Pulsed blue light, saliva and curcumin significantly inactivate human coronavirus.

In a recent study, we showed that pulsed blue light (PBL) inactivates as much as 52.3% of human beta coronavirus HCoV-OC43, a surrogate of SARS-CoV-2, and one of the major strains of viruses responsible for the annual epidemic of the common cold. Since curcumin and saliva are similarly antiviral and curcumin acts as blue light photosensitizer, we used Qubit fluorometry and WarmStart RT-LAMP assays to study the effect of combining 405 nm, 410 nm, 425 nm or 450 nm wavelengths of PBL with curcumin, saliva or a combination of curcumin and saliva against human beta coronavirus HCoV-OC43. The results showed that PBL, curcumin and saliva independently and collectively inactivate HCoV-OC43. Without saliva or curcumin supplementation 21.6 J/cm2 PBL reduced HCoV-OC43 RNA concentration a maximum of 32.8% (log10 = 2.13). Saliva supplementation alone inactivated the virus, reducing its RNA concentration by 61% (log10 = 2.23); with irradiation the reduction was as much as 79.1%. Curcumin supplementation alone decreased viral RNA 71.1%, and a maximum of 87.8% with irradiation. The combination of saliva and curcumin reduced viral RNA to 83.1% and decreased the RNA up to 90.2% with irradiation. The reduced levels could not be detected with qPCR. These findings show that PBL in the range of 405 nm to 450 nm wavelength is antiviral against human coronavirus HCoV-OC43, a surrogate of the COVID-19 virus. Further, it shows that with curcumin as a photosensitizer, it is possible to photodynamically inactivate the virus beyond qPCR detectable level using PBL. Since HCoV-OC43 is of the same beta coronavirus family as SARS-CoV-2, has the same genomic size, and is often used as its surrogate, these findings heighten the prospect of similarly inactivating novel coronavirus SARS-CoV-2, the virus responsible for COVID-19 pandemic.

Pulsed blue light inactivates two strains of human coronavirus.

Emerging evidence suggests that blue light has the potential to inactivate viruses. Therefore, we investigated the effect of 405 nm, 410 nm, 425 nm and 450 nm pulsed blue light (PBL) on human alpha coronavirus HCoV-229 E and human beta coronavirus HCoV-OC43, using Qubit fluorometry and RT-LAMP to quantitate the amount of nucleic acid in irradiated and control samples. Like SARS-CoV-2, HCoV-229E and HCoV-OC43 are single stranded RNA viruses transmitted by air and direct contact; they have similar genomic sizes as SARS-CoV-2, and are used as surrogates for SARS-CoV-2. Irradiation was carried out either at 32.4 J cm-2 using 3 mW cm-2 irradiance or at 130 J cm-2 using 12 mW cm-2 irradiance. Results: (1) At each wavelength tested, PBL was antiviral against both coronaviruses. (2) 405 nm light gave the best result, yielding 52.3% (2.37 log10) inactivation against HCoV-OC43 (p < .0001), and a significant 1.46 log 10 (44%) inactivation of HCoV-229E (p < .01). HCoV-OC43, which like SARS-CoV-2 is a beta coronavirus, was more susceptible to PBL irradiation than alpha coronavirus HCoV-229E. The latter finding suggests that PBL is potentially antiviral against multiple coronavirus strains, and that, while its potency may vary from one virus to another, it seems more antiviral against beta coronaviruses, such as HCoV-OC43. (3) Further, the antiviral effect of PBL was better at a higher irradiance than a lower irradiance, and this indicates that with further refinement, a protocol capable of yielding 100% inactivation of viruses is attainable.

Structural membrane changes induced by pulsed blue light on methicillin-resistant Staphylococcus aureus (MRSA).

BACKGROUND: In a recent study we showed that blue light inactivates methicillin-resistant Staphylococcus aureus (MRSA) by perturbing, depolarizing, and disrupting its cell membrane. PURPOSE: The current study presents visual evidence that the observed biochemical changes also result in cell metabolic changes and structural alteration of the cell membrane. METHODS: Cultures of MRSA were treated with 450 nm pulsed blue light (PBL) at 3 mW/cm2 irradiance, using a sub lethal dose of 2.7 J/cm2 radiant exposure three times at 30-min intervals. Following 24 h incubation at 37 °C, irradiated colonies and control non-irradiated colonies were processed for light and transmission electron microscopy. RESULTS: The images obtained revealed three major effects of PBL; (1) disruption of MRSA cell membrane, (2) alteration of membrane structure, and (3) disruption of cell replication. CONCLUSION: These signs of bacterial inactivation at a dose deliberately selected to be sub-lethal supports our previous finding that rapid depolarization of bacterial cell membrane and disruption of cellular function comprise another mechanism underlying photo-inactivation of bacteria. Further, it affirms the potency of PBL.

Rare mutations in Pfmdr1 gene of Plasmodium falciparum detected in clinical isolates from patients treated with anti-malarial drug in Nigeria.

BACKGROUND: Plasmodium falciparum, the deadliest causative agent of malaria, has high prevalence in Nigeria. Drug resistance causing failure of previously effective drugs has compromised anti-malarial treatment. On this basis, there is need for a proactive surveillance for resistance markers to the currently recommended artemisinin-based combination therapy (ACT), for early detection of resistance before it become widespread. METHODS: This study assessed anti-malarial resistance genes polymorphism in patients with uncomplicated P. falciparum malaria in Lagos, Nigeria. Sanger and Next Generation Sequencing (NGS) methods were used to screen for mutations in thirty-seven malaria positive blood samples targeting the P. falciparum chloroquine-resistance transporter (Pfcrt), P. falciparum multidrug-resistance 1 (Pfmdr1), and P. falciparum kelch 13 (Pfk13) genes, which have been previously associated with anti-malarial resistance. RESULTS: Expectedly, the NGS method was more proficient, detecting six Pfmdr1, seven Pfcrt and three Pfk13 mutations in the studied clinical isolates from Nigeria, a malaria endemic area. These mutations included rare Pfmdr1 mutations, N504K, N649D, F938Y and S967N, which were previously unreported. In addition, there was moderate prevalence of the K76T mutation (34.6%) associated with chloroquine and amodiaquine resistance, and high prevalence of the N86 wild type allele (92.3%) associated with lumefantrine resistance. CONCLUSION: Widespread circulation of mutations associated with resistance to current anti-malarial drugs could potentially limit effective malaria therapy in endemic populations.

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.

Blue/violet laser inactivates methicillin-resistant Staphylococcus aureus by altering its transmembrane potential.

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 photo-acceptor molecules. Bacterial cultures irradiated with one or two doses of 405nm laser light (each consisting of 121J/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 5min 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.

Spectrally resolved infrared microscopy and chemometric tools to reveal the interaction between blue light (470nm) 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.

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.

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

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. Lasers Surg. Med. 47:595-601, 2015. © 2015 Wiley Periodicals, Inc.

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

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.

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

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 × 10(6) and 5 × 10(6) 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 × 10(6) 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/cm(2) , either once, twice, or thrice. RESULTS: Irradiation with either wavelength significantly reduced the bacterial colonies regardless of bacterial density (P < 0.05). At 3 × 10(6) 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 × 10(6) 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 × 10(6) 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 × 10(6) CFU/ml.

Molecular typing reveals substantial Plasmodium vivax infection in asymptomatic adults in a rural area of Cameroon.

BACKGROUND: Malaria in Cameroon is due to infections by Plasmodium falciparum and, to a lesser extent, Plasmodium malariae and Plasmodium ovale, but rarely Plasmodium vivax. A recent report suggested "Plasmodium vivax-like" infections around the study area that remained unconfirmed. Therefore, molecular and antigenic typing was used to investigate the prevalence of P. vivax and Duffy in asymptomatic adults resident in Bolifamba. METHODS: A cross-sectional study was conducted from July 2008 to October 2009. The status of all parasite species was determined by nested PCR in 269 blood samples collected. The P. falciparum and P. vivax anti-MSP/CSP antibody status of each subject was also determined qualitatively by a rapid card assay. Parasite DNA was extracted from a sample infected with three parasite species, purified and sequenced. The Duffy antigen status of 12 subjects infected with P. vivax was also determined by sequencing. In silico web-based tools were used to analyse sequence data for similarities and matches to reference sequences in public DNA databases. RESULTS: The overall malaria parasite prevalence in 269 individuals was 32.3% (87) as determined by PCR. Remarkably, 14.9% (13/87) of infections were caused either exclusively or concomitantly by P. vivax, established both by PCR and microscopic examination of blood smears, in individuals both positive (50%, 6/12) and negative (50%, 6/12) for the Duffy receptor. A triple infection by P. falciparum, P. vivax and P. malariae, was detected in one infected individual. Anti-MSP/CSP antibodies were detected in 72.1% (194/269) of samples, indicating high and continuous exposure to infection through mosquito bites. DISCUSSION: These data provide the first molecular evidence of P. vivax in Duffy positive and negative Cameroonians and suggest that there may be a significant prevalence of P. vivax infection than expected in the study area. Whether the P. vivax cases were imported or due to expansion of a founder effect was not investigated. Notwithstanding, the presence of P. vivax may complicate control efforts if these parasites become hypnozoitic or latent as the liver stage. CONCLUSIONS: These data strongly suggest that P. vivax is endemic to the south-west region of Cameroon and should be taken into account when designing malaria control strategies.

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.