Blue Light Compromises Bacterial ß-Lactamases Activity to Overcome ß-Lactam Resistance.

OBJECTIVE: In this study, we evaluated the effectiveness of antimicrobial blue light (aBL; 410 nm wavelength) against ß-lactamase-carrying bacteria and the effect of aBL on the activity of ß-lactamases. METHODS: Pseudomonas aeruginosa, Escherichia coli, and Klebsiella pneumoniae strains carrying ß-lactamases as well as a purified ß-lactamase enzymes were studied. ß-lactamase activity was assessed using a chromogenic cephalosporin hydrolysis assay. Additionally, we evaluated the role of porphyrins in the photoreaction, as well as protein degradation by sodium dodecyl-sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Finally, we investigated the bactericidal effect of combined aBL-ceftazidime exposure against a metallo-ß-lactamase expressing P. aeruginosa strain. RESULTS: Our study demonstrated that aBL effectively killed ß-lactamase-producing bacteria and reduced ß-lactamase activity. After an aBL exposure of 1.52 J/cm2, a 50% reduction in enzymatic activity was observed in P. aeruginosa. Additionally, we found a 40% decrease in the photoreaction activity of porphyrins following an aBL exposure of 64.8 J/cm2. We also revealed that aBL reduced ß-lactamase activity via protein degradation (after 136.4 J/cm2). Additionally, aBL markedly improved the bactericidal effect of ceftazidime (by >4-log10) in the metallo-ß-lactamase P. aeruginosa strain. CONCLUSION: Our results provide evidence that aBL compromises bacterial ß-lactamase activity, offering a potential approach to overcome ß-lactam resistance in bacteria.

Efficacy of Preemptive Analgesia with Amantadine for Controlling Postoperative Pain in Cats Undergoing Ovariohysterectomy.

This study aimed to evaluate the effect of the preemptive administration of amantadine on postoperative analgesia in cats undergoing ovariohysterectomy and its influence on the physiological parameters. Twenty healthy domestic cats scheduled to undergo ovariohysterectomy at the Santa Cruz State University, Ilhéus, were divided into two groups: the control group (Group C; n = 10) and the amantadine group (Group A; n = 10). The cats in Group C received placebo capsules 30 min prior to the standard anesthetic protocol, whereas those in Group A received 5 mg/kg of amantadine orally 30 min prior to the standard anesthetic protocol. Postoperative pain was assessed using the visual analog scale and the UNESP-Botucatu multidimensional scale for the evaluation of postoperative pain in cats. The administration of amantadine had no effect on the physiological parameters evaluated. The pain scores in Group A were lower than those in Group C, indicating that the frequency of rescue analgesic administration cats in Group A was lower. That way, preemptive oral administration of amantadine at a dose of 5 mg/kg was effective at controlling postoperative pain in cats undergoing ovariohysterectomy. Moreover, no adverse effects or alterations in the physiological patterns were observed in the treated animals.

Antimicrobial Resistance: Is There a 'Light' at the End of the Tunnel?

In recent years, with the increases in microorganisms that express a multitude of antimicrobial resistance (AMR) mechanisms, the threat of antimicrobial resistance in the global population has reached critical levels. The introduction of the COVID-19 pandemic has further contributed to the influx of infections caused by multidrug-resistant organisms (MDROs), which has placed significant pressure on healthcare systems. For over a century, the potential for light-based approaches targeted at combatting both cancer and infectious diseases has been proposed. They offer effective killing of microbial pathogens, regardless of AMR status, and have not typically been associated with high propensities of resistance development. To that end, the goal of this review is to describe the different mechanisms that drive AMR, including intrinsic, phenotypic, and acquired resistance mechanisms. Additionally, the different light-based approaches, including antimicrobial photodynamic therapy (aPDT), antimicrobial blue light (aBL), and ultraviolet (UV) light, will be discussed as potential alternatives or adjunct therapies with conventional antimicrobials. Lastly, we will evaluate the feasibility and requirements associated with integration of light-based approaches into the clinical pipeline.

Haemoproteus jenniae (Haemoproteidae, Haemosporida) infects gulls (Larus spp.) in South Africa, with redescription of Haemoproteus skuae.

Haemoproteus spp. are dipteran-borne protozoa that infect erythrocytes and reticulo-endothelial cells of birds. These parasites are not usually transmitted between birds belonging to different orders. The suborder Lari (order Charadriiformes) comprises ~170 avian species, the majority of which are aquatic, including gulls, terns, auklets, murres and skuas, among others. In spite of the diversity of this avian group, there is limited known diversity of haemosporidian parasites, with only 4 recorded Haemoproteus morphospecies thus far. We examined the blood smears of 21 kelp gulls (Larus dominicanus) captured at a breeding colony in South Africa, as well as Haemoproteus-positive archival blood smears of 15 kelp gulls and 1 Hartlaub's gull (Larus hartlaubii) sampled while under care at seabird rehabilitation facilities in South Africa. Haemoproteus sp. infection was detected in 19% of wild-caught kelp gulls. All parasites from the gulls were morphologically identified as Haemoproteus jenniae, a species previously recorded in Lari birds at the Galapagos Islands (Ecuador), Rocas Atoll (Brazil) and Poland. Gene sequencing uncovered a new cytochrome b lineage, LARDOM01, which was closely related to the previously reported H. jenniae lineage CREFUR01. Additionally, we evaluated a hapantotype blood smear of Haemoproteus skuae, which had been described infecting a brown skua (Catharacta antarctica) in South Africa. We provide a redescription of H. skuae and discuss the morphological characters distinguishing it from H. jenniae. Further research is necessary to improve our knowledge about the host and geographic distribution, health effects and phylogeny of H. jenniae and H. skuae.

New Insights into the Bacterial Targets of Antimicrobial Blue Light.

Antimicrobial blue light (aBL) offers efficacy and safety in treating infections. However, the bacterial targets for aBL are still poorly understood and may be dependent on bacterial species. Here, we investigated the biological targets of bacterial killing by aBL (λ = 410 nm) on three pathogens: Staphylococcus aureus, Escherichia coli, and Pseudomonas aeruginosa. Initially, we evaluated the killing kinetics of bacteria exposed to aBL and used this information to calculate the lethal doses (LD) responsible for killing 90 and 99.9% of bacteria. We also quantified endogenous porphyrins and assessed their spatial distribution. We then quantified and suppressed reactive oxygen species (ROS) production in bacteria to investigate their role in bacterial killing by aBL. We also assessed aBL-induced DNA damage, protein carbonylation, lipid peroxidation, and membrane permeability in bacteria. Our data showed that P. aeruginosa was more susceptible to aBL (LD99.9 = 54.7 J/cm2) relative to S. aureus (LD99.9 = 158.9 J/cm2) and E. coli (LD99.9 = 195 J/cm2). P. aeruginosa exhibited the highest concentration of endogenous porphyrins and level of ROS production relative to the other species. However, unlike other species, DNA degradation was not observed in P. aeruginosa. Sublethal doses of blue light (LD99.9). We conclude that the primary targets of aBL depend on the species, which are probably driven by variable antioxidant and DNA-repair mechanisms. IMPORTANCE Antimicrobial-drug development is facing increased scrutiny following the worldwide antibiotic crisis. Scientists across the world have recognized the urgent need for new antimicrobial therapies. In this sense, antimicrobial blue light (aBL) is a promising option due to its antimicrobial properties. Although aBL can damage different cell structures, the targets responsible for bacterial inactivation have still not been completely established and require further exploration. In our study, we conducted a thorough investigation to identify the possible aBL targets and gain insights into the bactericidal effects of aBL on three relevant pathogens: Staphylococcus aureus, Escherichia coli, and Pseudomonas aeruginosa. This research not only adds new content to blue light studies but opens new perspectives to antimicrobial applications.

The Biochemical Mechanisms of Antimicrobial Photodynamic Therapy†.

The unbridled dissemination of multidrug-resistant pathogens is a major threat to global health and urgently demands novel therapeutic alternatives. Antimicrobial photodynamic therapy (aPDT) has been developed as a promising approach to treat localized infections regardless of drug resistance profile or taxonomy. Even though this technique has been known for more than a century, discussions and speculations regarding the biochemical mechanisms of microbial inactivation have never reached a consensus on what is the primary cause of cell death. Since photochemically generated oxidants promote ubiquitous reactions with various biomolecules, researchers simply assumed that all cellular structures are equally damaged. In this study, biochemical, molecular, biological and advanced microscopy techniques were employed to investigate whether protein, membrane or DNA damage correlates better with dose-dependent microbial inactivation kinetics. We showed that although mild membrane permeabilization and late DNA damage occur, no correlation with inactivation kinetics was found. On the other hand, protein degradation was analyzed by three different methods and showed a dose-dependent trend that matches microbial inactivation kinetics. Our results provide a deeper mechanistic understanding of aPDT that can guide the scientific community toward the development of optimized photosensitizing drugs and also rationally propose synergistic combinations with antimicrobial chemotherapy.

Beta diversity, prevalence, and specificity of avian haemosporidian parasites throughout the annual cycle of Chilean Elaenia (Elaenia chilensis), a Neotropical austral migrant.

Migratory birds are implicated in dispersing haemosporidian parasites over great geographic distances. However, their role in sharing these vector-transmitted blood parasites with resident avian host species along their migration flyway is not well understood. We studied avian haemosporidian parasites in 10 localities where Chilean Elaenia, a long-distance Neotropical austral migrant species, spends part of its annual cycle to determine local parasite transmission among resident sympatric host species in the elaenia's distributional range across South America. We sampled 371 Chilean Elaenias and 1,818 birds representing 243 additional sympatric species from Brazilian wintering grounds to Argentinian breeding grounds. The 23 haemosporidian lineages found in Chilean Elaenias exhibited considerable variation in distribution, specialization, and turnover across the 10 avian communities in South America. Parasite lineage dissimilarity increased with geographic distance, and infection probability by Parahaemoproteus decreased in localities harbouring a more diverse haemosporidian fauna. Furthermore, blood smears from migrating Chilean Elaenias and local resident avian host species did not contain infective stages of Leucocytozoon, suggesting that transmission did not take place in the Brazilian stopover site. Our analyses confirm that this Neotropical austral migrant connects avian host communities and transports haemosporidian parasites along its distributional range in South America. However, the lack of transmissive stages at stopover site and the infrequent parasite lineage sharing between migratory host populations and residents at breeding and wintering grounds suggest that Chilean Elaenias do not play a significant role in dispersing haemosporidian parasites, nor do they influence local transmission across South America.

Antimicrobial Blue Light for Prevention and Treatment of Highly Invasive Vibrio vulnificus Burn Infection in Mice.

Vibrio vulnificus is an invasive marine bacterium that causes a variety of serious infectious diseases. With the increasing multidrug-resistant variants, treatment of V. vulnificus infections is becoming more difficult. In this study, we explored antimicrobial blue light (aBL; 405 nm wavelength) for the treatment of V. vulnificus infections. We first assessed the efficacy of aBL against five strains of V. vulnificus in vitro. Next, we identified and quantified intracellular porphyrins in V. vulnificus to provide mechanistic insights. Additionally, we measured intracellular reactive oxygen species (ROS) production and bacterial membrane permeabilization following aBL exposures. Lastly, we conducted a preclinical study to investigate the efficacy and safety of aBL for the prevention and treatment of burn infections caused by V. vulnificus in mice. We found that aBL effectively killed V. vulnificus in vitro in both planktonic and biofilm states, with up to a 5.17- and 4.57-log10 CFU reduction being achieved, respectively, following an aBL exposure of 216 J/cm2. Protoporphyrin IX and coproporphyrins were predominant in all the strains. Additionally, intracellular ROS was significantly increased following aBL exposures (P < 0.01), and there was evidence of aBL-induced permeabilization of the bacterial membrane (P < 0.0001). In the preclinical studies, we found that female mice treated with aBL 30 min after bacterial inoculation showed a survival rate of 81% following 7 days of observation, while only 28% survival was observed in untreated female mice (P < 0.001). At 6 h post-inoculation, an 86% survival was achieved in aBL-treated female mice (P = 0.0002). For male mice, 86 and 63% survival rates were achieved when aBL treatment was given 30 min and 6 h after bacterial inoculation, respectively, compared to 32% survival in the untreated mice (P = 0.0004 and P = 0.04). aBL did not reduce cellular proliferation or induce apoptosis. We found five cytokines were significantly upregulated in the males after aBL treatment, including MCSF (P < 0.001), MCP-5 (P < 0.01), TNF RII (P < 0.01), CXCL1 (P < 0.01), and TIMP-1 (P < 0.05), and one in the females (TIMP-1; P < 0.05), suggesting that aBL may induce certain inflammatory processes. In conclusion, aBL may potentially be applied to prevent and treat V. vulnificus infections.

A new haemosporidian parasite from the Red-legged Seriema Cariama cristata (Cariamiformes, Cariamidae).

Haemoproteids (Haemosporida, Haemoproteidae) are a diverse group of avian blood parasites that are transmitted by hematophagous dipterans. In this study, we describe Haemoproteus pulcher sp. nov. from a Red-legged Seriema (Cariama cristata) in southeast Brazil. Analysis of the mitochondrial cytb gene indicates this parasite is closely related to Haemoproteus catharti (from Turkey Vulture, Cathartes aura) and the unidentified haemosporidian lineages PSOOCH01 (from Pale-winged Trumpeter, Psophia leucoptera) and MYCAME08 (from Wood Stork, Mycteria americana). This group of parasites appears to represent an evolutionary lineage that is distinct from other Haemoproteus spp., being instead more closely related to Haemocystidium spp. (from reptiles), Plasmodium spp. (from reptiles, birds, and mammals) and other mammal-infecting haemosporidians (Nycteria, Polychromophilus, and Hepatocystis). Current evidence suggests that parasites of this newly discovered evolutionary lineage may be endemic to the Americas, but further studies are necessary to clarify their taxonomy, life cycle, vectors, hosts, geographic distribution and host health effects. Additionally, it should be borne in mind that some PCR protocols targeting the cytb gene might not reliably detect H. pulcher due to low primer affinity.

Antimicrobial blue light: A 'Magic Bullet' for the 21st century and beyond?

Over the past decade, antimicrobial blue light (aBL) at 400 - 470 nm wavelength has demonstrated immense promise as an alternative approach for the treatment of multidrug-resistant infections. Since our last review was published in 2017, there have been numerous studies that have investigated aBL in terms of its, efficacy, safety, mechanism, and propensity for resistance development. In addition, researchers have looked at combinatorial approaches that exploit aBL and other traditional and non-traditional therapeutics. To that end, this review aims to update the findings from numerous studies that capitalize on the antimicrobial effects of aBL, with a focus on: efficacy of aBL against different microbes, identifying endogenous chromophores and targets of aBL, Resistance development to aBL, Safety of aBL against host cells, and Synergism of aBL with other agents. We will also discuss our perspective on the future of aBL.

How can biophotonics help dentistry to avoid or minimize cross infection by SARS-CoV-2?

Biophotonics is defined as the combination of biology and photonics (the physical science of the light). It is a general term for all techniques that deal with the interaction between biological tissues/cells and photons (light). Biophotonics offers a great variety of techniques that can facilitate the early detection of diseases and promote innovative theragnostic approaches. As the COVID-19 infection can be transmitted due to the face-to-face communication, droplets and aerosol inhalation and the exposure to saliva, blood, and other body fluids, as well as the handling of sharp instruments, dental practices are at increased risk of infection. In this paper, a literature review was performed to explore the application of Biophotonics approaches in Dentistry focusing on the COVID-19 pandemic and how they can contribute to avoid or minimize the risks of infection in a dental setting. For this, search-related papers were retrieved from PubMED, Scielo, Google Schoolar, and American Dental Association and Centers for Disease Control and Prevention databases. The body of evidence currently available showed that Biophotonics approaches can reduce microorganism load, decontaminate surfaces, air, tissues, and minimize the generation of aerosol and virus spreading by minimally invasive, time-saving, and alternative techniques in general. However, each clinical situation must be individually evaluated regarding the benefits and drawbacks of these approaches, but always pursuing less-invasive and less aerosol-generating procedures, especially during the COVID-19 pandemic.

First Molecular Detection of Polychromophilus Parasites in Brazilian Bat Species.

Blood parasites of the Haemosporida order, such as the Plasmodium spp. responsible for malaria, have become the focus of many studies in evolutionary biology. However, there is a lack of molecular investigation of haemosporidian parasites of wildlife, such as the genus Polychromophilus. Species of this neglected genus exclusively have been described in bats, mainly in Europe, Asia, and Africa, but little is known about its presence and genetic diversity on the American continent. Here, we investigated 406 bats from sites inserted in remnant fragments of the Atlantic Forest and Cerrado biomes and urbanized areas from southern Brazil for the presence of Polychromophilus species by PCR of the mitochondrial cytochrome b encoding gene. A total of 1.2% of bats was positive for Polychromophilus, providing the first molecular information of these parasites in Myotis riparius and Eptesicus diminutus, common vespertilionid bats widely distributed in different Brazilian biomes, and Myotis ruber, an endangered species. A Bayesian analysis was conducted to reconstruct the phylogenetic relationships between Polychromophilus recovered from Brazilian bats and those identified elsewhere. Sequences of Brazilian Polychromophilus lineages were placed with P. murinus and in a clade distinct from P. melanipherus, mainly restricted to bats in the family Vespertilionidae. However, the sequences were split into two minor clades, according to the genus of hosts, indicating that P. murinus and a distinct species may be circulating in Brazil. Morphological observations combined with additional molecular studies are needed to conclude and describe these Polychromophilus species.

Loss of forest cover and host functional diversity increases prevalence of avian malaria parasites in the Atlantic Forest.

Host phylogenetic relatedness and ecological similarity are thought to contribute to parasite community assembly and infection rates. However, recent landscape level anthropogenic changes may disrupt host-parasite systems by impacting functional and phylogenetic diversity of host communities. We examined whether changes in host functional and phylogenetic diversity, forest cover, and minimum temperature influence the prevalence, diversity, and distributions of avian haemosporidian parasites (genera Haemoproteus and Plasmodium) across 18 avian communities in the Atlantic Forest. To explore spatial patterns in avian haemosporidian prevalence and taxonomic and phylogenetic diversity, we surveyed 2241 individuals belonging to 233 avian species across a deforestation gradient. Mean prevalence and parasite diversity varied considerably across avian communities and parasites responded differently to host attributes and anthropogenic changes. Avian malaria prevalence (termed herein as an infection caused by Plasmodium parasites) was higher in deforested sites, and both Plasmodium prevalence and taxonomic diversity were negatively related to host functional diversity. Increased diversity of avian hosts increased local taxonomic diversity of Plasmodium lineages but decreased phylogenetic diversity of this parasite genus. Temperature and host phylogenetic diversity did not influence prevalence and diversity of haemosporidian parasites. Variation in the diversity of avian host traits that promote parasite encounter and vector exposure (host functional diversity) partially explained the variation in avian malaria prevalence and diversity. Recent anthropogenic landscape transformation (reduced proportion of native forest cover) had a major influence on avian malaria occurrence across the Atlantic Forest. This suggests that, for Plasmodium, host phylogenetic diversity was not a biotic filter to parasite transmission as prevalence was largely explained by host ecological attributes and recent anthropogenic factors. Our results demonstrate that, similar to human malaria and other vector-transmitted pathogens, prevalence of avian malaria parasites will likely increase with deforestation.

Effective Treatment of Cutaneous Mold Infections by Antimicrobial Blue Light That Is Potentiated by Quinine.

BACKGROUND: Cutaneous mold infections commonly result from an array of traumatic injuries that involve direct inoculation of contaminated soil into wounds. Here, we explored the use of antimicrobial blue light (aBL; 405 nm wavelength) and the combination of aBL with quinine hydrochloride (aBL + Q-HCL) for the treatment of cutaneous mold infections. METHODS: Efficacy of aBL and aBL + Q-HCL in killing clinically important pathogenic molds (Aspergillus fumigatus, Aspergillus flavus, and Fusarium oxyprorum) was investigated. Ultraperformance liquid chromatography identified and quantified endogenous porphyrins in the mold conidia. Finally, a mouse model of dermabrasion wound infected with a bioluminescent variant of A. fumigatus was developed to investigate the efficacy of aBL in treating cutaneous mold infections. RESULTS: We demonstrated that mold conidia are tolerant to aBL, but Q-HCL enhances efficacy. Transmission electron microscopy revealed intracellular damage by aBL. aBL + Q-HCL resulted in intracellular and cell wall damage. Porphyrins were observed in all mold strains, with A. fumigatus having the highest concentration. aBL and aBL + Q-HCL effectively reduced the burden of A. fumigatus within an established dermabrasion infection and limited recurrence posttreatment. CONCLUSIONS: aBL and aBL + Q-HCL may offer a novel approach for the treatment of mold infections.

Antimicrobial blue light and photodynamic therapy inhibit clinically relevant ß-lactamases with extended-spectrum (ESBL) and carbapenemase activity.

INTRODUCTION: The production of ß-lactamases by Gram-negative bacteria is among the most important factors of resistance to antibiotics, which has contributed to therapeutic failures that currently threaten human and veterinary medicine worldwide. Antimicrobial photodynamic therapy and antimicrobial blue light have a broad-spectrum antibacterial activity against multidrug-resistant and hypervirulent pathogens. OBJECTIVE: To investigate the ability of antimicrobial blue light to inhibit the hydrolytic activity of clinically relevant ß-lactamase enzymes (i.e., KPC, IMP, OXA, CTX-M, and SHV), with further comparison of the inhibitory effects of antimicrobial blue light with methylene blue-mediated antimicrobial photodynamic therapy. METHODS: Blue LED light (λ = 410 ± 10 nm) alone or red LED light (λ = 660 ± 10 nm) in combination with methylene blue were used to inactivate, in vitro, suspensions of Klebsiella pneumoniae strains producing clinically important ß-lactamase enzymes assigned to the A, B and D Ambler molecular classes. Furthermore, ß-lactamase activity inhibition mediated by antimicrobial blue light and methylene blue-mediated antimicrobial photodynamic therapy was measured by using the chromogenic ß-lactam substrate nitrocefin. RESULTS: ß-lactamase activities were effectively inactivated by both visible light-based approaches. In this regard, antimicrobial blue light and methylene blue-antimicrobial photodynamic therapy led to a significant reduction in the hydrolysis of nitrocefin (81-98 %). CONCLUSION: Sublethal doses of antimicrobial blue light and methylene blue-mediated antimicrobial photodynamic therapy are equally effective to inhibit clinically significant ß-lactamases, including extended-spectrum ß-lactamases and carbapenemases.

Instrumental validity and intra/inter-rater reliability of a novel low-cost digital pressure algometer.

BACKGROUND: Pain assessment is a key measure that accompanies treatments in a wide range of clinical settings. A low-cost valid and reliable pressure algometer would allow objective assessment of pressure pain to assist a variety of health professionals. However, the pressure algometer is often expensive, which limits its daily use in both clinical and research settings. OBJECTIVES: This study aimed to assess the instrumental validity, and the intra- and inter-rater reliability of an inexpensive digital adapted pressure algometer. METHODS: A single rater applied 60 random compressions on a force platform. The pressure pain thresholds of 20 volunteers were collected twice (3 days apart) by two raters. The main outcome measurements were as follows: the maximal peak force (in kPa) and the pressure pain threshold (adapted pressure algometer vs. force platform). Cronbach's α test was used to assess internal consistency. The standard error of measurement provided estimates of measurement error, and the measurement bias was estimated with the Bland-Altman method, with lower and upper limits of agreement. RESULTS: No differences were observed when comparing the compression results (P = 0.51). The validity and internal intra-rater consistencies ranged from 0.84 to 0.99, and the standard error of measurement from 0.005 to 0.04 kPa. Very strong (r = 0.73-0.74) to near-perfect (r = 0.99) correlations were found, with a low risk of bias for all measurements. The results demonstrated the validity and intra-rater reliability of the digitally adapted pressure algometer. Inter-rater reliability results were moderate (r = 0.55-0.60; Cronbach's α = 0.71-0.75). CONCLUSION: The adapted pressure algometer provide valid and reliable measurements of pressure pain threshold. The results support more widespread use of the pressure pain threshold method among clinicians.

Global priority multidrug-resistant pathogens do not resist photodynamic therapy.

Microbial drug-resistance demands immediate implementation of novel therapeutic strategies. Antimicrobial photodynamic therapy (aPDT) combines the administration of a photosensitizer (PS) compound with low-irradiance light to induce photochemical reactions that yield reactive oxygen species (ROS). Since ROS react with nearly all biomolecules, aPDT offers a powerful multitarget method to avoid selection of drug-resistant strains. In this study, we assayed photodynamic inactivation under a standardized method, combining methylene blue (MB) as PS and red light, against global priority pathogens. The species tested include Acinetobacter baumannii, Klebsiella aerogenes, Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, Enterococcus faecium, Enterococcus faecalis, Staphylococcus aureus, Candida albicans and Cryptococcus neoformans. Our strain collection presents resistance to all tested antimicrobials (>50). All drug-resistant strains were compared to their drug-sensitive counterparts. Regardless of resistance phenotype, MB-aPDT presented species-specific dose-response kinetics. More than 5log10 reduction was observed within less than 75 s of illumination for A. baumannii, E. coli, E. faecium, E. faecalis and S. aureus and within less than 7 min for K. aerogenes, K. pneumoniae, P. aeruginosa, C. albicans and C. neoformans. No signs of correlations in between drug-resistance profiles and aPDT sensitivity were observed. Therefore, MB-aPDT can provide effective therapeutic protocols for a very broad spectrum of pathogens. Hence, we believe that this study represents a very important step to bring aPDT closer to implementation into mainstream medical practices.

Hypervirulent and hypermucoviscous strains of Klebsiella pneumoniae challenged by antimicrobial strategies using visible light.

INTRODUCTION: Infections caused by hypervirulent and/or hypermucoviscous Klebsiella pneumoniae (K. pneumoniae) strains are frequently reported worldwide. Since convergence of hypervirulence and drug-resistance emerged as a serious clinical problem, novel therapeutic strategies are worthy of investigation. In this regard, antimicrobial photodynamic therapy and blue light have proven to be effective against a broad-spectrum of clinically relevant pathogens but have never been tested for hypervirulent/hypermucoviscous strains. Thus, this study investigated the influence of hypermucoviscosity and hypervirulence over the photoinactivation efficacy of blue light alone or antimicrobial photodynamic therapy mediated by methylene blue and red light. METHODS: Five clinical isolates of K. pneumoniae were screened for hypermucoviscosity by string test and for hypervirulence by a Galleria mellonella model of systemic infection. Strains were then challenged by both photoinactivation methods performed in vitro. All tests also included a non-hypervirulent/hypermucoviscous control strain for comparison. RESULTS: All K. pneumoniae strains were effectively inactivated by both light-based antimicrobial strategies. Hypervirulent/hypermucoviscous strains exposed to photodynamic therapy presented rapid and consistent inactivation kinetics, while blue light led to slower and more variable inactivation kinetics. CONCLUSION: Hypermucoviscosity and hypervirulence does not confer tolerance in K. pneumoniae against photoinactivation. Antimicrobial photodynamic therapy represents an interesting alternative to treat localised infections because it is a fast procedure with high effectiveness. On the other hand, antimicrobial blue light could be used to decontaminate hospital environments since no photosensitiser administration is required and harmful effects of ultraviolet light are avoided. Therefore, visible light-based strategies present great potential for the development of safe and effective antimicrobial technologies against such aggressive pathogens.

Inactivation kinetics and lethal dose analysis of antimicrobial blue light and photodynamic therapy.

BACKGROUND: Antimicrobial Photodynamic therapy (A-PDT) has been used to treat infections. Currently, microbial inactivation data is reported presenting survival fraction averages and standard errors as discrete points instead of a continuous curve of inactivation kinetics. Standardization of this approach would allow clinical protocols to be introduced globally, instead of the piecemeal situation which currently applies. METHODS: To this end, we used a power-law function to fit inactivation kinetics and directly report values of lethal doses (LD) and a tolerance factor (T) that informs if inactivation rate varies along the irradiation procedure. A deduced formula was also tested to predict LD for any given survival fraction value. We analyzed the photoantimicrobial effect caused by red light activation of methylene blue (MB-APDT) and by blue light (BL) activation of endogenous microbial pigments against 5 clinically relevant pathogens. RESULTS: Following MB- APDT, Escherichia coli and Staphylococcus aureus cells become increasingly more tolerant to inactivation along the irradiation process (T < 1). Klebsiella pneumoniae presents opposite behavior, i.e., more inactivation is observed towards the end of the process (T > 1). P. aeruginosa and Candida albicans present constant inactivation rate (T˜1). In contrast, all bacterial species presented similar behavior during inactivation caused by BL, i.e., continuously becoming more sensitive to blue light exposure (T > 1). CONCLUSION: The power-law function successfully fit all experimental data. Our proposed method precisely predicted LD and T values. We expect that these analytical models may contribute to more standardized methods for comparisons of photodynamic inactivation efficiencies.

Antimicrobial blue light inactivation of international clones of multidrug-resistant Escherichia coli ST10, ST131 and ST648.

BACKGROUND: International clones of multidrug-resistant Escherichia coli have been a leading cause of human and animal infections worldwide. Microbial inactivation by blue light has been proposed as an effective treatment for superficial infections and surface contamination. AIM: To evaluate the inactivation efficacy of blue light irradiation against high-risk multidrug-resistant strains of E. coli. METHODS: Blue LED light (λ = 410 nm) was used to inactivate in vitro suspensions of colistin- broad-spectrum cephalosporin-, or carbapenem-resistant E. coli strains belonging to sequence types (STs) ST10, ST131 and ST648, carrying mcr-1, blaCTX-M or blaKPC-2 genes. RESULTS: Our results showed that all E. coli strains were susceptible to blue light irradiation, independently of antibiotic resistance and virulence profiles. In addition, blue light irradiation induced a strain-specific and dose-dependent bacterial effect. CONCLUSION: Our results support use of blue light as a promising antimicrobial option against MDR pathogens, including high-risk clones of E. coli displaying resistance to polymyxins or broad-spectrum ß-lactam antibiotics.