Can antimicrobial blue light contribute to resistance development? Genome-wide analysis revealed aBL-protective genes in Escherichia coli.

IMPORTANCE: Increasing antibiotic resistance and the lack of new antibiotic-like compounds to combat bacterial resistance are significant problems of modern medicine. The development of new alternative therapeutic strategies is extremely important. Antimicrobial blue light (aBL) is an innovative approach to combat multidrug-resistant microorganisms. aBL has a multitarget mode of action; however, the full mechanism of aBL antibacterial action requires further investigation. In addition, the potential risk of resistance development to this treatment should be considered.

Activity of Cinnamic Acid Derivatives with 4-Chloro-2-mercaptobenzenesulfonamide Moiety against Clinical HLAR and VRE Enterococcus spp.

The rapid increase in strains that are resistant to antibiotics requires new active compounds to be found whose mechanism of action on bacteria is different to those that are currently known. Of particular interest are compounds that occur in plants as secondary metabolites. The focus of this study concerns the examination of the effects of synthetic cinnamic acid derivatives, with 4-chloro-2-mercaptobenzenesulfonamide moiety on Enterococcus spp. with HLAR (high-level aminoglycoside resistance) and VRE (vancomycin-resistant Enterococcus) mechanisms. The minimum inhibitory concentration (MIC) values of the tested compounds were determined using the serial dilution method for Enterococcus spp. groups, and the most active compounds were as follows: 16d, 17c, 16a, 16c and 16f (2-4 µg/mL). These compounds, at a concentration of 4 × MIC, inhibited the biofilm formation of HLAR strains (70 to 94%). At concentrations of 2 × MIC and 4 × MIC, they also inhibited the growth of VRE strains (42 to 96%). The best effect produced on the formed biofilm was demonstrated by compound 16f (from 62% MIC concentration to 89% 4 × MIC concentration) on the tested HLAR strains. In vitro studies, using the peripheral blood of domestic sheep, demonstrated the stable bacteriostatic activity of the tested compounds against Enterococcus spp. The compounds 16a, 16c, 16d, 16f and 17c showed synergism and additivity with ampicillin, streptomycin, gentamicin and vancomycin against resistant strains of Enterococcus spp. The tested compounds, when combined, reduce the MIC for antibiotics by 800 to 10,000 times for HLAR strains and by 8 to 10,000 times for VRE strains. The MIC of the tested compounds, in combination with antibiotics, is reduced 2-16-fold for HLAR strains and 2-32-fold for VRE strains. These studies demonstrate the potential for the therapeutic use of synthetic, cinnamic acid derivatives, with 4-chloro-2-mercaptobenzenesulfonamide moiety, to work against clinical strains of Enterococcus spp.

Can Gram-Negative Bacteria Develop Resistance to Antimicrobial Blue Light Treatment?

Antimicrobial blue light (aBL) treatment is considered low risk for the development of bacterial resistance and tolerance due to its multitarget mode of action. The aim of the current study was to demonstrate whether tolerance development occurs in Gram-negative bacteria. We evaluated the potential of tolerance/resistance development in Escherichia coli, Klebsiella pneumoniae, and Pseudomonas aeruginosa and demonstrated that representative Gram-negative bacteria may develop tolerance to aBL. The observed adaption was a stable feature. Assays involving E. coli K-12 tolC-, tolA-, umuD-, and recA-deficient mutants revealed some possible mechanisms for aBL tolerance development.

Factors Determining the Susceptibility of Bacteria to Antibacterial Photodynamic Inactivation.

Photodynamic inactivation of microorganisms (aPDI) is an excellent method to destroy antibiotic-resistant microbial isolates. The use of an exogenous photosensitizer or irradiation of microbial cells already equipped with endogenous photosensitizers makes aPDI a convenient tool for treating the infections whenever technical light delivery is possible. Currently, aPDI research carried out on a vast repertoire of depending on the photosensitizer used, the target microorganism, and the light delivery system shows efficacy mostly on in vitro models. The search for mechanisms underlying different responses to photodynamic inactivation of microorganisms is an essential issue in aPDI because one niche (e.g., infection site in a human body) may have bacterial subpopulations that will exhibit different susceptibility. Rapidly growing bacteria are probably more susceptible to aPDI than persister cells. Some subpopulations can produce more antioxidant enzymes or have better performance due to efficient efflux pumps. The ultimate goal was and still is to identify and characterize molecular features that drive the efficacy of antimicrobial photodynamic inactivation. To this end, we examined several genetic and biochemical characteristics, including the presence of individual genetic elements, protein activity, cell membrane content and its physical properties, the localization of the photosensitizer, with the result that some of them are important and others do not appear to play a crucial role in the process of aPDI. In the review, we would like to provide an overview of the factors studied so far in our group and others that contributed to the aPDI process at the cellular level. We want to challenge the question, is there a general pattern of molecular characterization of aPDI effectiveness? Or is it more likely that a photosensitizer-specific pattern of molecular characteristics of aPDI efficacy will occur?

Development of Antimicrobial Phototreatment Tolerance: Why the Methodology Matters.

Due to rapidly growing antimicrobial resistance, there is an urgent need to develop alternative, non-antibiotic strategies. Recently, numerous light-based approaches, demonstrating killing efficacy regardless of microbial drug resistance, have gained wide attention and are considered some of the most promising antimicrobial modalities. These light-based therapies include five treatments for which high bactericidal activity was demonstrated using numerous in vitro and in vivo studies: antimicrobial blue light (aBL), antimicrobial photodynamic inactivation (aPDI), pulsed light (PL), cold atmospheric plasma (CAP), and ultraviolet (UV) light. Based on their multitarget activity leading to deleterious effects to numerous cell structures-i.e., cell envelopes, proteins, lipids, and genetic material-light-based treatments are considered to have a low risk for the development of tolerance and/or resistance. Nevertheless, the most recent studies indicate that repetitive sublethal phototreatment may provoke tolerance development, but there is no standard methodology for the proper evaluation of this phenomenon. The statement concerning the lack of development of resistance to these modalities seem to be justified; however, the most significant motivation for this review paper was to critically discuss existing dogma concerning the lack of tolerance development, indicating that its assessment is more complex and requires better terminology and methodology.

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 (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.

Antimicrobials Are a Photodynamic Inactivation Adjuvant for the Eradication of Extensively Drug-Resistant Acinetobacter baumannii.

The worldwide emergence of extensively drug resistant (XDR) Acinetobacter baumannii has reduced the number of antimicrobials that exert high bactericidal activity against this pathogen. This is the reason why many scientists are focusing on investigations concerning novel non-antibiotic strategies such as antimicrobial photodynamic inactivation (aPDI) or the use of antimicrobial blue light (aBL). Therefore, the aim of the current study was to screen for antimicrobial synergies of routinely used antibiotics and phototherapies, including both aPDI involving exogenously administered photosensitizing molecules, namely, rose bengal, and aBL, involving excitation of endogenously produced photoactive compounds. The synergy testing was performed in accordance with antimicrobial susceptibility testing (AST) standards, including various methodological approaches, i.e., antibiotic diffusion tests, checkerboard assays, CFU counting and the evaluation of postantibiotic effects (PAEs). We report that combining antimicrobials and aPDI/aBL treatment led to a new strategy that overcomes drug resistance in XDR A. baumannii, rendering this pathogen susceptible to various categories of antibiotics. Sublethal aPDI/aBL treatment in the presence of sub-MIC levels of antimicrobials effectively killed A. baumannii expressing drug resistance to studied antibiotics when treated with only antibiotic therapy. The susceptibility of XDR A. baumannii to a range of antibiotics was enhanced following sublethal aPDI/aBL. Furthermore, 3'-(p-aminophenyl) fluorescein (APF) testing indicated that significantly increased reactive oxygen species production upon combined treatment could explain the observed synergistic activity. This result represents a conclusive example of the synergistic activity between photodynamic inactivation and clinically used antimicrobials leading to effective eradication of XDR A. baumannii isolates and indicates a potent novel therapeutic approach.

Photoinactivation of ESKAPE pathogens: overview of novel therapeutic strategy.

The emergence of antimicrobial drug resistance requires development of alternative therapeutic options. Multidrug-resistant strains of Enterococcus spp., Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumanii, Pseudomonas aeruginosa and Enterobacter spp. are still the most commonly identified antimicrobial-resistant pathogens. These microorganisms are part of the so-called 'ESKAPE' pathogens to emphasize that they currently cause the majority of hospital acquired infections and effectively 'escape' the effects of antibacterial drugs. Thus, alternative, safer and more efficient antimicrobial strategies are urgently needed, especially against 'ESKAPE' superbugs. Antimicrobial photodynamic inactivation is a therapeutic option used in the treatment of infectious diseases. It is based on a combination of a photosensitizer, light and oxygen to remove highly metabolically active cells.

Fine-tuning recA expression in Staphylococcus aureus for antimicrobial photoinactivation: importance of photo-induced DNA damage in the photoinactivation mechanism.

Bacterial cell envelope is generally accepted as the primary target for a photo-induced oxidative stress. It is plausible that DNA damage occurs during the antimicrobial photoinactivation. Here we investigate the correlation between DNA damage and photoinactivation by evaluating the level of RecA-based DNA repair system in Staphylococcus aureus. By using exogenous photosensitizers (new methylene blue (NMB), toluidine blue O (TBO), 5,10,15,20-tetrakis(1-methyl-4-pyridinio)porphyrin tetra(p-toluenesulfonate) (TMPyP), zinc phthalocyanine (ZnPc), Rose Bengal (RB)) and ALA-induced endogenous porphyrin-dependent blue light (405 nm), several outcomes were observed: (i) an increase of DNA damage (from gel electrophoresis in DNA damage assay), (ii) an increase of recA expression (luminescence assay in recA-lux strain), and (iii) an increase of RecA protein level (Western blotting). When recA expression was repressed by novobiocin, or abolished by deleting the gene, S. aureus susceptibility towards photoinactivation was increased at approximately a hundred-fold. The absence of RecA increases DNA damage to yield bactericidal effect. In novobiocin-resistant mutant (gyrB), as opposed to wild type, neither RecA protein level nor cell's susceptibility was affected by photoinactivation (when novobiocin is present). This is to suggest that GyrB-dependent inhibition mediated recA repression. Therefore, we have established the role of RecA in DNA damage during photoinactivation. With the use of rifampicin mutation frequency and Ames tests, we demonstrated that photoinactivation did not increase S. aureus mutagenesis and potentially is not mutagenic toward eukaryotic cells. The results suggest that the treatment is considered safe. In conclusion, we provide an evidence that recA inhibitor may serve as therapeutic adjuvant for antimicrobial photoinactivation. Clinical relevance of our findings warrants further investigations.

Multiresistant strains are as susceptible to photodynamic inactivation as their naïve counterparts: protoporphyrin IX-mediated photoinactivation reveals differences between methicillin-resistant and methicillin-sensitive Staphylococcus aureus strains.

OBJECTIVE: The current study was aimed at the investigation of differences in response to photoinactivation between methicillin-resistant Staphylococcus aureus (MRSA) and methicillin-sensitive S. aureus (MSSA) isolates. Moreover, we aimed to elucidate if the observed variation resulted from antimicrobial resistance mechanisms and strains' susceptibility to antibiotic therapy. BACKGROUND DATA: Because of the emergence of multidrug resistance, the development of alternative antimicrobial strategies seems to be required. The concept of photodynamic inactivation (PDI) involves cell exposure to appropriate wavelength light that leads to the excitation of photosensitizer molecules, resulting in the production of reactive oxygen species responsible for cell inactivation and death. Recently, we have demonstrated a strain-dependent response of S. aureus to photoinactivation, and observed elevated resistance to PDI among MRSA strains. Nevertheless, the mechanism underlying this phenomenon remains unexplained. METHODS: S. aureus response to protoporphyrin IX (PPIX)-mediated photoinactivation was studied for 424 MRSA/MSSA isolates. VITEK 2 Advanced Expert System was used to detect antimicrobial resistance mechanisms and strains' susceptibility to antibiotictherapy. RESULTS: Data obtained demonstrated that MRSA are significantly more resistant to photoinactivation than MSSA strains; however, the difference observed did not result from antimicrobial susceptibility or resistance mechanisms. Furthermore, regardless of the strains' origin, a similar effectiveness of PDI could be achieved. Moreover, it was determined that the ability to form biofilms in vitro, and the presence of mec element, does not explain the observed differences between MRSA and MSSA strains. CONCLUSIONS: PDI could be highly effective against multidrug resistant pathogens as well as their naïve counterparts. Nevertheless, regardless of the antimicrobial resistance mechanism, the difference in response to PDI between MRSA and MSSA exists.

The agr function and polymorphism: impact on Staphylococcus aureus susceptibility to photoinactivation.

Staphylococcus aureus is an important human pathogen that causes healthcare-associated and community-acquired infections. Moreover, the growing prevalence of multiresistant strains requires the development of alternative methods to antibiotic therapy. One effective therapeutic option may be antimicrobial photodynamic inactivation (aPDI). Recently, S. aureus strain-dependent response to PDI was demonstrated, although the mechanism underlying this phenomenon remains unexplained. The aim of the current study was to investigate statistically relevant correlations between the functionality and polymorphisms of agr gene determined for 750 methicillin-susceptible and methicillin-resistant S. aureus strains and their responses to photodynamic inactivation using protoporphyrin IX. An AluI and RsaI digestion of the agr gene PCR product revealed existing correlations between the determined digestion profiles (designations used for the first time) and the PDI response. Moreover, the functionality of the agr system affected S. aureus susceptibility to PDI. Based on our results, we conclude that the agr gene may be a genetic factor affecting the strain dependent response to PDI.

Sub-lethal photodynamic inactivation renders Staphylococcus aureus susceptible to silver nanoparticles.

Staphylococcus aureus is a common etiological factor in infections of burns and other chronic wounds. The development of an effective and fast-acting treatment would be enormously beneficial and is highly desired. We focused on testing the bactericidal efficacy of photoinactivation using a known photosensitizer (protoporphyrin IX, PPIX) in sequential combination with silver nanoparticles against S. aureus. Using PPIX-based photoinactivation followed by silver nanoparticles we obtained a high bactericidal effect (7 log10 units reduction) with limited harmful effects on human epidermal keratinocytes. Moreover, we observed that the use of silver nanoparticles prevents bacterial re-growth 24 h post-PDI treatment. A sequential combination of photoinactivation and silver nanoparticles represents a potentially effective antibacterial approach.