Daniela Masson-Meyers | University of Wisconsin Milwaukee (original) (raw)

Papers by Daniela Masson-Meyers

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

PLoS ONE, 12 (10), e0186375, 2017

Disinfectants and biocidal products have been widely used to combat Methicillin-resistant Staphyl... more 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 37ÊC (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, andlabel-free tool for evaluating the macromolecular changes involved in disinfectant-treated MRSA.

Research paper thumbnail of Skin changes in streptozotocin-induced diabetic rats

Biochemical and Biophysical Research Communications, 490 (4), 1154-1161, 2017.

Diabetes can cause serious health complications, which can affect every organ of the body, includ... more Diabetes can cause serious health complications, which can affect every organ of the body, including the skin. The molecular etiology has not yet been clarified for all diabetic skin conditions. Thus, this study aimed to investigate the changes of diabetes in skin compared to non-diabetic skin in rats. Fifteen days after establishing the diabetic status, skin samples from the dorsum-cervical region were harvested for subsequent analysis of alterations caused by diabetes. Our results demonstrate that diabetes stimulated higher inflammation and oxidative stress in skin, but antioxidant defense levels were lower compared to the non-diabetic group (p < 0.05). This could have been related to a decreased number of blood vessels and low expression of VEGF, eNOS and TGF-b1. Finally, insulin signaling proteins IRS, Akt, Shc and ERK showed a low expression in the diabetic group. Thus, our study shows that the pathology of diabetes induced immunohistopathological and biochemical skin changes compared to non-diabetic skin in rats.

Research paper thumbnail of Blue/violet laser inactivates methicillin-resistant Staphylococcus aureus by altering its transmembrane potential

Journal of Photochemistry and Photobiology B: Biology, 170, 118-124, 2017, 2017

The resistance of methicillin-resistant Staphylococcus aureus to antibiotics presents serious cli... more The resistance of methicillin-resistant Staphylococcus aureus to antibiotics presents serious clinical problems that
prompted the need for finding alternative or combination therapies. One such therapy is irradiation with blue
light. To determine the alterations in metabolic processes implicated in the observed antimicrobial effects of blue
light, we investigated the changes in membrane potential and the presence of free-radical-producing photoacceptor
molecules. Bacterial cultures irradiated with one or two doses of 405 nm laser light (each consisting of
121 J/cm2) were imaged with spectrally resolved laser-scanning microscopes to detect endogenous fluorescent
species as well as the voltage sensitive dye 3,3′-Diethyloxacarbocyanine iodide. The endogenous fluorescence
indicated the presence of photosensitizers (i.e., porphyrins, NADH, FAD) in the cells, while the exogenous signal
allowed us to monitor rapid changes in transmembrane potential following treatment with light. The changes
were drastic within the first 5 min after irradiation with the first dose and continued slowly after the second
irradiation. These results suggest that the early antimicrobial activity of blue light results from alteration of
membrane integrity with a consequent decrease in membrane polarization and rapid alteration of vital cellular
functions. The observation of an early antimicrobial activity of light is very encouraging, as it suggests that
treatment does not necessarily have to be administered over a long period of time.

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

Journal of Photochemistry and Photobiology B: Biology, 167, 150–157, 2017

Blue light inactivates methicillin-resistant Staphylococcus aureus (MRSA), a Gram-positive antibi... more 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 PO(2-) 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.

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

Journal of Pharmacological and Toxicological Methods, 79, 15–22, 2016, 2016

Several tests are available for assessing the viability of cells; however, there is a dearth of s... more 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. Cells were irradiated at 3, 55, 110 and 220J/cm(2), incubated for 24h and viability assessed using each test. 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&lt;0.0001, compared to control); likewise the trypan blue assay showed 42% and 46% viable cells (p&lt;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. (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.

Research paper thumbnail of Blue light does not impair wound healing in vitro

Irradiation with red or near infrared light promotes tissue repair, while treatment with blue lig... more 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 470 nm 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 48 h until confluent. Then a linear scratch wound was created and irradiated with 3, 5, 10 or 55 J/cm2. Control plates were not irradiated. Following 24 h 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 10 J/cm2, with a slight improvement with the 5 J/cm2 dose, and slower closure with 55 J/cm2 p < 0.001). Total protein concentration increased
after irradiation with 3, 5 and 10 J/cm2, reaching statistical significance at 5 J/cm2 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 470 nm light is anti-inflammatory.

Research paper thumbnail of The relative antimicrobial effect of blue 405 nm LED and blue 405 nm laser on methicillin-resistant Staphylococcus aureus in vitro

Lasers in Medical Sciences, Sep 11, 2015

It has long been argued that light from a laser diode is superior to light from a light-emitting ... more It has long been argued that light from a laser diode is superior to light from a light-emitting diode (LED) in terms of its effect on biological tissues. In order to shed light on this ongoing debate, we compared the antimicrobial effect of light emitted from a 405-nm LED with that of a 405-nm laser on methicillin-resistant Staphylococcus aureus (MRSA) at comparable fluences. We cultured 5 × 106 CFU/ml MRSA on tryptic soy agar and then irradiated culture plates once, twice, or thrice with either LED or laser light using 40, 54, 81, or 121 J/cm2 fluence at 15-, 30-, or 240-min time interval between irradiation. Cultures were incubated immediately after irradiation at 37 °C for 24 h before imaging and counting remnant bacterial colonies. Regardless of the device used, LED or laser, irradiation at each fluence resulted in statistically significant bacterial growth suppression compared to non-irradiated controls (p < 0.0001). The antimicrobial effect of both light sources, LED and laser, was not statistically different at each fluence in 35 of the 36 experimental trials. Bacterial growth suppression achieved with either source of light increased with repeated irradiation, particularly at the 15- or 30-min treatment time interval. Thus, we conclude that the antimicrobial effect of 405-nm laser and 405-nm LED on MRSA is similar; neither has a superior antimicrobial effect when compared to the other.
KEYWORDS: Antimicrobial therapy; Blue light; LED versus laser; Low-level light therapy; Methicillin-resistant Staphylococcus aureus; Phototherapy

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

Lasers in Surgery and Medicine, doi: 10.1002/lsm.22385. [Epub ahead of print], Jul 14, 2015

BACKGROUND AND OBJECTIVE: Emerging evidence suggests that blue light can photo-inactivate some ba... more BACKGROUND AND OBJECTIVE: 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. MATERIALS AND METHODS: We plated 5 × 106 and 7 × 106 CFU/ml USA300 strain of MRSA and 1 × 106 CFU/ml of Salmonella enterica serovars Typhimurium and Heidelberg. Plates were irradiated with 55, 110, 165 and 220 J/cm2 of blue light, incubated at 37°C for 24 hours and colony counts determined. RESULTS: Compared with controls, blue light irradiation produced a significant dose-dependent reduction in the number of colonies formed by each bacterial strain (P < 0.001). Irradiation of 5 × 106 and 7 × 106 CFU/ml MRSA with 55 J/cm2 produced 92% (4.6 × 106 CFU/ml) and 86% (6 × 106 CFU/ml) inactivation respectively, while 110 and 220 J/cm2 suppressed each MRSA density 100%. Irradiation of Salmonella Typhimurium with 55 and 110 J/cm2 suppressed bacterial growth 31% (3.1 × 105 CFU/ml) and 93% (9.3 × 105 CFU/ml) respectively; while Salmonella Heidelberg was inhibited 11% (1.1 × 105 CFU/ml) and 84% (8.4 × 105 CFU/ml) respectively by the two fluences. Complete inactivation of each Salmonella strain was achieved using 165 or 220 J/cm2 . CONCLUSION: The observed inhibition of Gram-positive (MRSA) and Gram-negative (Salmonella) bacteria suggests the versatility of blue light in bacteria eradication, making it a viable intervention strategy for decontamination of food and environments that harbor such bacteria.

Research paper thumbnail of Whole-genome sequence for methicillin-resistant Staphylococcus aureus strain ATCC BAA-1680

Genome Announc 3(2):e00011-15. doi:10.1128/genomeA.00011-15, Mar 2015

We report here the whole-genome sequence of the USA300 strain of methicillin-resistant Staphyloco... more 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.

Research paper thumbnail of The bactericidal effect of 470-nm light and hyperbaric oxygen on methicillin-resistant Staphylococcus aureus (MRSA)

Lasers in Medical Science, Feb 21 2015, doi:10.1007/s10103-015-1722-9

It has been shown that, in vitro, hyperbaric oxygen (HBO) suppresses 28 % bacterial growth, whil... more It has been shown that, in vitro, hyperbaric oxygen
(HBO) suppresses 28 % bacterial growth, while 470-nm blue
light alone suppresses up to 92 % methicillin-resistant
Staphylococcus aureus (MRSA) in one application in vitro.
Therefore, we determined if combined 470-nm light (55 J/
cm2) and HBO will yield 100 % bacterial suppression in experimental
simulation of mild, moderate or severe MRSA
infection. We cultured MRSA at 3×106, 5×106, 7×106, 8×
106, or 12×106 CFU/ml and treated each concentration in four
groups as follows: (1) control (no treatment) (2) photoirradiation
only, (3) photo-irradiation then HBO, (4) HBO
only, and (5) HBO then photo-irradiation. Bacteria colonies
were then quantified. The results showed that at each bacterial
concentration, HBO alone was significantly less effective in
suppressing MRSA than photo-irradiation or combined HBO
and photo-irradiation (p<0.0001). Similarly, at no bacterial
concentration did combined HBO and 470-nm light treatment
yield a statistically better result than 470-nm light alone
(p>0.05), neither did HBO treatment either before or after
irradiation make a difference. Furthermore, at no bacterial
concentration was 100 % MRSA suppression achieved.
Indeed, the maximum bacterial suppression attained was in
the mild infection model (3×106 CFU/ml), with blue light
producing 97.3±0.2 % suppression and HBO+55 J/cm2
yielding 97.5±2.5 % suppression. We conclude that (1)
HBO and 470-nm light individually suppress MRSA growth;
(2) 470-nm blue light is more effective in suppressing MRSA
than HBO; and (3) HBO did not act synergistically to heighten
the bactericidal effect of 470-nm light.

Research paper thumbnail of Optimization of the antimicrobial effect of blue light on Methicillin-resistant Staphylococcus aureus (MRSA) in vitro

Lasers in Surgery and Medicine. Article first published online: 12 JAN 2015 DOI: 10.1002/lsm.22327, Jan 12, 2015

Background and Objective: In previous studies, we showed that irradiation with 405 nm or 470 nm l... more Background and Objective: 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. Materials and Methods: We cultured 3 × 106 and 5 × 106 CFU/ml USA300 strain of MRSA and then irradiated each plate with varying fluences of 1–60 J/cm2 of 405 nm or 470 nm light, either once or twice at 6 hours intervals. Next, we plated 7 × 106 CFU/ml and irradiated it with 45, 50, 55, or 60 J/cm2 fluence, once, twice, or thrice at the same 6 hours intervals. In a third experiment, the same culture density was irradiated with 0, 165, 180, 220, or 240 J/cm2, either once, twice, or thrice. Results: Irradiation with either wavelength significantly reduced the bacterial colonies regardless of bacterial density (P < 0.05). At 3 × 106 CFU/ml density, nearly 40% and 50% growth of MRSA were suppressed with as little as 3 J/cm2 of 405 nm and 470 nm wavelengths, respectively. Moreover, 100% of the colonies were suppressed with a single exposure to 55 or 60 J/cm2 of 470 nm light or double treatment with 50, 55, or 60 J/cm2 of 405 nm wavelength. At 5 × 106 CFU/ml density, irradiating twice with 50, 55, or 60 J/cm2 of either wavelength suppressed bacterial growth completely, lower fluences did not. The denser 7 × 106 CFU/ml culture required higher doses to achieve 100% suppression, either one shot with 220 J/cm2 of 470 nm light or two shots of the same dose using 405 nm. Conclusion: The bactericidal effect of blue light can be optimized to yield 100% bacterial growth suppression, but with relatively high fluences for dense bacterial cultures, such as 7 × 106 CFU/ml.

Research paper thumbnail of Phototherapy promotes healing of cutaneous wounds in undernourished rat

Anais brasileiros de dermatologia, 89(6):899-904. , Dec 2014

BACKGROUND: Various studies have shown that phototherapy promotes the healing of cutaneous wounds... more 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.
Keywords: Nutritional marasmus; Phototherapy; Protein malnutrition; Wound healing

Research paper thumbnail of Preponderance of toxigenic Escherichia coli in stool pathogens correlates with toxin detection in accessible drinking-water sources

Epidemiology and Infection, May 1, 2014

Since early detection of pathogens and their virulence factors contribute to intervention and co... more Since early detection of pathogens and their virulence factors contribute to intervention and
control strategies, we assessed the enteropathogens in diarrhoea disease and investigated the
link between toxigenic strains of Escherichia coli from stool and drinking-water sources; and
determined the expression of toxin genes by antibiotic-resistant E. coli in Lagos, Nigeria. This
was compared with isolates from diarrhoeal stool and water from Wisconsin, USA. The new
Luminex xTAG GPP (Gastroplex) technique and conventional real-time PCR were used to
profile enteric pathogens and E. coli toxin gene isolates, respectively. Results showed the
pathogen profile of stool and indicated a relationship between E. coli toxin genes in water and
stool from Lagos which was absent in Wisconsin isolates. The Gastroplex technique was efficient
for multiple enteric pathogens and toxin gene detection. The co-existence of antibiotic resistance
with enteroinvasive E. coli toxin genes suggests an additional prognostic burden on patients.

Research paper thumbnail of Topical treatment with Copaifera langsdorffii oleoresin improves wound healing in rats

International Journal of Phytomedicine, v.5, n.3, 378-386

Copaifera langsdorffii oleoresin (copaiba) has been used in the Amazon as traditional wound heali... more Copaifera langsdorffii oleoresin (copaiba) has been used in the Amazon as traditional wound healing remedy for centuries. Since its mechanisms of action remain unclear, we investigated its effects on excisional wounds in rats. Wounds were made on the dorsum of animals assigned to three groups: saline, control cream and 10% copaiba cream, and assessed on days 2, 7 and 14 post-wounding morphometrically, histologically and biochemically. Wound healing rate was faster in copaiba than in saline or cream groups. This was corroborated by matrix metalloproteinase (MMP)-2 activity which rose progressively throughout in copaiba group. MMP-9, a marker of inflammation, was not detectable at day 14 in copaiba group, but persisted in the other groups. Moreover, histology showed early population of copaiba-treated wounds by inflammatory cells, and by day 14 this group had less fibroblasts and more organized collagen. Further, copaiba group synthesized collagen faster than saline and cream groups, as evidenced by progressive increases in the amounts of hydroxyproline at days 7 and 14 (p < 0.012). These findings suggest that 10% copaiba oleoresin cream promotes wound healing in rats by regulating MMP-2 and MMP-9 activities, stimulating collagen synthesis and promoting tissue remodeling and reepithelialization.

Research paper thumbnail of Antimicrobial effects of Copaifera langsdorffii oleoresin in infected rat wounds

International Journal of Applied Microbiology Sciences 2013; 2(3):9-20

We determined the effect of Copaifera langsdorffii (copaiba) cream on wounds infected with Strept... more We determined the effect of Copaifera langsdorffii (copaiba) cream on wounds infected with Streptococcus pyogenes or Staphylococcus aureus. Wounds were created on the dorsum of Wistar rats, then inoculated with either pathogen, and observed through 14 days as they were treated either with saline, control cream or 10% copaiba cream. Wounds were assessed for healing on days 2, 7 and 14 post-surgery, and then swabbed and biopsied to quantify bacteria. By day 14, treatment with 10% copaiba decreased S. pyogenes infection to 0.02%; saline treatment reduced infection to 17.2%, while control cream increased infection 87.4%. Copaiba similarly decreased S. aureus infection to 0.3% by day 14, compared with 26.9% for saline and 12.7% for control cream. Consistent with these findings, copaiba treated S. pyogenes infected wounds re-epithelized 29% by day 2, compared with 15.8% for saline (p = 0.025) and 18.4% for control cream treated wounds; maintaining a higher rate of re-epithelialization (67.5%) over the control cream group (54.9%) on day 7 (p = 0.002). Similarly, copaiba enhanced healing of S. aureus infected wounds, 74.4% by day 7, when compared with saline and control cream wounds. These findings suggest that copaiba retards bacterial infection resulting in better healing.

Research paper thumbnail of Cytotoxicity and wound healing properties of Copaifera langsdorffii oleoresin in rabbits

"" Plants of the Copaifera genus (copaiba) are documented to have healing potential; however, t... more ""
Plants of the Copaifera genus (copaiba) are documented to have healing potential; however, their cytotoxicity and therapeutic mechanisms remain unclear. This study assessed in vitro cytotoxicity of copaiba oleoresin from Copaifera langsdorffii on 3T3 fibroblasts and investigated its wound healing potential in rabbits. Cell viability and proliferation were assessed by MTT and H3-Thymidine assays, respectively. Wounds were created on rabbits’ ears and topically treated with saline (S), control cream (Cr), 10% (C10) and 25% (C25) copaiba cream, or pure oleoresin (OR) over 21 days, and assessed 2, 7, 14 and 21 days post-wounding by wound healing rates and histology. Fibroblasts treated with up to 100 μg/mL of copaiba yielded greater than 80% of viability and proliferation compared to control culture, decreasing significantly (p<0.001) at 500 and 1000 μg/mL. Wounds were re-epithelialized by the 21st day, with some OR-treated wounds showing evidence of sustained inflammation on day 7. C10 and C25-treated wounds presented better clinical outcomes, confirmed by histology with evidence of fibroblastic activity by day 7 and organized collagen fibers observed from day 14. These findings suggest that C. langsdorffii oleoresin shows no cytotoxic effects on fibroblasts at 100 μg/mL and at appropriate doses enhances wound healing.
""

Research paper thumbnail of 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-... more 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 5×106 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/cm2. 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 3×106, 5×106, and 7×106 CFU/mL cultures, respectively. Both wavelengths had similar effects on the sparser 3×106 and 5×106 CFU/mL cultures, but in the denser 7×106 CFU/mL culture, 405 nm light cleared more bacteria at each fluence (p<0.001). To determine the effect of beam penetration, denser 8×106 and 12×106 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.

Research paper thumbnail of Antimicrobial activity of copaiba (Copaifera langsdorffii) oleoresin on bacteria of clinical significance in cutaneous wounds

The present study was designed to evaluate the in vitro antimicrobial activity of Copaifera langs... more The present study was designed to evaluate the in vitro antimicrobial activity of Copaifera langsdorffii oleoresin, which has been used in folk medicine as an anti-inflammatory, antibacterial, healing among others. The oleoresin was tested against Gram-positive (Staphylococcus aureus, Streptococcus pyogenes, Enterococcus faecalis) and Gram-negative (Pseudomonas aeruginosa and Escherichia coli) bacteria related to infections in cutaneous wounds. Antimicrobial activity was determined by the minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) assays. Copaiba oleoresin showed antimicrobial activity only against the Gram-positive bacteria with MIC of 200 μg/mL, 400 μg/mL and 1100 μg/mL for S. aureus, S. pyogenes and E. faecalis, respectively. MBC values were the same as MIC for S. aureus and S. pyogenes and for E. faecalis it was 1200 μg/mL. Considering that infection significantly impairs the wound healing process, we believe that the use of copaiba oleoresin as a component of a topical formulation could be a valuable adjunct in the treatment of infected wounds, mainly in the case of wounds infected by Gram-positive microorganisms.

Research paper thumbnail of Experimental models of malnutrition and its effect on skin trophism

The skin requires adequate levels of nutrients to function properly. To analyze skin trophism i... more The skin requires adequate levels of nutrients to function properly.
To analyze skin trophism in well-nourished and undernourished rats using two models of malnutrition. In the marasmus model, 60 Wistar rats were kept on a controlled diet, 30 being randomly selected to receive half the established diet for 60 days. In the gelatin model, 60 rats were used, 30 of which received a diet consisting of poor quality protein (gelatin) for 30 days. The nutritional status of the animals was evaluated according to body mass index, clinical signs and serum albumin measurement. After the period of malnutrition, histology was performed on the animals' skin to analyze the thickness of the dermis and epidermis using the Leica Application Suite software. Collagen was analyzed on slides stained with Gömöri trichrome using the ImageJ software program.
The body mass index of the malnourished animals in the marasmus and gelatin groups was significantly lower than that of the well-nourished animals in the two groups (p<0.0001 in both models). With respect to serum albumin, there was no difference between the groups in either of the two models. In relation to the histological analysis of skin thickness, the dermis of the malnourished animals was significantly thinner compared to that of the well-nourished animals (p<0.0001 in both models). The percentage of collagen was lower in the malnourished animals compared to the well-nourished animals (p<0.0005 and p<0.003 in the marasmus and gelatin model, respectively).
Skin thickness measurements were lower in the malnourished animals in both models, and this finding was histologically confirmed by the lower percentage of collagen, showing the negative effect of malnutrition on skin trophism

Research paper thumbnail of Polyhydroxy alcohols and peach oil addition influence in liquid crystal formation and rheological behaviour of O/W emulsions

The aim of this study was to developed O/W emulsions with Self-Emulsifying Base (SEB) cointainnin... more The aim of this study was to developed O/W emulsions with Self-Emulsifying Base (SEB) cointainning liquid crystals (LC) and evaluate the influence of polyhydroxy alcohols and peach oil addition in liquid crystals formation as well as to analyze the rheological behaviour of these systems. Formulations containing SEB, were supplemented or not, with glycerin or sorbitol or propylene glycol, and/or peach oil. All formulations studied had lamellar crystalline phases. Peach oil addition increased the viscosity of SEB, while humectants had different behaviour, glycerin enhanced the viscosity of the studied formulations supplemented or not with peach oil, however, propylene glycol decreased the viscosity of all formulations studied. On the other hand, sorbitol enhanced the viscosity of formulations containing peach oil. All emulsions presented pseudoplastic behaviour and tixotropy.

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

PLoS ONE, 12 (10), e0186375, 2017

Disinfectants and biocidal products have been widely used to combat Methicillin-resistant Staphyl... more 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 37ÊC (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, andlabel-free tool for evaluating the macromolecular changes involved in disinfectant-treated MRSA.

Research paper thumbnail of Skin changes in streptozotocin-induced diabetic rats

Biochemical and Biophysical Research Communications, 490 (4), 1154-1161, 2017.

Diabetes can cause serious health complications, which can affect every organ of the body, includ... more Diabetes can cause serious health complications, which can affect every organ of the body, including the skin. The molecular etiology has not yet been clarified for all diabetic skin conditions. Thus, this study aimed to investigate the changes of diabetes in skin compared to non-diabetic skin in rats. Fifteen days after establishing the diabetic status, skin samples from the dorsum-cervical region were harvested for subsequent analysis of alterations caused by diabetes. Our results demonstrate that diabetes stimulated higher inflammation and oxidative stress in skin, but antioxidant defense levels were lower compared to the non-diabetic group (p < 0.05). This could have been related to a decreased number of blood vessels and low expression of VEGF, eNOS and TGF-b1. Finally, insulin signaling proteins IRS, Akt, Shc and ERK showed a low expression in the diabetic group. Thus, our study shows that the pathology of diabetes induced immunohistopathological and biochemical skin changes compared to non-diabetic skin in rats.

Research paper thumbnail of Blue/violet laser inactivates methicillin-resistant Staphylococcus aureus by altering its transmembrane potential

Journal of Photochemistry and Photobiology B: Biology, 170, 118-124, 2017, 2017

The resistance of methicillin-resistant Staphylococcus aureus to antibiotics presents serious cli... more The resistance of methicillin-resistant Staphylococcus aureus to antibiotics presents serious clinical problems that
prompted the need for finding alternative or combination therapies. One such therapy is irradiation with blue
light. To determine the alterations in metabolic processes implicated in the observed antimicrobial effects of blue
light, we investigated the changes in membrane potential and the presence of free-radical-producing photoacceptor
molecules. Bacterial cultures irradiated with one or two doses of 405 nm laser light (each consisting of
121 J/cm2) were imaged with spectrally resolved laser-scanning microscopes to detect endogenous fluorescent
species as well as the voltage sensitive dye 3,3′-Diethyloxacarbocyanine iodide. The endogenous fluorescence
indicated the presence of photosensitizers (i.e., porphyrins, NADH, FAD) in the cells, while the exogenous signal
allowed us to monitor rapid changes in transmembrane potential following treatment with light. The changes
were drastic within the first 5 min after irradiation with the first dose and continued slowly after the second
irradiation. These results suggest that the early antimicrobial activity of blue light results from alteration of
membrane integrity with a consequent decrease in membrane polarization and rapid alteration of vital cellular
functions. The observation of an early antimicrobial activity of light is very encouraging, as it suggests that
treatment does not necessarily have to be administered over a long period of time.

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

Journal of Photochemistry and Photobiology B: Biology, 167, 150–157, 2017

Blue light inactivates methicillin-resistant Staphylococcus aureus (MRSA), a Gram-positive antibi... more 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 PO(2-) 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.

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

Journal of Pharmacological and Toxicological Methods, 79, 15–22, 2016, 2016

Several tests are available for assessing the viability of cells; however, there is a dearth of s... more 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. Cells were irradiated at 3, 55, 110 and 220J/cm(2), incubated for 24h and viability assessed using each test. 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&lt;0.0001, compared to control); likewise the trypan blue assay showed 42% and 46% viable cells (p&lt;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. (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.

Research paper thumbnail of Blue light does not impair wound healing in vitro

Irradiation with red or near infrared light promotes tissue repair, while treatment with blue lig... more 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 470 nm 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 48 h until confluent. Then a linear scratch wound was created and irradiated with 3, 5, 10 or 55 J/cm2. Control plates were not irradiated. Following 24 h 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 10 J/cm2, with a slight improvement with the 5 J/cm2 dose, and slower closure with 55 J/cm2 p < 0.001). Total protein concentration increased
after irradiation with 3, 5 and 10 J/cm2, reaching statistical significance at 5 J/cm2 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 470 nm light is anti-inflammatory.

Research paper thumbnail of The relative antimicrobial effect of blue 405 nm LED and blue 405 nm laser on methicillin-resistant Staphylococcus aureus in vitro

Lasers in Medical Sciences, Sep 11, 2015

It has long been argued that light from a laser diode is superior to light from a light-emitting ... more It has long been argued that light from a laser diode is superior to light from a light-emitting diode (LED) in terms of its effect on biological tissues. In order to shed light on this ongoing debate, we compared the antimicrobial effect of light emitted from a 405-nm LED with that of a 405-nm laser on methicillin-resistant Staphylococcus aureus (MRSA) at comparable fluences. We cultured 5 × 106 CFU/ml MRSA on tryptic soy agar and then irradiated culture plates once, twice, or thrice with either LED or laser light using 40, 54, 81, or 121 J/cm2 fluence at 15-, 30-, or 240-min time interval between irradiation. Cultures were incubated immediately after irradiation at 37 °C for 24 h before imaging and counting remnant bacterial colonies. Regardless of the device used, LED or laser, irradiation at each fluence resulted in statistically significant bacterial growth suppression compared to non-irradiated controls (p < 0.0001). The antimicrobial effect of both light sources, LED and laser, was not statistically different at each fluence in 35 of the 36 experimental trials. Bacterial growth suppression achieved with either source of light increased with repeated irradiation, particularly at the 15- or 30-min treatment time interval. Thus, we conclude that the antimicrobial effect of 405-nm laser and 405-nm LED on MRSA is similar; neither has a superior antimicrobial effect when compared to the other.
KEYWORDS: Antimicrobial therapy; Blue light; LED versus laser; Low-level light therapy; Methicillin-resistant Staphylococcus aureus; Phototherapy

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

Lasers in Surgery and Medicine, doi: 10.1002/lsm.22385. [Epub ahead of print], Jul 14, 2015

BACKGROUND AND OBJECTIVE: Emerging evidence suggests that blue light can photo-inactivate some ba... more BACKGROUND AND OBJECTIVE: 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. MATERIALS AND METHODS: We plated 5 × 106 and 7 × 106 CFU/ml USA300 strain of MRSA and 1 × 106 CFU/ml of Salmonella enterica serovars Typhimurium and Heidelberg. Plates were irradiated with 55, 110, 165 and 220 J/cm2 of blue light, incubated at 37°C for 24 hours and colony counts determined. RESULTS: Compared with controls, blue light irradiation produced a significant dose-dependent reduction in the number of colonies formed by each bacterial strain (P < 0.001). Irradiation of 5 × 106 and 7 × 106 CFU/ml MRSA with 55 J/cm2 produced 92% (4.6 × 106 CFU/ml) and 86% (6 × 106 CFU/ml) inactivation respectively, while 110 and 220 J/cm2 suppressed each MRSA density 100%. Irradiation of Salmonella Typhimurium with 55 and 110 J/cm2 suppressed bacterial growth 31% (3.1 × 105 CFU/ml) and 93% (9.3 × 105 CFU/ml) respectively; while Salmonella Heidelberg was inhibited 11% (1.1 × 105 CFU/ml) and 84% (8.4 × 105 CFU/ml) respectively by the two fluences. Complete inactivation of each Salmonella strain was achieved using 165 or 220 J/cm2 . CONCLUSION: The observed inhibition of Gram-positive (MRSA) and Gram-negative (Salmonella) bacteria suggests the versatility of blue light in bacteria eradication, making it a viable intervention strategy for decontamination of food and environments that harbor such bacteria.

Research paper thumbnail of Whole-genome sequence for methicillin-resistant Staphylococcus aureus strain ATCC BAA-1680

Genome Announc 3(2):e00011-15. doi:10.1128/genomeA.00011-15, Mar 2015

We report here the whole-genome sequence of the USA300 strain of methicillin-resistant Staphyloco... more 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.

Research paper thumbnail of The bactericidal effect of 470-nm light and hyperbaric oxygen on methicillin-resistant Staphylococcus aureus (MRSA)

Lasers in Medical Science, Feb 21 2015, doi:10.1007/s10103-015-1722-9

It has been shown that, in vitro, hyperbaric oxygen (HBO) suppresses 28 % bacterial growth, whil... more It has been shown that, in vitro, hyperbaric oxygen
(HBO) suppresses 28 % bacterial growth, while 470-nm blue
light alone suppresses up to 92 % methicillin-resistant
Staphylococcus aureus (MRSA) in one application in vitro.
Therefore, we determined if combined 470-nm light (55 J/
cm2) and HBO will yield 100 % bacterial suppression in experimental
simulation of mild, moderate or severe MRSA
infection. We cultured MRSA at 3×106, 5×106, 7×106, 8×
106, or 12×106 CFU/ml and treated each concentration in four
groups as follows: (1) control (no treatment) (2) photoirradiation
only, (3) photo-irradiation then HBO, (4) HBO
only, and (5) HBO then photo-irradiation. Bacteria colonies
were then quantified. The results showed that at each bacterial
concentration, HBO alone was significantly less effective in
suppressing MRSA than photo-irradiation or combined HBO
and photo-irradiation (p<0.0001). Similarly, at no bacterial
concentration did combined HBO and 470-nm light treatment
yield a statistically better result than 470-nm light alone
(p>0.05), neither did HBO treatment either before or after
irradiation make a difference. Furthermore, at no bacterial
concentration was 100 % MRSA suppression achieved.
Indeed, the maximum bacterial suppression attained was in
the mild infection model (3×106 CFU/ml), with blue light
producing 97.3±0.2 % suppression and HBO+55 J/cm2
yielding 97.5±2.5 % suppression. We conclude that (1)
HBO and 470-nm light individually suppress MRSA growth;
(2) 470-nm blue light is more effective in suppressing MRSA
than HBO; and (3) HBO did not act synergistically to heighten
the bactericidal effect of 470-nm light.

Research paper thumbnail of Optimization of the antimicrobial effect of blue light on Methicillin-resistant Staphylococcus aureus (MRSA) in vitro

Lasers in Surgery and Medicine. Article first published online: 12 JAN 2015 DOI: 10.1002/lsm.22327, Jan 12, 2015

Background and Objective: In previous studies, we showed that irradiation with 405 nm or 470 nm l... more Background and Objective: 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. Materials and Methods: We cultured 3 × 106 and 5 × 106 CFU/ml USA300 strain of MRSA and then irradiated each plate with varying fluences of 1–60 J/cm2 of 405 nm or 470 nm light, either once or twice at 6 hours intervals. Next, we plated 7 × 106 CFU/ml and irradiated it with 45, 50, 55, or 60 J/cm2 fluence, once, twice, or thrice at the same 6 hours intervals. In a third experiment, the same culture density was irradiated with 0, 165, 180, 220, or 240 J/cm2, either once, twice, or thrice. Results: Irradiation with either wavelength significantly reduced the bacterial colonies regardless of bacterial density (P < 0.05). At 3 × 106 CFU/ml density, nearly 40% and 50% growth of MRSA were suppressed with as little as 3 J/cm2 of 405 nm and 470 nm wavelengths, respectively. Moreover, 100% of the colonies were suppressed with a single exposure to 55 or 60 J/cm2 of 470 nm light or double treatment with 50, 55, or 60 J/cm2 of 405 nm wavelength. At 5 × 106 CFU/ml density, irradiating twice with 50, 55, or 60 J/cm2 of either wavelength suppressed bacterial growth completely, lower fluences did not. The denser 7 × 106 CFU/ml culture required higher doses to achieve 100% suppression, either one shot with 220 J/cm2 of 470 nm light or two shots of the same dose using 405 nm. Conclusion: The bactericidal effect of blue light can be optimized to yield 100% bacterial growth suppression, but with relatively high fluences for dense bacterial cultures, such as 7 × 106 CFU/ml.

Research paper thumbnail of Phototherapy promotes healing of cutaneous wounds in undernourished rat

Anais brasileiros de dermatologia, 89(6):899-904. , Dec 2014

BACKGROUND: Various studies have shown that phototherapy promotes the healing of cutaneous wounds... more 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.
Keywords: Nutritional marasmus; Phototherapy; Protein malnutrition; Wound healing

Research paper thumbnail of Preponderance of toxigenic Escherichia coli in stool pathogens correlates with toxin detection in accessible drinking-water sources

Epidemiology and Infection, May 1, 2014

Since early detection of pathogens and their virulence factors contribute to intervention and co... more Since early detection of pathogens and their virulence factors contribute to intervention and
control strategies, we assessed the enteropathogens in diarrhoea disease and investigated the
link between toxigenic strains of Escherichia coli from stool and drinking-water sources; and
determined the expression of toxin genes by antibiotic-resistant E. coli in Lagos, Nigeria. This
was compared with isolates from diarrhoeal stool and water from Wisconsin, USA. The new
Luminex xTAG GPP (Gastroplex) technique and conventional real-time PCR were used to
profile enteric pathogens and E. coli toxin gene isolates, respectively. Results showed the
pathogen profile of stool and indicated a relationship between E. coli toxin genes in water and
stool from Lagos which was absent in Wisconsin isolates. The Gastroplex technique was efficient
for multiple enteric pathogens and toxin gene detection. The co-existence of antibiotic resistance
with enteroinvasive E. coli toxin genes suggests an additional prognostic burden on patients.

Research paper thumbnail of Topical treatment with Copaifera langsdorffii oleoresin improves wound healing in rats

International Journal of Phytomedicine, v.5, n.3, 378-386

Copaifera langsdorffii oleoresin (copaiba) has been used in the Amazon as traditional wound heali... more Copaifera langsdorffii oleoresin (copaiba) has been used in the Amazon as traditional wound healing remedy for centuries. Since its mechanisms of action remain unclear, we investigated its effects on excisional wounds in rats. Wounds were made on the dorsum of animals assigned to three groups: saline, control cream and 10% copaiba cream, and assessed on days 2, 7 and 14 post-wounding morphometrically, histologically and biochemically. Wound healing rate was faster in copaiba than in saline or cream groups. This was corroborated by matrix metalloproteinase (MMP)-2 activity which rose progressively throughout in copaiba group. MMP-9, a marker of inflammation, was not detectable at day 14 in copaiba group, but persisted in the other groups. Moreover, histology showed early population of copaiba-treated wounds by inflammatory cells, and by day 14 this group had less fibroblasts and more organized collagen. Further, copaiba group synthesized collagen faster than saline and cream groups, as evidenced by progressive increases in the amounts of hydroxyproline at days 7 and 14 (p < 0.012). These findings suggest that 10% copaiba oleoresin cream promotes wound healing in rats by regulating MMP-2 and MMP-9 activities, stimulating collagen synthesis and promoting tissue remodeling and reepithelialization.

Research paper thumbnail of Antimicrobial effects of Copaifera langsdorffii oleoresin in infected rat wounds

International Journal of Applied Microbiology Sciences 2013; 2(3):9-20

We determined the effect of Copaifera langsdorffii (copaiba) cream on wounds infected with Strept... more We determined the effect of Copaifera langsdorffii (copaiba) cream on wounds infected with Streptococcus pyogenes or Staphylococcus aureus. Wounds were created on the dorsum of Wistar rats, then inoculated with either pathogen, and observed through 14 days as they were treated either with saline, control cream or 10% copaiba cream. Wounds were assessed for healing on days 2, 7 and 14 post-surgery, and then swabbed and biopsied to quantify bacteria. By day 14, treatment with 10% copaiba decreased S. pyogenes infection to 0.02%; saline treatment reduced infection to 17.2%, while control cream increased infection 87.4%. Copaiba similarly decreased S. aureus infection to 0.3% by day 14, compared with 26.9% for saline and 12.7% for control cream. Consistent with these findings, copaiba treated S. pyogenes infected wounds re-epithelized 29% by day 2, compared with 15.8% for saline (p = 0.025) and 18.4% for control cream treated wounds; maintaining a higher rate of re-epithelialization (67.5%) over the control cream group (54.9%) on day 7 (p = 0.002). Similarly, copaiba enhanced healing of S. aureus infected wounds, 74.4% by day 7, when compared with saline and control cream wounds. These findings suggest that copaiba retards bacterial infection resulting in better healing.

Research paper thumbnail of Cytotoxicity and wound healing properties of Copaifera langsdorffii oleoresin in rabbits

"" Plants of the Copaifera genus (copaiba) are documented to have healing potential; however, t... more ""
Plants of the Copaifera genus (copaiba) are documented to have healing potential; however, their cytotoxicity and therapeutic mechanisms remain unclear. This study assessed in vitro cytotoxicity of copaiba oleoresin from Copaifera langsdorffii on 3T3 fibroblasts and investigated its wound healing potential in rabbits. Cell viability and proliferation were assessed by MTT and H3-Thymidine assays, respectively. Wounds were created on rabbits’ ears and topically treated with saline (S), control cream (Cr), 10% (C10) and 25% (C25) copaiba cream, or pure oleoresin (OR) over 21 days, and assessed 2, 7, 14 and 21 days post-wounding by wound healing rates and histology. Fibroblasts treated with up to 100 μg/mL of copaiba yielded greater than 80% of viability and proliferation compared to control culture, decreasing significantly (p<0.001) at 500 and 1000 μg/mL. Wounds were re-epithelialized by the 21st day, with some OR-treated wounds showing evidence of sustained inflammation on day 7. C10 and C25-treated wounds presented better clinical outcomes, confirmed by histology with evidence of fibroblastic activity by day 7 and organized collagen fibers observed from day 14. These findings suggest that C. langsdorffii oleoresin shows no cytotoxic effects on fibroblasts at 100 μg/mL and at appropriate doses enhances wound healing.
""

Research paper thumbnail of 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-... more 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 5×106 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/cm2. 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 3×106, 5×106, and 7×106 CFU/mL cultures, respectively. Both wavelengths had similar effects on the sparser 3×106 and 5×106 CFU/mL cultures, but in the denser 7×106 CFU/mL culture, 405 nm light cleared more bacteria at each fluence (p<0.001). To determine the effect of beam penetration, denser 8×106 and 12×106 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.

Research paper thumbnail of Antimicrobial activity of copaiba (Copaifera langsdorffii) oleoresin on bacteria of clinical significance in cutaneous wounds

The present study was designed to evaluate the in vitro antimicrobial activity of Copaifera langs... more The present study was designed to evaluate the in vitro antimicrobial activity of Copaifera langsdorffii oleoresin, which has been used in folk medicine as an anti-inflammatory, antibacterial, healing among others. The oleoresin was tested against Gram-positive (Staphylococcus aureus, Streptococcus pyogenes, Enterococcus faecalis) and Gram-negative (Pseudomonas aeruginosa and Escherichia coli) bacteria related to infections in cutaneous wounds. Antimicrobial activity was determined by the minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) assays. Copaiba oleoresin showed antimicrobial activity only against the Gram-positive bacteria with MIC of 200 μg/mL, 400 μg/mL and 1100 μg/mL for S. aureus, S. pyogenes and E. faecalis, respectively. MBC values were the same as MIC for S. aureus and S. pyogenes and for E. faecalis it was 1200 μg/mL. Considering that infection significantly impairs the wound healing process, we believe that the use of copaiba oleoresin as a component of a topical formulation could be a valuable adjunct in the treatment of infected wounds, mainly in the case of wounds infected by Gram-positive microorganisms.

Research paper thumbnail of Experimental models of malnutrition and its effect on skin trophism

The skin requires adequate levels of nutrients to function properly. To analyze skin trophism i... more The skin requires adequate levels of nutrients to function properly.
To analyze skin trophism in well-nourished and undernourished rats using two models of malnutrition. In the marasmus model, 60 Wistar rats were kept on a controlled diet, 30 being randomly selected to receive half the established diet for 60 days. In the gelatin model, 60 rats were used, 30 of which received a diet consisting of poor quality protein (gelatin) for 30 days. The nutritional status of the animals was evaluated according to body mass index, clinical signs and serum albumin measurement. After the period of malnutrition, histology was performed on the animals' skin to analyze the thickness of the dermis and epidermis using the Leica Application Suite software. Collagen was analyzed on slides stained with Gömöri trichrome using the ImageJ software program.
The body mass index of the malnourished animals in the marasmus and gelatin groups was significantly lower than that of the well-nourished animals in the two groups (p<0.0001 in both models). With respect to serum albumin, there was no difference between the groups in either of the two models. In relation to the histological analysis of skin thickness, the dermis of the malnourished animals was significantly thinner compared to that of the well-nourished animals (p<0.0001 in both models). The percentage of collagen was lower in the malnourished animals compared to the well-nourished animals (p<0.0005 and p<0.003 in the marasmus and gelatin model, respectively).
Skin thickness measurements were lower in the malnourished animals in both models, and this finding was histologically confirmed by the lower percentage of collagen, showing the negative effect of malnutrition on skin trophism

Research paper thumbnail of Polyhydroxy alcohols and peach oil addition influence in liquid crystal formation and rheological behaviour of O/W emulsions

The aim of this study was to developed O/W emulsions with Self-Emulsifying Base (SEB) cointainnin... more The aim of this study was to developed O/W emulsions with Self-Emulsifying Base (SEB) cointainning liquid crystals (LC) and evaluate the influence of polyhydroxy alcohols and peach oil addition in liquid crystals formation as well as to analyze the rheological behaviour of these systems. Formulations containing SEB, were supplemented or not, with glycerin or sorbitol or propylene glycol, and/or peach oil. All formulations studied had lamellar crystalline phases. Peach oil addition increased the viscosity of SEB, while humectants had different behaviour, glycerin enhanced the viscosity of the studied formulations supplemented or not with peach oil, however, propylene glycol decreased the viscosity of all formulations studied. On the other hand, sorbitol enhanced the viscosity of formulations containing peach oil. All emulsions presented pseudoplastic behaviour and tixotropy.