Photoactivated Gallium Porphyrin Reduces Staphylococcus aureus Colonization on the Skin and Suppresses Its Ability to Produce Enterotoxin C and TSST-1.

Staphylococcus aureus is a key pathogen in atopic dermatitis (AD) pathogenicity. Over half of AD patients are carriers of S. aureus. Clinical isolates derived from AD patients produce various staphylococcal enterotoxins, such as staphylococcal enterotoxin C or toxic shock syndrome toxin. The production of these virulence factors is correlated with more severe AD. In this study, we propose cationic heme-mimetic gallium porphyrin (Ga3+CHP), a novel gallium metalloporphyrin, as an anti-staphylococcal agent that functions through dual mechanisms: a light-dependent mechanism (antimicrobial photodynamic inactivation, aPDI) and a light-independent mechanism (suppressing iron metabolism). Ga3+CHP has two additive quaternary ammonium groups that increase its water solubility. Furthermore, Ga3+CHP is an efficient generator of singlet oxygen and can be recognized by heme-target systems such as Isd, which improves the intracellular accumulation of this compound. Ga3+CHP activated with green light effectively reduced the survival of clinical S. aureus isolates derived from AD patients (>5 log10 CFU/mL) and affected their enterotoxin gene expression. Additionally, there was a decrease in the biological functionality of studied toxins regarding their superantigenicity. In aPDI conditions, there was no pronounced toxicity in HaCaT keratinocytes with both normal and suppressed filaggrin gene expression, which occurs in ∼50% of AD patients. Additionally, no mutagenic activity was observed. Green light-activated gallium metalloporphyrins may be a promising chemotherapeutic to reduce S. aureus colonization on the skin of AD patients.

Dual Spinneret Electrospun Polyurethane/PVA-Gelatin Nanofibrous Scaffolds Containing Cinnamon Essential Oil and Nanoceria for Chronic Diabetic Wound Healing: Preparation, Physicochemical Characterization and In-Vitro Evaluation.

In this study, a dual spinneret electrospinning technique was applied to fabricate a series of polyurethane (PU) and polyvinyl alcohol-gelatin (PVA/Gel) nanofibrous scaffolds. The study aims to enhance the properties of PU/PVA-Gel NFs loaded with a low dose of nanoceria through the incorporation of cinnamon essential oil (CEO). The as-prepared nCeO2 were embedded into the PVA/Gel nanofibrous layer, where the cinnamon essential oil (CEO) was incorporated into the PU nanofibrous layer. The morphology, thermal stability, mechanical properties, and chemical composition of the produced NF mats were investigated by STEM, DSC, and FTIR. The obtained results showed improvement in the mechanical, and thermal stability of the dual-fiber scaffolds by adding CEO along with nanoceria. The cytotoxicity evaluation revealed that the incorporation of CEO to PU/PVA-Gel loaded with a low dose of nanoceria could enhance the cell population compared to using pure PU/PVA-Gel NFs. Moreover, the presence of CEO could inhibit the growth rate of S. aureus more than E. coli. To our knowledge, this is the first time such nanofibrous membranes composed of PU and PVA-Gel have been produced. The first time was to load the nanofibrous membranes with both CEO and nCeO2. The obtained results indicate that the proposed PU/PVA-Gel NFs represent promising platforms with CEO and nCeO2 for effectively managing diabetic wounds.

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.

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.

Photoinactivation of Staphylococcus aureus using protoporphyrin IX: the role of haem-regulated transporter HrtA.

Light- and photosensitiser-based antimicrobial photodynamic therapy is a very promising approach to the control of microbial infections. How the phenotypic features of a microorganism affect its response to photosensitiser-based photokilling represents an area of substantial research interest. To understand the mechanisms governing the phenomenon of a strain-dependent response to photodynamic inactivation (PDI), we analysed the possible role of the membrane-located haem transporter HrtA in Staphylococcus aureus. We used a S. aureus strains with an inactivated component of the haem-regulated transporter, HrtA, along with its wild-type counterpart to determine differences in PDI outcome and photosensitiser uptake between the studied isogenic strains. We observed that a lack of HrtA protein potentiates the phototoxic effect towards S. aureus but only when extracellular protoporphyrin IX is used. The observed effect may depend on the function of the HrtA transporter but is likely to result from changed membrane properties following the absence of the protein in the membrane. This indicates that disturbing the membrane properties is an attractive method for improving the efficacy of the photodynamic inactivation of microorganisms.

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.

Antimicrobial photodynamic therapy with fulleropyrrolidine: photoinactivation mechanism of Staphylococcus aureus, in vitro and in vivo studies.

A family of N-methylpyrrolidinium fullerene iodide salts has been intensively studied to determine their applicability in antimicrobial photodynamic therapy (APDT). This study examined in vitro the efficacy of a C60 fullerene functionalized with one methylpyrrolidinium group to kill upon irradiation with white light gram-negative and gram-positive bacteria, as well as fungal cells, and the corresponding mechanism of the fullerene bactericidal action. The in vitro studies revealed that the high antistaphylococcal efficacy of functionalized fullerene could be linked to their ability to photogenerate singlet oxygen and superoxide anion. Following Staphylococcus aureus photoinactivation, no modifications of its genomic DNA were detected. In contrast, photodamage of the cell envelope seemed to be a dominant mechanism of bactericidal action. In in vivo studies, a 2 log10 reduction in the average bioluminescent radiance between treated and non-treated mice was reached. One day post APDT treatment, moist and abundant growth of bacteria could be observed on wounds of non-fulleropyrrolidine and dark control mice. APDT-treated wounds stayed visibly clear up to the third day. Moreover, cytotoxicity test on human dermal keratinocytes revealed great safety of using the sensitizer toward eukaryotic cells. These data indicate potential application of functionalized fullerene as antistaphylococcal sensitizer for superficial infections.

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.

Decrease of ESKAPE virulence with a cationic heme-mimetic gallium porphyrin photosensitizer: The Trojan horse strategy that could help address antimicrobial resistance.

INTRODUCTION: Emerging antibiotic resistance among bacterial pathogens has forced an urgent need for alternative non-antibiotic strategies development that could combat drug resistant-associated infections. Suppression of virulence of ESKAPE pathogens' by targeting multiple virulence traits provides a promising approach. OBJECTIVES: Here we propose an iron-blocking antibacterial therapy based on a cationic heme-mimetic gallium porphyrin (GaCHP), which antibacterial efficacy could be further enhanced by photodynamic inactivation. METHODS: We used gallium heme mimetic porphyrin (GaCHP) excited with light to significantly reduce microbial viability and suppress both the expression and biological activity of several virulence traits of both Gram-positive and Gram-negative ESKAPE representatives, i.e., S. aureus and P. aeruginosa. Moreover, further improvement of the proposed strategy by combining it with routinely used antimicrobials to resensitize the microbes to antibiotics and provide enhanced bactericidal efficacy was investigated. RESULTS: The proposed strategy led to substantial inactivation of critical priority pathogens and has been evidenced to suppress the expression and biological activity of multiple virulence factors in S. aureus and P. aeruginosa. Finally, the combination of GaCHP phototreatment and antibiotics resulted in promising strategy to overcome antibiotic resistance of the studied microbes and to enhance disinfection of drug resistant pathogens. CONCLUSION: Lastly, considering high safety aspects of the proposed treatment toward host cells, i.e., lack of mutagenicity, no dark toxicity and mild phototoxicity, we describe an efficient alternative that simultaneously suppresses the functionality of multiple virulence factors in ESKAPE pathogens.

Imidazoacridinone derivatives as efficient sensitizers in photoantimicrobial chemotherapy.

The objective of this study was to investigate a new potential photosensitizer (PS) in the photodynamic inactivation (PDI) of microorganisms in vitro (11 reference strains and 13 clinical isolates, representing common Gram-positive and Gram-negative human pathogens), with special emphasis on Candida albicans. We studied the light-induced cytotoxicity of the imidazoacridinone derivative C1330 toward fungal cells grown in planktonic form. We examined the influence of various parameters (time of incubation, PDI quencher effect, and C1330 accumulation in C. albicans cells) on the efficacy of light-dependent cytotoxicity. Additionally, we checked for the potential cyto- and phototoxic activity of C1330 against human dermal keratinocytes. In our research, we used a broadband incoherent blue light source (380 to 470 nm) with an output power of 100 mW/cm(2). In vitro studies showed that the C1330 action against C. albicans was a light-dependent process. C1330 was an efficient photosensitizer in the photodynamic inactivation of C. albicans, which reduced the growth of planktonic cells by 6.1 log10 units. Efficient accumulation of PS in the nucleus and vacuoles was observed after 30 min of incubation, which correlated with the highest photokilling efficacy. Significant changes in intracellular structure were observed upon illumination of C1330-incubated C. albicans cells. In the case of the human HaCaT cell line, approximately 40% of cells survived the treatment, which indicates the potential benefit of further study of the application of C1330 in photoantimicrobial chemotherapy. These data suggest that PDI may be a viable approach for the treatment of localized C. albicans infections.