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Although conventional antimicrobial drugs have been viewed as miraculous cure-alls for the past 80 years, increasing antimicrobial drug resistance requires a major and rapid intervention. However, the development of novel but still conventional systemic antimicrobial agents, having only a single mode or site of action, will not alleviate the situation because it is probably only a matter of time until any such agents will also become ineffective. To continue to produce new agents based on this notion is unacceptable, and there is an increasing need for alternative approaches to the problem. By contrast, light-activated molecules called photoantimicrobials act locally via the in-situ production of highly reactive oxygen species, which simultaneously attack various biomolecular sites in the pathogenic target and therefore offer both multiple and variable sites of action. This non-specificity at the target circumvents conventional mechanisms of resistance and inhibits the development of resistance to the agents themselves. Photoantimicrobial therapy is safe and easy to implement and, unlike conventional agents, the activity spectrum of photoantimicrobials covers bacteria, fungi, viruses, and protozoa. However, clinical trials of these new, truly broad-spectrum, and minimally toxic agents have been few, and the funding for research and development is almost non-existent. Photoantimicrobials constitute one of the few ways forward through the morass of drug-resistant infectious disease and should be fully explored. In this Personal View, we raise awareness of the novel photoantimicrobial technologies that offer a viable alternative to conventional drugs in many relevant application fields, and could thus slow the pace of resistance development.

Accelerating growth and global expansion of antimicrobial resistance has deepened the need for discovery of novel antimicrobial agents. Antimicrobial peptides have clear advantages over conventional antibiotics which include slower emergence of resistance, broad-spectrum antibiofilm activity, and the ability to favourably modulate the host immune response. Broad bacterial susceptibility to antimicrobial peptides offers an additional tool to expand knowledge about the evolution of antimicrobial resistance. Structural and functional limitations, combined with a stricter regulatory environment, have hampered the clinical translation of antimicrobial peptides as potential therapeutic agents. Existing computational and experimental tools attempt to ease the preclinical and clinical development of antimicrobial peptides as novel therapeutics. This Review identifies the benefits, challenges, and opportunities of using antimicrobial peptides against multidrug-resistant pathogens, highlights advances in the deployment of novel promising antimicrobial peptides, and underlines the needs and priorities in designing focused development strategies taking into account the most advanced tools available.

Resistance of bacteria to antibiotics is a public health concern worldwide due to the increasing failure of standard antibiotic therapies. Antimicrobial photodynamic inactivation (aPDI) is a promising non-antibiotic alternative for treating localized bacterial infections that uses non-toxic photosensitizers and harmless visible light to produce reactive oxygen species and kill microbes. Phenothiazinium photosensitizers like methylene blue (MB) and toluidine blue O are hydrophobic cations that are naturally expelled from bacterial cells by multidrug efflux pumps, which reduces their effectiveness. We recently reported the discovery of a NorA efflux pump inhibitor-methylene blue (EPI-MB) hybrid compound INF55-(Ac)en-MB that shows enhanced photodynamic inactivation of the Gram-positive bacterium methicillin-resistant Staphylococcus aureus (MRSA) relative to MB, both in vitro and in vivo. Here, we report the surprising observation that INF55-(Ac)en-MB and two related hybrids bearing the NorA efflux pump inhibitors INF55 and INF271 also show enhanced aPDI activity in vitro (relative to MB) against the Gram-negative bacteria Escherichia coli and Acinetobacter baumannii, despite neither species expressing the NorA pump. Two of the hybrids showed superior effects to MB in murine aPDI infection models. The findings motivate wider exploration of aPDI with EPI-MB hybrids against Gram-negative pathogens and more detailed studies into the molecular mechanisms underpinning their activity.

Methicillin-resistant Staphylococcus aureus (MRSA) has become the most important drug-resistant microbial pathogen in countries throughout the world. Morbidity and mortality due to MRSA infections continue to increase despite efforts to improve infection control measures and to develop new antibiotics. Therefore alternative antimicrobial strategies that do not give rise to development of resistance are urgently required. A group of therapeutic interventions has been developed in the field of photomedicine with the common theme that they rely on electromagnetic radiation with wavelengths between 200 and 1000 nm broadly called "light". These techniques all use simple absorption of photons by specific chromophores to deliver the killing blow to microbial cells while leaving the surrounding host mammalian cells relatively unharmed. Photodynamic inactivation uses dyes called photosensitizers (PS) that bind specifically to MRSA cells and not host cells, and generate reactive oxygen species including singlet oxygen and singlet oxygen upon illumination. Sophisticated molecular strategies to target the PS to MRSA cells have been designed. Ultraviolet C radiation can damage microbial DNA without unduly harming host DNA. Blue light can excite endogenous porphyrins and flavins in MRSA cells that are not present in host cells. Near-infrared lasers can interfere with microbial membrane potentials without raising the temperature of the tissue. Taken together these innovative approaches towards harnessing the power of light suggest that the ongoing threat of MRSA may eventually be defeated.

Antimicrobial photodynamic inactivation (aPDI) uses photosensitizers (PSs) and harmless visible light to generate reactive oxygen species (ROS) and kill microbes. Multidrug efflux systems can moderate the phototoxic effects of PSs by expelling the compounds from cells. We hypothesized that increasing intracellular concentrations of PSs by inhibiting efflux with a covalently attached efflux pump inhibitor (EPI) would enhance bacterial cell phototoxicity and reduce exposure of neighboring host cells to damaging ROS. In this study, we tested the hypothesis by linking NorA EPIs to methylene blue (MB) and examining the photoantimicrobial activity of the EPI−MB hybrids against the human pathogen methicillin-resistant Staphylococcus aureus (MRSA). Photochemical/photophysical and in vitro microbiological evaluation of 16 hybrids carrying four different NorA EPIs attached to MB via four linker types identified INF55-(Ac)en−MB 12 as a lead. Compound 12 showed increased uptake into S. aureus cells and enhanced aPDI activity and wound healing effects (relative to MB) in a murine model of an abrasion wound infected by MRSA. The study supports a new approach for treating localized multidrug-resistant MRSA infections and paves the way for wider exploration of the EPI−PS hybrid strategy in aPDI.

Conventional antimicrobial strategies have become increasingly ineffective due to the emergence of multidrug resistance among pathogenic microorganisms. The need to overcome these deficiencies has triggered the exploration of alternative treatments and unconventional approaches towards controlling microbial infections. Photodynamic therapy (PDT) was originally established as an anticancer modality and is currently used in the treatment of age-related macular degeneration. The concept of photodynamic inactivation requires cell exposure to light energy, typically wavelengths in the visible region that causes the excitation of photosensitizer molecules either exogenous or endogenous, which results in the production of reactive oxygen species (ROS). ROS produce cell inactivation and death through modification of intracellular components. The versatile characteristics of PDT prompted its investigation as an anti-infective discovery platform. Advances in understanding of microbial physiology have shed light on a series of pathways, and phenotypes that serve as putative targets for antimicrobial drug discovery. Investigations of these phenotypic elements in concert with PDT have been reported focused on multidrug efflux systems, biofilms, virulence and pathogenesis determinants. In many instances the results are promising but only preliminary and require further investigation. This review discusses the different antimicrobial PDT strategies and highlights the need for highly informative and comprehensive discovery approaches.

The story of prevention and control of infectious diseases remains open and a series of highly virulent pathogens are emerging both in and beyond the hospital setting. Antibiotics were an absolute success story for a previous era. The academic and industrial biomedical communities have now come together to formulate consensus beliefs regarding the pursuit of novel and effective alternative anti-infective countermeasures. Photodynamic therapy was established and remains a successful modality for malignancies but photodynamic inactivation has been transformed recently to an antimicrobial discovery and development platform. The concept of photodynamic inactivation is quite straightforward and requires microbial exposure to visible light energy, typically wavelengths in the visible region, that causes the excitation of photosensitizer molecules (either exogenous or endogenous), which results in the production of singlet oxygen and other reactive oxygen species that react with intracellular components, and consequently produce cell inactivation. It is an area of increasing interest, as research is advancing i) to identify the photochemical and photophysical mechanisms involved in inactivation; ii) to develop potent and clinically compatible photosensitizer; iii) to understand how photoinactivation is affected by key microbial phenotypic elements (multidrug resistance and efflux, virulence and pathogenesis determinants, biofilms); iv) to explore novel delivery platforms inspired by current trends in pharmacology and nanotechnology; and v) to identify photoinactivation applications beyond the clinical setting such as environmental disinfectants.

Chitosan and nanoparticle silver are both materials with demonstrated antimicrobial properties and have been proposed singly or in combination as constituents of antimicrobial burn dressings. Here, we show that they combine synergistically to inhibit the in vitro growth of Gram-positive methicillin-resistant Staphylococcus aureus (MRSA) and Gram-negative bacteria ( Pseudomonas aeruginosa , Proteus mirabilis , and Acinetobacter baumannii ), as judged by bioluminescence monitoring and isobolographic analysis, and also produce synergistic killing after 30 min of incubation, as measured by a CFU assay. The hypothesized explanation involves chitosan-mediated permeabilization of bacterial cells, allowing better penetration of silver ions into the cell. A dressing composed of freeze-dried chitosan acetate incorporating nanoparticle silver was compared with a dressing of chitosan acetate alone in an in vivo burn model infected with bioluminescent P. aeruginosa . The survival rates of mice treated with silver-chitosan or regular chitosan or left untreated were 64.3% ( P = 0.0082 versus regular chitosan and P = 0.0003 versus the control), 21.4%, and 0%, respectively. Most of the fatalities occurred between 2 and 5 days postinfection. Silver-chitosan dressings effectively controlled the development of systemic sepsis, as shown by blood culture. These data suggest that a dressing combining chitosan acetate with silver leads to improved antimicrobial efficacy against fatal burn infections.

Traditional antimicrobials are increasingly suffering from the emergence of multidrug resistance among pathogenic microorganisms. To overcome these deficiencies, a range of novel approaches to control microbial infections are under investigation as potential alternative treatments. Multidrug efflux is a key target of these efforts. Efflux mechanisms are broadly recognized as major components of resistance to many classes of chemotherapeutic agents as well as antimicrobials. Efflux occurs due to the activity of membrane transporter proteins widely known as Multidrug Efflux Systems (MES). They are implicated in a variety of physiological roles other than efflux and identifying natural substrates and inhibitors is an active and expanding research discipline. One plausible alternative is the combination of conventional antimicrobial agents/antibiotics with small molecules that block MES known as multidrug efflux pump inhibitors (EPIs). An array of approaches in academic and industrial research settings, varying from high-throughput screening (HTS) ventures to bioassay guided purification and determination, have yielded a number of promising EPIs in a series of pathogenic systems. This synergistic discovery platform has been exploited in translational directions beyond the potentiation of conventional antimicrobial treatments. This venture attempts to highlight different tactical elements of this platform, identifying the need for highly informative and comprehensive EPI-discovery strategies. Advances in assay development genomics, proteomics as well as the accumulation of bioactivity and structural information regarding MES facilitates the basis for a new discovery era. This platform is expanding drastically. A combination of chemogenomics and chemoinformatics approaches will integrate data mining with virtual and physical HTS ventures and populate the chemical-biological interface with a plethora of novel chemotypes. This comprehensive step will expedite the preclinical development of lead EPIs.

Antimicrobial photodynamic therapy (aPDT) is an emerging alternative to antibiotics motivated by growing problems with multi-drug resistant pathogens. aPDT uses non-toxic dyes or photosensitizers (PS) in combination with harmless visible of the correct wavelength to be absorbed by the PS. The excited state PS can form a long-lived triplet state that can interact with molecular oxygen to produce reactive oxygen species such as singlet oxygen and hydroxyl radical that kill the microbial cells. To obtain effective PS for treatment of infections it is necessary to use cationic PS with positive charges that are able to bind to and penetrate different classes of microbial cells. Other drug design criteria require PS with high absorption coefficients in the red/near infra-red regions of the spectrum where light penetration into tissue is maximum, high photostability to minimize photobleaching, and devising compounds that will selectively bind to microbial cells rather than host mammalian cells. Several molecular classes fulfill many of these requirements including phenothiazinium dyes, cationic tetrapyrroles such as porphyrins, phthalocyanines and bacteriochlorins, cationic fullerenes and cationic derivatives of other known PS. Larger structures such as conjugates between PS and cationic polymers, cationic nanoparticles and cationic liposomes that contain PS are also effective. In order to demonstrate in vivo efficacy it is necessary to use animal models of localized infections in which both PS and light can be effectively delivered into the infected area. This review will cover a range of mouse models we have developed using bioluminescent pathogens and a sensitive low light imaging system to non-invasively monitor the progress of the infection in real time. Effective aPDT has been demonstrated in acute lethal infections and chronic biofilm infections; in infections caused by Gram-positive, Gram-negative bacteria and fungi; in infections in wounds, third degree burns, skin abrasions and soft-tissue abscesses. This range of animal models also represents a powerful aid in antimicrobial drug discovery.

To investigate whether the major fungal multidrug efflux systems (MESs) affect the efficiency of methylene blue (MB)-mediated antimicrobial photodynamic inactivation (APDI) in pathogenic fungi and test specific inhibitors of these efflux systems to potentiate APDI.Candida albicans wild-type and mutants that overexpressed two classes of MESs [ATP-binding cassette (ABC) and major facilitator superfamily (MFS)] were tested for APDI using MB as the photosensitizer with and without addition of MES inhibitors. The uptake and cytoplasm localization of photosensitizer were achieved using laser confocal microscopy.ABC MES overexpression reduced MB accumulation and APDI killing more than MFS MES overexpression. Furthermore, by combining MB APDI with the ABC inhibitor verapamil, fungal killing and MB uptake were potentiated, while by combining MB APDI with the MFS inhibitor INF(271), fungal killing and MB uptake were inhibited. This latter surprising finding may be explained by the hypothesis that the MFS channel can also serve as an uptake mechanism for MB.The ABC pumps are directly implicated in MB efflux from the cell cytoplasm. Both the influx and efflux of MB may be regulated by MFS systems, and blocking this gate before incubation with MB can decrease the uptake and APDI effects. An ABC inhibitor could be usefully combined with MB APDI for treating C. albicans infections.

Aims: Fullerenes are under intensive study for potential biomedical applications. We have previously reported that a C60 fullerene functionalized with three dimethylpyrrolidinium groups (BF6) is a highly active broad-spectrum antimicrobial photosensitizer in vitro when combined with white-light illumination. We asked whether this high degree of in vitro activity would translate into an in vivo therapeutic effect in two potentially lethal mouse models of infected wounds. Materials & methods: We used stable bioluminescent bacteria and a low light imaging system to follow the progress of the infection noninvasively in real time. An excisional wound on the mouse back was contaminated with one of two bioluminescent Gram-negative species, Proteus mirabilis (2.5 × 107 cells) and Pseudomonas aeruginosa (5 × 106 cells). A solution of BF6 was placed into the wound followed by delivery of up to 180 J/cm2 of broadband white light (400–700 nm). Results: In both cases there was a light-dose-dependent reduction of bioluminescence from the wound not observed in control groups (light alone or BF6 alone). Fullerene-mediated photodynamic therapy of mice infected with P. mirabilis led to 82% survival compared with 8% survival without treatment (p < 0.001). Photodynamic therapy of mice infected with highly virulent P. aeruginosa did not lead to survival, but when photodynamic therapy was combined with a suboptimal dose of the antibiotic tobramycin (6 mg/kg for 1 day) there was a synergistic therapeutic effect with a survival of 60% compared with a survival of 20% with tobramycin alone (p < 0.01). Conclusion: These data suggest that cationic fullerenes have clinical potential as an antimicrobial photosensitizer for superficial infections where red light is not needed to penetrate tissue.

Microbial biofilm architecture contains numerous protective features, including extracellular polymeric material that render biofilms impermeable to conventional antimicrobial agents. This study evaluated the efficacy of antimicrobial photodynamic inactivation (aPDI) of Enterococcus faecalis biofilms. The ability of a cationic, phenothiazinium photosensitizer, methylene blue (MB) and an anionic, xanthene photosensitizer, rose bengal (RB) to inactivate biofilms of E. faecalis (OG1RF and FA 2-2) and disrupt the biofilm structure was evaluated. Bacterial cells were tested as planktonic suspensions, intact biofilms and biofilm-derived suspensions obtained by the mechanical disruption of biofilms. The role of a specific microbial efflux pump inhibitor (EPI), verapamil hydrochloride in the MB-mediated aPDI of E. faecalis biofi l ms was also investigated. The results showed that E. faecalis biofilms exhibited significantly higher resistance to aPDI when compared with E. faecalis in suspension (P < 0.001). aPDI with cationic MB produced superior inactivation of E. faecalis strains in a biofilm along with significant destruction of biofilm structure when compared with anionic RB (P < 0.05). The ability to inactivate biofilm bacteria was further enhanced when the EPI was used with MB (P < 0.001). These experiments demonstrated the advantage of a cationic phenothiazinium photosensitizer combined with an EPI to inactivate biofilm bacteria and disrupt biofilm structure.

HemCon s bandage is an engineered chitosan acetate preparation designed as a hemostatic dressing, and is under investigation as a topical antimicrobial dressing. We studied its effects on healing of excisional wounds that were or were not infected with Staphylococcus aureus, in normal mice or mice previously pretreated with cyclophosphamide (CY). CY significantly suppressed wound healing in both the early and later stages, while S. aureus alone significantly stimulated wound healing in the early stages by preventing the initial wound expansion. CY plus S. aureus showed an advantage in early stages by preventing expansion, but a significant slowing of wound healing in later stages. In order to study the conflicting clamping and stimulating effects of chitosan acetate bandage on normal wounds, we removed the bandage from wounds at times after application ranging from 1 hour to 9 days. Three days application gave the earliest wound closure, and all application times gave a faster healing slope after removal compared with control wounds. Chitosan acetate bandage reduced the number of inflammatory cells in the wound at days 2 and 4, and had an overall beneficial effect on wound healing especially during the early period where its antimicrobial effect is most important.

HemCon® bandage is an engineered chitosan acetate preparation used as a hemostatic control dressing, and its chemical structure suggests that it should also be antimicrobial. We tested its ability to rapidly kill bacteria in vitro and in mouse models of infected wounds. We used the Gram-negative species Pseudomonas aeruginosa and Proteus mirabilis and the Gram-positive Staphylococcus aureus that had all been stably transduced with the entire bacterial lux operon to allow in vivo bioluminescence imaging. An excisional wound in Balb/c mice was inoculated with 50–250 million cells followed after 30 min by application of HemCon bandage, alginate sponge bandage, silver sulfadiazine cream or no treatment. HemCon was more adhesive to the wound and conformed well to the injury compared to alginate. Animal survival was followed over 15 days with observations of bioluminescence emission and animal activity daily. Chitosan acetate treated mice infected with P. aeruginosa and P. mirabilis all survived while those receiving no treatment, alginate and silver sulfadiazine demonstrated 25–100% mortality. Chitosan acetate was much more effective than other treatments in rapidly reducing bioluminescence in the wound consistent with its rapid bactericidal activity in vitro as well as its light-scattering properties. S. aureus formed only non-lethal localized infections after temporary immunosuppression of the mice but HemCon was again more effective in reducing bioluminescence. The data suggest that chitosan acetate rapidly kills bacteria in the wound before systemic invasion can take place, and is superior to alginate bandage and silver sulfadiazine that may both encourage bacterial growth in the short term.

This special issue of Current Opinion in Pharmacology is concerned with new developments in antimicrobial drugs and covers innovative strategies for dealing with microbial infection in the age of multi-antibiotic resistance. Despite widespread fears that many infectious diseases may become untreatable, disruptive innovations are in the process of being discovered and developed that may go some way to leading the fight-back against the rising threat. Natural products, quorum sensing inhibitors, biofilm disruptors, gallium-based drugs, cyclodextrin inhibitors of pore-forming toxins, anti-fungals that deal with biofilms, and light based antimicrobial strategies are specifically addressed. New non-vertebrate animal models of infection may facilitate high-throughput screening (HTS) of novel anti-infectives.

Reactive oxygen species (ROS) can attack a diverse range of targets to exert antimicrobial activity, which accounts for their versatility in mediating host defense against a broad range of pathogens. Most ROS are formed by the partial reduction in molecular oxygen. Four major ROS are recognized comprising superoxide (O2•-), hydrogen peroxide (H2O2), hydroxyl radical (•OH), and singlet oxygen ((1)O2), but they display very different kinetics and levels of activity. The effects of O2•- and H2O2 are less acute than those of •OH and (1)O2, because the former are much less reactive and can be detoxified by endogenous antioxidants (both enzymatic and nonenzymatic) that are induced by oxidative stress. In contrast, no enzyme can detoxify •OH or (1)O2, making them extremely toxic and acutely lethal. The present review will highlight the various methods of ROS formation and their mechanism of action. Antioxidant defenses against ROS in microbial cells and the use of ROS by antimicrobial host defense systems are covered. Antimicrobial approaches primarily utilizing ROS comprise both bactericidal antibiotics and nonpharmacological methods such as photodynamic therapy, titanium dioxide photocatalysis, cold plasma, and medicinal honey. A brief final section covers reactive nitrogen species and related therapeutics, such as acidified nitrite and nitric oxide-releasing nanoparticles.

Microbial biofilms are responsible for a variety of microbial infections in different parts of the body, such as urinary tract infections, catheter infections, middle-ear infections, gingivitis, caries, periodontitis, orthopedic implants, and so on. The microbial biofilm cells have properties and gene expression patterns distinct from planktonic cells, including phenotypic variations in enzymic activity, cell wall composition and surface structure, which increase the resistance to antibiotics and other antimicrobial treatments. There is consequently an urgent need for new approaches to attack biofilm-associated microorganisms, and antimicrobial photodynamic therapy (aPDT) may be a promising candidate. aPDT involves the combination of a nontoxic dye and low-intensity visible light which, in the presence of oxygen, produces cytotoxic reactive oxygen species. It has been demonstrated that many biofilms are susceptible to aPDT, particularly in dental disease. This review will focus on aspects of aPDT that are designed to increase efficiency against biofilms modalities to enhance penetration of photosensitizer into biofilm, and a combination of aPDT with biofilm-disrupting agents.

Background: Candida spp. are recognized as a primary agent of severe fungal infection in immunocompromised patients, and are the fourth most common cause of bloodstream infections. Our study explores treatment with photodynamic therapy (PDT) as an innovative antimicrobial technology that employs a nontoxic dye, termed a photosensitizer (PS), followed by irradiation with harmless visible light. After photoactivation, the PS produces either singlet oxygen or other reactive oxygen species (ROS) that primarily react with the pathogen cell wall, promoting permeabilization of the membrane and cell death. The emergence of antifungal-resistant Candida strains has motivated the study of antimicrobial PDT (aPDT) as an alternative treatment of these infections. We employed the invertebrate wax moth Galleria mellonella as an in vivo model to study the effects of aPDT against C. albicans infection. The effects of aPDT combined with conventional antifungal drugs were also evaluated in G. mellonella. Results: We verified that methylene blue-mediated aPDT prolonged the survival of C. albicans infected G. mellonella larvae. The fungal burden of G. mellonella hemolymph was reduced after aPDT in infected larvae. A fluconazole-resistant C. albicans strain was used to test the combination of aPDT and fluconazole. Administration of fluconazole either before or after exposing the larvae to aPDT significantly prolonged the survival of the larvae compared to either treatment alone. Conclusions: G. mellonella is a useful in vivo model to evaluate aPDT as a treatment regimen for Candida infections. The data suggests that combined aPDT and antifungal therapy could be an alternative approach to antifungal-resistant Candida strains., National Institutes of Health (U.S.) (Grant R01 AI050875), United States. Air Force. Medical Free Electron Laser Program (FA9550-04-1-0079)

Blue light has attracted increasing attention due to its intrinsic antimicrobial effect without the addition of exogenous photosensitizers. However, the use of blue light for wound infections has not been established yet. In this study, we demonstrated the efficacy of blue light at 415 nm for the treatment of acute, potentially lethal Pseudomonas aeruginosa burn infections in mice. Our in vitro studies demonstrated that the inactivation rate of P. aeruginosa cells by blue light was approximately 35-fold higher than that of keratinocytes ( P = 0.0014). Transmission electron microscopy revealed blue light-mediated intracellular damage to P. aeruginosa cells. Fluorescence spectroscopy suggested that coproporphyrin III and/or uroporphyrin III are possibly the intracellular photosensitive chromophores associated with the blue light inactivation of P. aeruginosa . In vivo studies using an in vivo bioluminescence imaging technique and an area-under-the-bioluminescence-time-curve (AUBC) analysis showed that a single exposure of blue light at 55.8 J/cm 2 , applied 30 min after bacterial inoculation to the infected mouse burns, reduced the AUBC by approximately 100-fold in comparison with untreated and infected mouse burns ( P < 0.0001). Histological analyses and terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling (TUNEL) assays indicated no significant damage in the mouse skin exposed to blue light at the effective antimicrobial dose. Survival analyses revealed that blue light increased the survival rate of the infected mice from 18.2% to 100% ( P < 0.0001). In conclusion, blue light therapy might offer an effective and safe alternative to conventional antimicrobial therapy for P. aeruginosa burn infections.

Submitted by Vitor Silverio Rodrigues (vitorsrodrigues@reitoria.unesp.br) on 2014-05-27T11:28:27Z No. of bitstreams: 0Bitstream added on 2014-05-27T14:48:09Z : No. of bitstreams: 1 2-s2.0-84874006080.pdf: 650584 bytes, checksum: 7c5b49e68362d89881ef802f5396e712 (MD5) Made available in DSpace on 2014-05-27T11:28:27Z (GMT). No. of bitstreams: 0 Previous issue date: 2013-02-14 Enterococcus faecium has emerged as one of the most important pathogens in healthcare-associated infections worldwide due to its intrinsic and acquired resistance to many antibiotics, including vancomycin. Antimicrobial photodynamic therapy (aPDT) is an alternative therapeutic platform that is currently under investigation for the control and treatment of infections. PDT is based on the use of photoactive dye molecules, widely known as photosensitizer (PS). PS, upon irradiation with visible light, produces reactive oxygen species that can destroy lipids and proteins causing cell death. We employed Galleria mellonella (the greater wax moth) caterpillar fatally infected with E. faecium to develop an invertebrate host model system that can be used to study the antimicrobial PDT (alone or combined with antibiotics). In the establishment of infection by E. faecium in G. mellonella, we found that the G. mellonella death rate was dependent on the number of bacterial cells injected into the insect hemocoel and all E. faecium strains tested were capable of infecting and killing G. mellonella. Antibiotic treatment with ampicillin, gentamicin or the combination of ampicillin and gentamicin prolonged caterpillar survival infected by E. faecium (P = 0.0003, P = 0.0001 and P = 0.0001, respectively). In the study of antimicrobial PDT, we verified that methylene blue (MB) injected into the insect followed by whole body illumination prolonged the caterpillar survival (P = 0.0192). Interestingly, combination therapy of larvae infected with vancomycin-resistant E. faecium, with antimicrobial PDT followed by vancomycin, significantly prolonged the survival of the caterpillars when compared to either antimicrobial PDT (P = 0.0095) or vancomycin treatment alone (P = 0.0025), suggesting that the aPDT made the vancomycin resistant E. faecium strain more susceptible to vancomycin action. In summary, G. mellonella provides an invertebrate model host to study the antimicrobial PDT and to explore combinatorial aPDT-based treatments. Department of Biosciences and Oral Diagnosis Univ Estadual Paulista/UNESP, São José dos Campos, São Paulo Division of Infectious Diseases Massachusetts General Hospital, Boston, MA Department of Restorative Dentistry Faculty of Pindamonhangaba, Pindamonhangaba, São Paulo Wellman Center for Photomedicine Massachusetts General Hospital, Boston, MA Center for Lasers and Applications Nuclear and Energy Research Institute, São Paulo, São Paulo Massachusetts Eye and Ear Infirmary Harvard Medical School, Boston, MA Warren Alpert Medical School Brown University/Rhode Island and Miriam Hospitals, Providence, RI Department of Dermatology Harvard Medical School, Boston, MA Department of Pathology and Center for Molecular Discovery University of New Mexico, Albuquerque, NM Division of Health Sciences and Technology Harvard-Massachusetts Institute of Technology, Cambridge, MA Department of Biosciences and Oral Diagnosis Univ Estadual Paulista/UNESP, São José dos Campos, São Paulo

The objective of this study was to evaluate whether Candida albicans exhibits altered pathogenicity characteristics following sublethal antimicrobial photodynamic inactivation (APDI) and if such alterations are maintained in the daughter cells. C. albicans was exposed to sublethal APDI by using methylene blue (MB) as a photosensitizer (0.05 mM) combined with a GaAlAs diode laser (λ 660 nm, 75 mW/cm 2 , 9 to 27 J/cm 2 ). In vitro , we evaluated APDI effects on C. albicans growth, germ tube formation, sensitivity to oxidative and osmotic stress, cell wall integrity, and fluconazole susceptibility. In vivo , we evaluated C. albicans pathogenicity with a mouse model of systemic infection. Animal survival was evaluated daily. Sublethal MB-mediated APDI reduced the growth rate and the ability of C. albicans to form germ tubes compared to untreated cells ( P < 0.05). Survival of mice systemically infected with C. albicans pretreated with APDI was significantly increased compared to mice infected with untreated yeast ( P < 0.05). APDI increased C. albicans sensitivity to sodium dodecyl sulfate, caffeine, and hydrogen peroxide. The MIC for fluconazole for C. albicans was also reduced following sublethal MB-mediated APDI. However, none of those pathogenic parameters was altered in daughter cells of C. albicans submitted to APDI. These data suggest that APDI may inhibit virulence factors and reduce in vivo pathogenicity of C. albicans . The absence of alterations in daughter cells indicates that APDI effects are transitory. The MIC reduction for fluconazole following APDI suggests that this antifungal could be combined with APDI to treat C. albicans infections.

Antimicrobial photodynamic inactivation (APDI) is based on the utilization of substances that can photosensitize biological tissues and are capable of being activated in the presence of light. Cryptococcus neoformans is an yeast surrounded by a capsule composed primarily of glucoronoxylomannan that plays an important role in its virulence. This yeast causes infection on skin, lungs and brain that can be associated with neurological sequelae and neurosurgical interventions, and its conventional treatment requires prolonged antifungal therapy, which presents important adverse effects. The aim of this study was to evaluate the protective effect of Cryptococcus neoformans capsule against reactive oxygen species generated by APDI. Cryptococcus neoformans KN99 D , which is a strain able to produce capsule, and CAP59 that does not present capsule production were submitted to APDI using methylene blue (MB), rose bengal (RB), and pL-ce6 as photosensitizers (PS). Then microbial inactivation was evaluated by counting colony form units following APDI and confocal laser scanni ng microscopy (CLSM) illustrated localization as well as the preferential accumulation of PS into the fungal cells. C. neoformans KN99 D was more resistant to APDI than CAP59 for all PSs tested. CLSM showed incorporation of MB and RB into the cytoplasm and a preferential uptake in mitochondria. A nuclear accumulation of MB was also observed. Contrarily, pL-ce6 appears accumulated in cell wall and cell membrane and minimal florescence was observed inside the fungal cells. In conclusion, the ability of C. neoformans to form capsule enhances survival following APDI . Keywords: Antimicrobial photodynamic chemotherapy; Conjugate Chlorine; Methylene Blue; Cryptococcus neoformans ; Photodynamic therapy; Photosensitizer; Rose Bengal.

Photosensitive dyes or photo sensitizers (PS) in combination with visible light and oxygen produce reactive oxygen species that kill cells in the process known as photodynamic therapy (PDT). Antimicrobial PDT employs PS that is selective for microbial cells and is a new treatment for infections. Most antimicrobial PS is based on tetrapyrrole or phenothiazinium structures that have been synthesized to carry quaternary cationic charges or basic amino groups. However we recently showed that cationic-substituted fullerene derivative were highly effective in killing a broad spectrum of microbial cells after illumination with white light. In the present report we compared a new group of synthetic fullerene derivatives that possessed either basic or quaternary amino groups as antimicrobial PS against Gram-positive (Staphylococcus aureus), Gram-negative bacteria (Escherichia coli) and fungi (Candida albicans). Quantitative structure-function relationships were derived with LogP and hydrophilic lipophilic balance parameters. Compounds with non-quaternary amino groups tended to form nanoaggregates in water and were only effective against S. aureus. The most important determinant of effectiveness was an increased number of quaternary cationic groups that were widely dispersed around the fullerene cage to minimize aggregation. S. aureus was most susceptible; E. coli was intermediate, while C. albicans was the most resistant species tested. The high effectiveness of antimicrobial PDT with quaternized fullerenes suggest they may have applications in treatment of superficial infections (for instance in wounds and burns) where light penetration into tissue is not problematic.

Photodynamic inactivation is a rapidly developing antimicrobial treatment that employs a nontoxic photoactivatable dye or photosensitizer in combination with harmless visible light to generate reactive oxygen species that are toxic to cells. Tetrapyrroles (e.g., porphyrins, chlorins, bacteriochlorins) are a class of photosensitizers that exhibit promising characteristics to serve as broad-spectrum antimicrobials. In order to bind to and efficiently penetrate into all classes of microbial cells, tetrapyrroles should have structures that contain (i) one or more cationic charge(s) or (ii) a basic group. In this report, we investigate the use of new stable synthetic bacteriochlorins that have a strong absorption band in the range 720 to 740 nm, which is in the near-infrared spectral region. Four bacteriochlorins with 2, 4, or 6 quaternized ammonium groups or 2 basic amine groups were compared for light-mediated killing against a Gram-positive bacterium ( Staphylococcus aureus ), a Gram-negative bacterium ( Escherichia coli ), and a dimorphic fungal yeast ( Candida albicans ). Selectivity was assessed by determining phototoxicity against human HeLa cancer cells under the same conditions. All four compounds were highly active (6 logs of killing at 1 μM or less) against S. aureus and showed selectivity for bacteria over human cells. Increasing the cationic charge increased activity against E. coli . Only the compound with basic groups was highly active against C. albicans. Supporting photochemical and theoretical characterization studies indicate that (i) the four bacteriochlorins have comparable photophysical features in homogeneous solution and (ii) the anticipated redox characteristics do not correlate with cell-killing ability. These results support the interpretation that the disparate biological activities observed stem from cellular binding and localization effects rather than intrinsic electronic properties. These findings further establish cationic bacteriochlorins as extremely active and selective near-infrared activated antimicrobial photosensitizers, and the results provide fundamental information on structure-activity relationships for antimicrobial photosensitizers.

Multidrug-resistant Acinetobacter baumannii infections represent a growing problem, especially in traumatic wounds and burns suffered by military personnel injured in Middle Eastern conflicts. Effective treatment with traditional antibiotics can be extremely difficult, and new antimicrobial approaches are being investigated. One of these alternatives to antimicrobials could be the combination of nontoxic photosensitizers (PSs) and visible light, known as photodynamic therapy (PDT). We report on the establishment of a new mouse model of full-thickness thermal burns infected with a bioluminescent derivative of a clinical Iraqi isolate of A. baumannii and its PDT treatment by topical application of a PS produced by the covalent conjugation of chlorin(e6) to polyethylenimine, followed by illumination of the burn surface with red light. Application of 10 8 A. baumannii cells to the surface of 10-s burns made on the dorsal surface of shaved female BALB/c mice led to chronic infections that lasted, on average, 22 days and that were characterized by a remarkably stable bacterial bioluminescence. PDT carried out on day 0 soon after application of the bacteria gave over 3 log units of loss of bacterial luminescence in a light exposure-dependent manner, while PDT carried out on day 1 and day 2 gave an approximately 1.7-log reduction. The application of PS dissolved in 10% or 20% dimethyl sulfoxide without light gave only a modest reduction in the bacterial luminescence from mouse burns. Some bacterial regrowth in the treated burn was observed but was generally modest. It was also found that PDT did not lead to the inhibition of wound healing. The data suggest that PDT may be an effective new treatment for multidrug-resistant localized A. baumannii infections.

Multi-drug resistant Acinetobacter baumanii infections represent a growing problem, especially in traumatic wounds and burns suffered by military personnel injured in Middle Eastern conflicts. Effective treatment using traditional antibiotics can be extremely difficult and new antimicrobial approaches are being investigated. One of these antimicrobial alternatives could be the combination of non-toxic photosensitizers (PS) and visible light known as photodynamic therapy (PDT). We report on the establishment of a new mouse model of full thickness thermal burns infected with a bioluminescent derivative of a clinical Iraqi isolate of A. baumannii and its PDT treatment by topical application of a PS produced by covalent conjugation chlorin(e6) to polyethylenimine followed by illumination of the burn surface with red light. Application of 10 8 A. baumannii cells to the surface of 10-second burns made on the dorsal surface of shaved female BALB/c mice led to chronic infections that lasted on average 22 days characterized by a remarkably stable bacterial bioluminescence. PDT carried out on day 0 soon after applying bacteria gave over three logs of loss of bacterial luminescence in a light exposure dependent manner, while PDT carried out on day 1 and day 2 gave approximately a 1.7-log reduction. Application of PS dissolved in 10% or 20% DMSO without light gave only modest reduction in bacterial luminescence from mouse burns. Some bacterial regrowth in the treated burn was observed but was generally modest. It was also found that PDT did not lead to inhibition of wound healing. The data suggest that PDT may be an effective new treatment for multi-drug resistant localized A. baumannii infections.

Photodynamic therapy has emerged as a possible supplement to the existing protocols for endodontic disinfection. Microbes are known to gain significant ecological advantage when they survive as coaggregates and biofilms in an infected tissue. Such microbial coaggregates and biofilms have been confirmed to play a key role in the pathogenicity of many infections. So far, not many studies have correlated the efficacy of antimicrobial photodynamic inactivation (APDI) to the different modes of bacterial growth. This study aims to evaluate the APDI of 3 strains of Enterococcus faecalis in planktonic phase, in a co-aggregated suspension and in a 4-day old biofilm. The results showed that the biofilm mode of growth offered the greatest resistance to APDI and the inclusion of an efflux pump inhibitor significantly increased the APDI of biofilm bacteria. From this study, we conclude that APDI of bacteria in biofilms is the most challenging and that the use of bacterial efflux pump inhibitors enhances its photodynamic antibiofilm efficacy.

An engineered chitosan acetate bandage preparation (HemCon) is used as a hemostatic dressing, and its chemical structure suggests that it should also be antimicrobial. We previously showed that when a chitosan acetate bandage was applied to full-thickness excisional wounds in mice that had been infected with pathogenic bioluminescent bacteria (Pseudomonas aeruginosa, Proteus mirabilis, andStaphylococcus aureus), it was able to rapidly kill the bacteria and save the mice from developing fatal infections. Wound healing was also stimulated. In the present study, we asked whether a chitosan acetate bandage could act as a topical antimicrobial dressing when it was applied to third-degree burns in mice contaminated with two of these bacterial species (P. aeruginosaandP. mirabilis). Preliminary experiments established the length of burn time and the number of bacteria needed to produce fatal infections in untreated mice and established that the chitosan acetate bandage could adhere to the infected burn for up to 21 days. In the case ofP. aeruginosainfections, the survival rate of mice treated with the chitosan acetate bandage was 73.3% (whereas the survival rate of mice treated with a nanocrystalline silver dressing was 27.3% [P= 0.0055] and that of untreated mice was 13.3% [P< 0.0002]). ForP. mirabilisinfections, the comparable survival rates were 66.7%, 62.5%, and 23.1% respectively. Quantitative bioluminescent signals showed that the chitosan acetate bandage effectively controlled the growth of bacteria in the burn and prevented the development of systemic sepsis, as shown by blood culture. These data suggest that chitosan acetate bandage is efficacious in preventing fatal burn infections.

Antimicrobial photodynamic inactivation (APDI) combines a nontoxic photoactivatable dye or photosensitizer (PS) with harmless visible light to generate singlet oxygen and reactive oxygen species that kill microbial cells. Cationic phenothiazinium dyes, such as toluidine blue O (TBO), are the only PS used clinically for APDI, and we recently reported that this class of PS are substrates of multidrug efflux pumps in both gram-positive and gram-negative bacteria. We now report that APDI can be significantly potentiated by combining the PS with an efflux pump inhibitor (EPI). Killing of Staphylococcus aureus mediated by TBO and red light is greatly increased by coincubation with known inhibitors of the major facilitator pump (NorA): the diphenyl urea INF271, reserpine, 5′-methoxyhydnocarpin, and the polyacylated neohesperidoside, ADH7. The potentiation effect is greatest in the case of S. aureus mutants that overexpress NorA and least in NorA null cells. Addition of the EPI before TBO has a bigger effect than addition of the EPI after TBO. Cellular uptake of TBO is increased by EPI. EPI increased photodynamic inactivation killing mediated by other phenothiazinium dyes, such as methylene blue and dimethylmethylene blue, but not that mediated by nonphenothiazinium PS, such as Rose Bengal and benzoporphyrin derivative. Killing of Pseudomonas aeruginosa mediated by TBO and light was also potentiated by the resistance nodulation division pump (MexAB-OprM) inhibitor phenylalanine-arginine beta-naphthylamide but to a lesser extent than for S. aureus . These data suggest that EPI could be used in combination with phenothiazinium salts and light to enhance their antimicrobial effect against localized infections.

Background and Objective: To compare the effectiveness of antimicrobial photodynamic therapy (PDT), standard endodontic treatment and the combined treatment to eliminate bacterial biofilms present in infected root canals. Study Design/Materials and Methods: Ten singlerooted freshly extracted human teeth wereinoculated with stable bioluminescent Gram-negative bacteria, Proteus mirabilis and Pseudomonas aeruginosa to form 3-day biofilms in prepared root canals. Bioluminescence imaging was used to serially quantify bacterial burdens. PDT employed a conjugate between polyethylenimine and chlorin(e6) as the photosensitizer (PS) and 660-nm diode laserlightdeliveredintotherootcanalviaa200-mfiber,and thiswascomparedandcombinedwithstandardendodontic treatment using mechanical debridement and antiseptic irrigation. Results: Endodontic therapy alone reduced bacterial bioluminescence by 90% while PDT alone reduced bioluminescence by 95%. The combination reduced bioluminescence by >98%, and importantly the bacterial regrowth observed 24 hours after treatment was much less for the combination (P

Two new 2-arylbenzofuran aldehydes (1 and 2) and three known phenolic compounds (3-5) were isolated from organic extracts of Dalea spinosa. These compounds were evaluated for their intrinsic antimicrobial activity and their ability to perform as multidrug-resistance inhibitors by potentiating the activity of known antimicrobials against a variety of pathogenic microorganisms. Compound 1 and its acetate derivative 6 exhibited no direct antimicrobial activity but enhanced the effect of the weak plant antimicrobial berberine when tested against Staphylococcus aureus. Additional potentiation assays with S. aureus overexpression and knockout isogenic efflux mutants for the NorA pump were done in order to assess whether the potentiating effects were associated with inhibition of this known pump mechanism.

Antimicrobial photodynamic therapy (PDT) combines a nontoxic photoactivatable dye, or photosensitizer (PS), with harmless visible light to generate singlet oxygen and free radicals that kill microbial cells. Although the light can be focused on the diseased area, the best selectivity is achieved by choosing a PS that binds and penetrates microbial cells. Cationic phenothiazinium dyes, such as methylene blue and toluidine blue O, have been studied for many years and are the only PSs used clinically for antimicrobial PDT. Multidrug resistance pumps (MDRs) are membrane-localized proteins that pump drugs out of cells and have been identified for a wide range of organisms. We asked whether phenothiazinium salts with structures that are amphipathic cations could potentially be substrates of MDRs. We used MDR-deficient mutants of Staphylococcus aureus (NorA), Escherichia coli (TolC), and Pseudomonas aeruginosa (MexAB) and found 2 to 4 logs more killing than seen with wild-type strains by use of three different phenothiazinium PSs and red light. Mutants that overexpress MDRs were protected from killing compared to the wild type. Effective antimicrobial PSs of different chemical structures showed no difference in light-mediated killing depending on MDR phenotype. Differences in uptake of phenothiazinium PS by the cells depending on level of MDR expression were found. We propose that specific MDR inhibitors could be used in combination with phenothiazinium salts to enhance their photodestructive efficiency.

Publisher Summary This chapter describes the rationale for the characterization of natural substrates of multidrug resistant (MDR) pumps, reporting the identification of the first MDR substrates and inhibitors from plants against bacteria and yeasts. It discusses the methodology for the isolation of these compounds as well as the specificity of their activity. The chapter analyzes the current and future perspectives to natural drug discovery based on the classification of plant antimicrobials. The experimental data available demonstrate and reassure that plants produce MDR inhibitors against gram-positive bacteria and yeasts. An effective plant MDR inhibitor of gram-negative bacteria is still a missing part of the puzzle. Currently, there is only preliminary evidence for this, but the identification of such a compound is more than plausible. A comprehensive library of extracts from the National Cancer Institute (NCI), United States of America that consists of over 50,000 extracts from approximately 35,000 plants from all over the world is currently being screened. The collection represents most genera of vascular plants found in the tropics.

Plant antimicrobials are not used as systemic antibiotics at present. The main reason for this is their low level of activity, especially against gram-negative bacteria. The reported MIC is often in the range of 100 to 1,000 μg/ml, orders of magnitude higher than those of common broad-spectrum antibiotics from bacteria or fungi. Major plant pathogens belong to the gram-negative bacteria, which makes the low level of activity of plant antimicrobials against this group of microorganisms puzzling. Gram-negative bacteria have an effective permeability barrier, comprised of the outer membrane, which restricts the penetration of amphipathic compounds, and multidrug resistance pumps (MDRs), which extrude toxins across this barrier. It is possible that the apparent ineffectiveness of plant antimicrobials is largely due to the permeability barrier. We tested this hypothesis in the present study by applying a combination of MDR mutants and MDR inhibitors. A panel of plant antimicrobials was tested by using a set of bacteria representing the main groups of plant pathogens. The human pathogens Pseudomonas aeruginosa , Escherichia coli , and Salmonella enterica serovar Typhimurium were also tested. The results show that the activities of the majority of plant antimicrobials were considerably greater against the gram-positive bacteria Staphylococcus aureus and Bacillus megaterium and that disabling of the MDRs in gram-negative species leads to a striking increase in antimicrobial activity. Thus, the activity of rhein, the principal antimicrobial from rhubarb, was potentiated 100- to 2,000-fold (depending on the bacterial species) by disabling the MDRs. Comparable potentiation of activity was observed with plumbagin, resveratrol, gossypol, coumestrol, and berberine. Direct measurement of the uptake of berberine, a model plant antimicrobial, confirmed that disabling of the MDRs strongly increases the level of penetration of berberine into the cells of gram-negative bacteria. These results suggest that plants might have developed means of delivering their antimicrobials into bacterial cells. These findings also suggest that plant antimicrobials might be developed into effective, broad-spectrum antibiotics in combination with inhibitors of MDRs.

SummaryFullerenes are soccer ball-shaped molecules composed of carbon atoms, and, when derivatized with functional groups, they become soluble and can act as photosensitizers. Antimicrobial photodynamic therapy combines a nontoxic photosensitizer with harmless visible light to generate reactive oxygen species that kill microbial cells. We have compared the antimicrobial activity of six functionalized C60 compounds with one, two, or three hydrophilic or cationic groups in combination with white light against gram-positive bacteria, gram-negative bacteria, and fungi. After a 10 min incubation, the bis- and tris-cationic fullerenes were highly active in killing all tested microbes (4–6 logs) under conditions in which mammalian cells were comparatively unharmed. These compounds performed significantly better than a widely used antimicrobial photosensitizer, toluidine blue O. The high selectivity and efficacy exhibited by these photosensitizers encourage further testing for antimicrobial applications.