possible mechanisms of developed tolerance. Increased expression upon sublethal aPDI treatment was reported for all but two genes, namely, ahpC and cylE. aPDI targeting cylE was further supported by colony morphology changes induced with 10 cycles of aPDI (increased SCV population, increased hemolysis, increased numbers of dark- and unpigmented colonies). In oxidant killing assays, aPDI-tolerant strains demonstrated no increased tolerance to hypochlorite, superoxide (paraquat), singlet oxygen (new methylene blue) or oxidative stress induced by aPDI employing a structurally different photosensitizer, i.e., zinc phthalocyanine, indicating a lack of cross resistance. The results indicate that S. agalactiae may develop stable aPDI tolerance but not resistance when subjected to multiple sublethal phototreatments, and this risk should be considered significant when defining efficient anti-S. agalactiae aPDI protocols.

Light- and photosensitizer-based antimicrobial photodynamic therapy (aPDI) is a very promising approach to control microbial infections. Other applications of aPDI are also being studied, for example, in water disinfection or food preservation. Because this technology is based on the use of light, the proper characterization of light sources is of prime importance. The aim of this study was to prepare and characterize three irradiation systems constructed for research purposes. These LED-based light sources produce visible light in the range of blue, green and red wavelengths. The efficacy and light distribution produced by the light sources were verified on a Staphylococcus aureus model sensitized with three photosensitizers: protoporphyrin IX diarginate, N-methylpyrrolidinium fullerene iodide (Full), and rose bengal. Antimicrobial blue light treatment was likewise investigated. The survival of bacterial cells after photoinactivation was determined for irradiance at three power densities. Based on the proper characterization of light emitted by each LED at each point of the illuminated area, a homogeneous distribution of light can be achieved, which is crucial for precise dosimetry.