Daniela S. Masson-Meyers

Wavelength and Bacterial Density Influence the Bactericidal Effect of Blue Light on Methicillin-Resistant Staphylococcus aureus (MRSA)

OBJECTIVE The purpose of this study was to investigate the effect of wavelength and methicillin-resistant Staphylococcus aureus (MRSA) density on the bactericidal effect of 405 and 470 nm light. BACKGROUND DATA It is recognized that 405 and 470 nm light-emitting diode (LED) light kill MRSA in standard 5 × 10(6) colony-forming units (CFU)/mL cultures; however, the effect of bacterial density on the bactericidal effect of each wavelength is not known. METHODS In three experiments, we cultured and plated US300 MRSA at four densities. Then, we irradiated each plate once with either wavelength at 0, 1, 3, 45, 50, 55, 60, and 220 J/cm(2). RESULTS Irradiation with either wavelength reduced bacterial colonies at each density (p<0.05). More bacteria were cleared as density increased; however, the proportion of colonies cleared, inversely decreased as density increased--the maximum being 100%, 96%, and 78% for 3 × 10(6), 5 × 10(6), and 7 × 10(6) CFU/mL cultures, respectively. Both wavelengths had similar effects on the sparser 3 × 10(6) and 5 × 10(6) CFU/mL cultures, but in the denser 7 × 10(6) CFU/mL culture, 405 nm light cleared more bacteria at each fluence (p<0.001). To determine the effect of beam penetration, denser 8 × 10(6) and 12 × 10(6) CFU/mL culture plates were irradiated either from the top, the bottom, or both directions. More colonies were eradicated from plates irradiated from top and bottom, than from plates irradiated from top or bottom at the same sum total fluences (p<0.001). CONCLUSIONS The bactericidal effect of LED blue light is limited more by light penetration of bacterial layers than by bacterial density per se.

Optimization of the antimicrobial effect of blue light on methicillin‐resistant Staphylococcus aureus (MRSA) in vitro

In previous studies, we showed that irradiation with 405 nm or 470 nm light suppresses up to 92% methicillin‐resistant Staphylococcus aureus (MRSA) growth in vitro and that the remaining bacteria re‐colonize. In this study, the aim was to develop a protocol that yields 100% MRSA growth suppression.

Blue 470 nm light suppresses the growth of Salmonella enterica and methicillin-resistant Staphylococcus aureus (MRSA) in vitro.

Emerging evidence suggests that blue light can photo‐inactivate some bacteria of clinical importance. Consequently, we tested the hypothesis that 470 nm light can suppress growth of two recalcitrant bacteria, MRSA and Salmonella.

Blue light does not impair wound healing in vitro.

Irradiation with red or near infrared light promotes tissue repair, while treatment with blue light is known to be antimicrobial. Consequently, it is thought that infected wounds could benefit more from combined blue and red/infrared light therapy; but there is a concern that blue light may slow healing. We investigated the effect of blue 470nm light on wound healing, in terms of wound closure, total protein and collagen synthesis, growth factor and cytokines expression, in an in vitro scratch wound model. Human dermal fibroblasts were cultured for 48h until confluent. Then a linear scratch wound was created and irradiated with 3, 5, 10 or 55J/cm(2). Control plates were not irradiated. Following 24h of incubation, cells were fixed and stained for migration and fluorescence analyses and the supernatant collected for quantification of total protein, hydroxyproline, bFGF, IL-6 and IL-10. The results showed that wound closure was similar for groups treated with 3, 5 and 10J/cm(2), with a slight improvement with the 5J/cm(2) dose, and slower closure with 55J/cm(2) p<0.001). Total protein concentration increased after irradiation with 3, 5 and 10J/cm(2), reaching statistical significance at 5J/cm(2) compared to control (p<0.0001). However, hydroxyproline levels did not differ between groups. Similarly, bFGF and IL-10 concentrations did not differ between groups, but IL-6 concentration decreased progressively as fluence increased (p<0.0001). Fluorescence analysis showed viable cells regardless of irradiation fluence. We conclude that irradiation with blue light at low fluence does not impair in vitro wound healing. The significant decrease in IL-6 suggests that 470nm light is anti-inflammatory.

Phototherapy promotes healing of cutaneous wounds in undernourished rats

BACKGROUND Various studies have shown that phototherapy promotes the healing of cutaneous wounds. OBJECTIVE To investigate the effect of phototherapy on healing of cutaneous wounds in nourished and undernourished rats. METHODS Forty rats, 20 nourished plus 20 others rendered marasmus with undernourishment, were assigned to four equal groups: nourished sham, nourished Light Emitting Diode treated, undernourished sham and undernourished Light Emitting Diode treated. In the two treated groups, two 8-mm punch wounds made on the dorsum of each rat were irradiated three times per week with 3 J/cm2 sq cm of combined 660 and 890nm light; wounds in the other groups were not irradiated. Wounds were evaluated with digital photography and image analysis, either on day 7 or day 14, with biopsies obtained on day 14 for histological studies. RESULTS Undernourishment retarded the mean healing rate of the undernourished sham wounds (p < 0.01), but not the undernourished Light emission diode treated wounds, which healed significantly faster (p < 0.001) and as fast as the two nourished groups. Histological analysis showed a smaller percentage of collagen in the undernourished sham group compared with the three other groups, thus confirming our photographic image analysis data. CONCLUSION Phototherapy reverses the adverse healing effects of undernourishment. Similar beneficial effects may be achieved in patients with poor nutritional status.

A comparison of four methods for determining viability in human dermal fibroblasts irradiated with blue light.

INTRODUCTION Several tests are available for assessing the viability of cells; however, there is a dearth of studies comparing the results obtained with each test. We compared the capability of four viability assays (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide (MTT), neutral red, trypan blue and live/dead fluorescence), to detect potential toxicity in fibroblasts irradiated with 470nm blue light. METHODS Cells were irradiated at 3, 55, 110 and 220J/cm(2), incubated for 24h and viability assessed using each test. RESULTS MTT assay showed significant decreases in viability when cells were irradiated with 110 and 220J/cm(2) energy fluence (dose) (89% and 57% viable cells, respectively; p<0.0001, compared to control); likewise the trypan blue assay showed 42% and 46% viable cells (p<0.0001). Neutral red assay revealed significant decrease in viability when cells were irradiated with 220J/cm(2) (84% viable cells; p=0.0008, compared to control). The live/dead fluorescence assay was less sensitive, evincing 91% and 95% viable cells after irradiation with 110 and 220J/cm(2) respectively. DISCUSSION (1) The four assays differed in their levels of sensitivity to cell viability. (2) The adverse effect of increasing doses seems to manifest as alteration of mitochondrial metabolism, followed by lysosomal dysfunction, membrane disruption and finally loss of cell membrane integrity. (3) Overall, irradiation with 3J/cm(2) or 55J/cm(2) did not adversely affect cell viability. Thus, doses below 110J/cm(2) appear safe.

Spectrally resolved infrared microscopy and chemometric tools to reveal the interaction between blue light (470 nm) and methicillin-resistant Staphylococcus aureus

Blue light inactivates methicillin-resistant Staphylococcus aureus (MRSA), a Gram-positive antibiotic resistant bacterium that leads to fatal infections; however, the mechanism of bacterial death remains unclear. In this paper, to uncover the mechanism underlying the bactericidal effect of blue light, a combination of Fourier transform infrared (FTIR) spectroscopy and chemometric tools is employed to detect the photoreactivity of MRSA and its distinctive pathway toward apoptosis after treatment. The mechanism of action of UV light and vancomycin against MRSA is also investigated to support the findings. Principal component analysis followed by linear discriminant analysis (PCA- LDA) is employed to reveal clustering of five groups of MRSA samples, namely untreated (control I), untreated and incubated at ambient air (control II), irradiated with 470nm blue light, irradiated with 253.5 UV light, and vancomycin-treated MRSA. Loadings plot from PCA-LDA analysis reveals important functional groups in proteins (1683, 1656, 1596, 1542cm-1), lipids (1743, 1409cm-1), and nucleic acids region of the spectrum (1060, 1087cm-1) that are responsible for the classification of blue light irradiated spectra and control spectra. Cluster vector plots and scores plot reveals that UV light-irradiated spectra are the most biochemically similar to blue light- irradiated spectra; however, some wavenumbers experience a shift. The shifts between blue light and UV light irradiated loadings plot at νasym PO2- band (from 1228 to 1238cm-1), DNA backbone (from 970 to 966cm-1) and base pairing vibration of DNA (from 1717 to 1712cm-1) suggest distinctive changes in DNA conformation in response to irradiation. Our findings indicate that irradiation of MRSA with 470nm light induces A-DNA cleavage and that B-DNA is more resistant to damage by blue light. Blue light and UV light treatment of MRSA are complementary and distinct from the known antimicrobial effect of vancomycin. Moreover, it is known that UV-induced cleavage of DNA predominantly targets B-DNA, which is in agreement with the FTIR findings. Overall the results suggest that the combination of light and vancomycin could be a more robust approach in treating MRSA infections.

Whole-Genome Sequence for Methicillin-Resistant Staphylococcus aureus Strain ATCC BAA-1680

ABSTRACT We report here the whole-genome sequence of the USA300 strain of methicillin-resistant Staphylococcus aureus (MRSA), designated ATCC BAA-1680, and commonly referred to as community-associated MRSA (CA-MRSA). This clinical MRSA isolate is commercially available from the American Type Culture Collection (ATCC) and is widely utilized as a control strain for research applications and clinical diagnosis. The isolate was propagated in ATCC medium 18, tryptic soy agar, and has been utilized as a model S. aureus strain in several studies, including MRSA genetic analysis after irradiation with 470-nm blue light.

Understanding the antimicrobial activity of selected disinfectants against methicillin-resistant Staphylococcus aureus (MRSA)

Disinfectants and biocidal products have been widely used to combat Methicillin-resistant Staphylococcus aureus (MRSA) infections in homes and healthcare environments. Although disruption of cytoplasmic membrane integrity has been documented as the main bactericidal effect of biocides, little is known about the biochemical alterations induced by these chemical agents. In this study, we used Fourier transform infrared (FT-IR) spectroscopy and chemometric tools as an alternative non-destructive technique to determine the bactericidal effects of commonly used disinfectants against MRSA USA-300. FTIR spectroscopy permits a detailed characterization of bacterial reactivity, allowing an understanding of the fundamental mechanism of action involved in the interaction between bacteria and disinfectants. The disinfectants studied were ethanol 70% (N = 5), isopropanol (N = 5), sodium hypochlorite (N = 5), triclosan (N = 5) and triclocarban (N = 5). Results showed less than 5% colony forming units growth of MRSA treated with triclocarban and no growth in the other groups. Nearly 70,000 mid-infrared spectra from the five treatments and the two control (untreated; N = 4) groups of MRSA (bacteria grown in TSB and incubated at 37°C (Control I) / at ambient temperature (Control II), for 24h) were pre-processed and analyzed using principal component analysis followed by linear discriminant analysis (PCA-LDA). Clustering of strains of MRSA belonging to five treatments and the discrimination between each treatment and two control groups in MRSA (untreated) were investigated. PCA-LDA discriminatory frequencies suggested that ethanol-treated spectra are the most similar to isopropanol-treated spectra biochemically. Also reported here are the biochemical alterations in the structure of proteins, lipid membranes, and phosphate groups of MRSA produced by sodium hypochlorite, triclosan, and triclocarban treatments. These findings provide mechanistic information involved in the interaction between MRSA strains and hygiene products; thereby demonstrating the potential of spectroscopic analysis as an objective, robust, and label-free tool for evaluating the macromolecular changes involved in disinfectant-treated MRSA.