RESUMO
Clostridioides difficile infection (CDI) is a life-threatening disease caused by the Gram-positive, opportunistic intestinal pathogen C. difficile. Despite the availability of antimicrobial drugs to treat CDI, such as vancomycin, metronidazole, and fidaxomicin, recurrence of infection remains a significant clinical challenge. The use of live commensal microorganisms, or probiotics, is one of the most investigated non-antibiotic therapeutic options to balance gastrointestinal (GI) microbiota and subsequently tackle dysbiosis. In this review, we will discuss major commensal probiotic strains that have the potential to prevent and/or treat CDI and its recurrence, reassess the efficacy of probiotics supplementation as a CDI intervention, delve into lessons learned from probiotic modulation of the immune system, explore avenues like genome-scale metabolic network reconstructions, genome sequencing, and multi-omics to identify novel strains and understand their functionality, and discuss the current regulatory framework, challenges, and future directions.
Assuntos
Clostridioides difficile , Infecções por Clostridium , Probióticos , Humanos , Antibacterianos/uso terapêutico , Clostridioides difficile/genética , Clostridioides , Vancomicina/uso terapêutico , Infecções por Clostridium/tratamento farmacológico , Infecções por Clostridium/prevenção & controle , Probióticos/uso terapêuticoRESUMO
Bacteria can form single- and multispecies biofilms exhibiting diverse features based upon the microbial composition of their community and microenvironment. The study of bacterial biofilm development has received great interest in the past 20 years and is motivated by the elegant complexity characteristic of these multicellular communities and their role in infectious diseases. Biofilms can thrive on virtually any surface and can be beneficial or detrimental based upon the community's interplay and the surface. Advances in the understanding of structural and functional variations and the roles that biofilms play in disease and host-pathogen interactions have been addressed through comprehensive literature searches. In this review article, a synopsis of the methodological landscape of biofilm analysis is provided, including an evaluation of the current trends in methodological research. We deem this worthwhile because a keyword-oriented bibliographical search reveals that less than 5% of the biofilm literature is devoted to methodology. In this report, we (i) summarize current methodologies for biofilm characterization, monitoring, and quantification; (ii) discuss advances in the discovery of effective imaging and sensing tools and modalities; (iii) provide an overview of tailored animal models that assess features of biofilm infections; and (iv) make recommendations defining the most appropriate methodological tools for clinical settings.
Assuntos
Fenômenos Fisiológicos Bacterianos , Biofilmes , Animais , Humanos , Técnicas Microbiológicas/normas , Modelos AnimaisRESUMO
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.
Assuntos
Acinetobacter baumannii/efeitos dos fármacos , Antibacterianos/farmacologia , Proteínas de Bactérias/antagonistas & inibidores , Escherichia coli/efeitos dos fármacos , Indóis/farmacologia , Azul de Metileno/farmacologia , Proteínas Associadas à Resistência a Múltiplos Medicamentos/antagonistas & inibidores , Antibacterianos/síntese química , Antibacterianos/química , Proteínas de Bactérias/metabolismo , Relação Dose-Resposta a Droga , Indóis/química , Azul de Metileno/química , Testes de Sensibilidade Microbiana , Estrutura Molecular , Proteínas Associadas à Resistência a Múltiplos Medicamentos/metabolismo , Relação Estrutura-AtividadeRESUMO
The mitochondrial ATP binding cassette transporter ABCB6 has been associated with a broad range of physiological functions, including growth and development, therapy-related drug resistance, and the new blood group system Langereis. ABCB6 has been proposed to regulate heme synthesis by shuttling coproporphyrinogen III from the cytoplasm into the mitochondria. However, direct functional information of the transport complex is not known. To understand the role of ABCB6 in mitochondrial transport, we developed an in vitro system with pure and active protein. ABCB6 overexpressed in HEK293 cells was solubilized from mitochondrial membranes and purified to homogeneity. Purified ABCB6 showed a high binding affinity for MgATP (Kd = 0.18 µM) and an ATPase activity with a Km of 0.99 mM. Reconstitution of ABCB6 into liposomes allowed biochemical characterization of the ATPase including (i) substrate-stimulated ATPase activity, (ii) transport kinetics of its proposed endogenous substrate coproporphyrinogen III, and (iii) transport kinetics of substrates identified using a high throughput screening assay. Mutagenesis of the conserved lysine to alanine (K629A) in the Walker A motif abolished ATP hydrolysis and substrate transport. These results suggest a direct interaction between mitochondrial ABCB6 and its transport substrates that is critical for the activity of the transporter. Furthermore, the simple immunoaffinity purification of ABCB6 to near homogeneity and efficient reconstitution of ABCB6 into liposomes might provide the basis for future studies on the structure/function of ABCB6.
Assuntos
Transportadores de Cassetes de Ligação de ATP/isolamento & purificação , Isoformas de Proteínas/isolamento & purificação , Transportadores de Cassetes de Ligação de ATP/química , Transportadores de Cassetes de Ligação de ATP/metabolismo , Trifosfato de Adenosina/metabolismo , Coproporfirinas/metabolismo , Células HEK293 , Humanos , Cinética , Lipídeos de Membrana/metabolismo , Mitocôndrias/metabolismo , Ligação Proteica , Conformação Proteica , Isoformas de Proteínas/química , Isoformas de Proteínas/metabolismoRESUMO
This review highlights the concepts, recent applications and limitations of High Throughput Screening (HTS) flow cytometry-based efflux inhibitory assays. This platform has been employed in mammalian and yeast efflux systems leading to the identification of small molecules with transporter inhibitory capabilities. This technology offers the possibility of substrate multiplexing and may promote novel strategies targeting microbial efflux systems. This platform can generate a comprehensive dataset that may support efforts to map the interface between chemistry and transporter biology in a variety of pathogenic systems.
Assuntos
Transportadores de Cassetes de Ligação de ATP/antagonistas & inibidores , Descoberta de Drogas/métodos , Citometria de Fluxo , Ensaios de Triagem em Larga Escala , Bibliotecas de Moléculas Pequenas/farmacologia , Animais , Transporte Biológico , Humanos , Bibliotecas de Moléculas Pequenas/química , Especificidade por SubstratoRESUMO
Antibiotic resistance is a pressing public health threat. Despite rising resistance, antibiotic development, especially for Gram-negative bacteria, has stagnated. As the traditional antibiotic research and development pipeline struggles to address this growing concern, alternative solutions become imperative. Synthetic molecular nanomachines (MNMs) are molecular structures that rotate unidirectionally in a controlled manner in response to a stimulus, such as light, resulting in a mechanical action that can propel molecules to drill into cell membranes, causing rapid cell death. Due to their broad destructive capabilities, clinical translation of MNMs remains challenging. Hence, here, we explore the ability of nonlethal visible-light-activated MNMs to potentiate conventional antibiotics against Gram-negative bacteria. Nonlethal MNMs enhanced the antibacterial activity of various classes of conventional antibiotics against Gram-negative bacteria, including those typically effective only against Gram-positive strains, reducing the antibiotic concentration required for bactericidal action. Our study also revealed that MNMs bind to the negatively charged phospholipids of the bacterial inner membrane, leading to permeabilization of the cell envelope and impairment of efflux pump activity following light activation of MNMs. The combined effects of MNMs on membrane permeability and efflux pumps resulted in increased antibiotic accumulation inside the cell, reversing antibiotic resistance and attenuating its development. These results identify nonlethal MNMs as pleiotropic antibiotic enhancers or adjuvants. The combination of MNMs with traditional antibiotics is a promising strategy against multidrug-resistant Gram-negative infections. This approach can reduce the amount of antibiotics needed and slow down antibiotic resistance development, thereby preserving the effectiveness of our current antibiotics.
Assuntos
Antibacterianos , Farmacorresistência Bacteriana Múltipla , Antibacterianos/metabolismo , Bactérias Gram-Negativas , Transporte Biológico , PermeabilidadeRESUMO
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.
Assuntos
Queimaduras/terapia , Infecções por Pseudomonas/terapia , Pseudomonas aeruginosa/efeitos da radiação , Infecção dos Ferimentos/terapia , Animais , Queimaduras/microbiologia , Queimaduras/patologia , Sobrevivência Celular/efeitos da radiação , Feminino , Marcação In Situ das Extremidades Cortadas , Queratinócitos/citologia , Queratinócitos/efeitos da radiação , Luz , Camundongos , Camundongos Endogâmicos BALB C , Viabilidade Microbiana/efeitos da radiação , Fototerapia , Infecções por Pseudomonas/microbiologia , Infecções por Pseudomonas/patologia , Pseudomonas aeruginosa/crescimento & desenvolvimento , Infecção dos Ferimentos/microbiologia , Infecção dos Ferimentos/patologiaRESUMO
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.
Assuntos
Candida albicans/efeitos dos fármacos , Candidíase/tratamento farmacológico , Azul de Metileno/farmacologia , Fármacos Fotossensibilizantes/farmacologia , Fatores de Virulência/antagonistas & inibidores , Animais , Antifúngicos/farmacologia , Candida albicans/crescimento & desenvolvimento , Candida albicans/patogenicidade , Candida albicans/efeitos da radiação , Candidíase/microbiologia , Candidíase/mortalidade , Parede Celular/efeitos dos fármacos , Parede Celular/efeitos da radiação , Feminino , Fluconazol/farmacologia , Padrões de Herança , Lasers Semicondutores , Luz , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Pressão Osmótica , Estresse Oxidativo , Análise de Sobrevida , Fatores de Virulência/metabolismoRESUMO
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.
Assuntos
Anti-Infecciosos/farmacologia , Candida albicans/efeitos da radiação , Lepidópteros/microbiologia , Luz , Azul de Metileno/farmacologia , Fotoquimioterapia/métodos , Fármacos Fotossensibilizantes/farmacologia , Animais , Anti-Infecciosos/administração & dosagem , Contagem de Colônia Microbiana , Quimioterapia Combinada/métodos , Fluconazol/administração & dosagem , Fluconazol/farmacologia , Hemolinfa/microbiologia , Larva/microbiologia , Modelos Animais , Fármacos Fotossensibilizantes/administração & dosagem , Análise de SobrevidaRESUMO
ATP binding cassette (ABC) transmembrane efflux pumps such as P-glycoprotein (ABCB1), multidrug resistance protein 1 (ABCC1), and breast cancer resistance protein (ABCG2) play an important role in anticancer drug resistance. A large number of structurally and functionally diverse compounds act as substrates or modulators of these pumps. In vitro assessment of the affinity of drug candidates for multidrug resistance proteins is central to predict in vivo pharmacokinetics and drug-drug interactions. The objective of this study was to identify and characterize new substrates for these transporters. As part of a collaborative project with Life Technologies, 102 fluorescent probes were investigated in a flow cytometric screen of ABC transporters. The primary screen compared substrate efflux activity in parental cell lines with their corresponding highly expressing resistant counterparts. The fluorescent compound library included a range of excitation/emission profiles and required dual laser excitation as well as multiple fluorescence detection channels. A total of 31 substrates with active efflux in one or more pumps and practical fluorescence response ranges were identified and tested for interaction with eight known inhibitors. This screening approach provides an efficient tool for identification and characterization of new fluorescent substrates for ABCB1, ABCC1, and ABCG2.
Assuntos
Transportadores de Cassetes de Ligação de ATP/antagonistas & inibidores , Transportadores de Cassetes de Ligação de ATP/metabolismo , Citometria de Fluxo/métodos , Corantes Fluorescentes/metabolismo , Subfamília B de Transportador de Cassetes de Ligação de ATP , Membro 1 da Subfamília B de Cassetes de Ligação de ATP/antagonistas & inibidores , Membro 1 da Subfamília B de Cassetes de Ligação de ATP/genética , Membro 1 da Subfamília B de Cassetes de Ligação de ATP/metabolismo , Membro 2 da Subfamília G de Transportadores de Cassetes de Ligação de ATP , Transportadores de Cassetes de Ligação de ATP/genética , Linhagem Celular , Humanos , Proteínas Associadas à Resistência a Múltiplos Medicamentos/antagonistas & inibidores , Proteínas Associadas à Resistência a Múltiplos Medicamentos/genética , Proteínas Associadas à Resistência a Múltiplos Medicamentos/metabolismo , Proteínas de Neoplasias/antagonistas & inibidores , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Ligação ProteicaRESUMO
Patients with severe burns are highly susceptible to bacterial infection. While immunosuppression facilitates infection, the contribution of soft tissues to infection beyond providing a portal for bacterial entry remains unclear. We showed previously that glutathione S-transferase S1 (gstS1), an enzyme with conjugating activity against the lipid peroxidation byproduct 4-hydroxynonenal (4HNE), is important for resistance against wound infection in Drosophila muscle. The importance of the mammalian functional counterpart of GstS1 in the context of wounds and infection has not been investigated. Here we demonstrate that the presence of a burn wound dramatically affects expression of both human (hGSTA4) and mouse (mGsta4) 4HNE scavengers. hGSTA4 is down-regulated significantly within 1 wk of thermal burn injury in the muscle and fat tissues of patients from the large-scale collaborative Inflammation and the Host Response to Injury multicentered study. Similarly, mGsta4, the murine GST with the highest catalytic efficiency for 4HNE, is down-regulated to approximately half of normal levels in mouse muscle immediately postburn. Consequently, 4HNE protein adducts are increased 4- to 5-fold in mouse muscle postburn. Using an open wound infection model, we show that deletion of mGsta4 renders mice more susceptible to infection with the prevalent wound pathogen Pseudomonas aeruginosa, while muscle hGSTA4 expression negatively correlates with burn wound infection episodes per patient. Our data suggest that hGSTA4 down-regulation and the concomitant increase in 4HNE adducts in human muscle are indicative of susceptibility to infection in individuals with severely thermal injuries.
Assuntos
Infecções Bacterianas/enzimologia , Infecções Bacterianas/etiologia , Queimaduras/complicações , Queimaduras/enzimologia , Glutationa Transferase/metabolismo , Músculo Esquelético/enzimologia , Infecção dos Ferimentos/enzimologia , Infecção dos Ferimentos/etiologia , Aldeídos/metabolismo , Animais , Infecções Bacterianas/genética , Sequência de Bases , Queimaduras/genética , Estudos de Casos e Controles , Primers do DNA/genética , Modelos Animais de Doenças , Suscetibilidade a Doenças , Regulação para Baixo , Feminino , Glutationa Transferase/deficiência , Glutationa Transferase/genética , Humanos , Estudos Longitudinais , Camundongos , Camundongos da Linhagem 129 , Camundongos Knockout , Estudos Prospectivos , Infecções por Pseudomonas/enzimologia , Infecções por Pseudomonas/etiologia , Infecções por Pseudomonas/genética , Infecção dos Ferimentos/genéticaRESUMO
Blue light, particularly in the wavelength range of 405-470 nm, has attracted increasing attention due to its intrinsic antimicrobial effect without the addition of exogenous photosensitizers. In addition, it is commonly accepted that blue light is much less detrimental to mammalian cells than ultraviolet irradiation, which is another light-based antimicrobial approach being investigated. In this review, we discussed the blue light sensing systems in microbial cells, antimicrobial efficacy of blue light, the mechanism of antimicrobial effect of blue light, the effects of blue light on mammalian cells, and the effects of blue light on wound healing. It has been reported that blue light can regulate multi-cellular behavior involving cell-to-cell communication via blue light receptors in bacteria, and inhibit biofilm formation and subsequently potentiate light inactivation. At higher radiant exposures, blue light exhibits a broad-spectrum antimicrobial effect against both Gram-positive and Gram-negative bacteria. Blue light therapy is a clinically accepted approach for Propionibacterium acnes infections. Clinical trials have also been conducted to investigate the use of blue light for Helicobacter pylori stomach infections and have shown promising results. Studies on blue light inactivation of important wound pathogenic bacteria, including Staphylococcus aureus and Pseudomonas aeruginosa have also been reported. The mechanism of blue light inactivation of P. acnes, H. pylori, and some oral bacteria is proved to be the photo-excitation of intracellular porphyrins and the subsequent production of cytotoxic reactive oxygen species. Although it may be the case that the mechanism of blue light inactivation of wound pathogens (e.g., S. aureus, P. aeruginosa) is the same as that of P. acnes, this hypothesis has not been rigorously tested. Limited and discordant results have been reported regarding the effects of blue light on mammalian cells and wound healing. Under certain wavelengths and radiant exposures, blue light may cause cell dysfunction by the photo-excitation of blue light sensitizing chromophores, including flavins and cytochromes, within mitochondria or/and peroxisomes. Further studies should be performed to optimize the optical parameters (e.g., wavelength, radiant exposure) to ensure effective and safe blue light therapies for infectious disease. In addition, studies are also needed to verify the lack of development of microbial resistance to blue light.
Assuntos
Acne Vulgar/terapia , Infecções por Bactérias Gram-Positivas/terapia , Infecções por Helicobacter/terapia , Helicobacter pylori/efeitos da radiação , Fototerapia/métodos , Propionibacterium acnes/efeitos da radiação , Animais , Infecções por Bactérias Gram-Positivas/microbiologia , Infecções por Helicobacter/microbiologia , Humanos , Luz , Cicatrização/efeitos da radiaçãoRESUMO
Invasive fungal infections are a growing public health threat. As fungi become increasingly resistant to existing drugs, new antifungals are urgently needed. Here, it is reported that 405-nm-visible-light-activated synthetic molecular machines (MMs) eliminate planktonic and biofilm fungal populations more effectively than conventional antifungals without resistance development. Mechanism-of-action studies show that MMs bind to fungal mitochondrial phospholipids. Upon visible light activation, rapid unidirectional drilling of MMs at ≈3 million cycles per second (MHz) results in mitochondrial dysfunction, calcium overload, and ultimately necrosis. Besides their direct antifungal effect, MMs synergize with conventional antifungals by impairing the activity of energy-dependent efflux pumps. Finally, MMs potentiate standard antifungals both in vivo and in an ex vivo porcine model of onychomycosis, reducing the fungal burden associated with infection.
Assuntos
Antifúngicos , Cálcio , Animais , Suínos , Antifúngicos/farmacologia , Antifúngicos/uso terapêutico , Antifúngicos/metabolismo , Cálcio/metabolismo , Fungos/metabolismoRESUMO
Antibiotic resistance is a growing health threat. There is an urgent and critical need to develop new antimicrobial modalities and therapies. Here, a set of hemithioindigo (HTI)-based molecular machines capable of specifically killing Gram-positive bacteria within minutes of activation with visible light (455 nm at 65 mW cm-2 ) that are safe for mammalian cells is described. Importantly, repeated exposure of bacteria to HTI does not result in detectable development of resistance. Visible light-activated HTI kill both exponentially growing bacterial cells and antibiotic-tolerant persister cells of various Gram-positive strains, including methicillin-resistant S. aureus (MRSA). Visible light-activated HTI also eliminate biofilms of S. aureus and B. subtilis in as little as 1 h after light activation. Quantification of reactive oxygen species (ROS) formation and protein carbonyls, as well as assays with various ROS scavengers, identifies oxidative damage as the underlying mechanism for the antibacterial activity of HTI. In addition to their direct antibacterial properties, HTI synergize with conventional antibiotics in vitro and in vivo, reducing the bacterial load and mortality associated with MRSA infection in an invertebrate burn wound model. To the best of the authors' knowledge, this is the first report on the antimicrobial activity of HTI-based molecular machines.
Assuntos
Staphylococcus aureus Resistente à Meticilina , Animais , Testes de Sensibilidade Microbiana , Staphylococcus aureus/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Antibacterianos/farmacologia , Antibacterianos/uso terapêutico , Bactérias , Luz , Estresse Oxidativo , Mamíferos/metabolismoRESUMO
The increasing occurrence of antibiotic-resistant bacteria and the dwindling antibiotic research and development pipeline have created a pressing global health crisis. Here, we report the discovery of a distinctive antibacterial therapy that uses visible (405 nanometers) light-activated synthetic molecular machines (MMs) to kill Gram-negative and Gram-positive bacteria, including methicillin-resistant Staphylococcus aureus, in minutes, vastly outpacing conventional antibiotics. MMs also rapidly eliminate persister cells and established bacterial biofilms. The antibacterial mode of action of MMs involves physical disruption of the membrane. In addition, by permeabilizing the membrane, MMs at sublethal doses potentiate the action of conventional antibiotics. Repeated exposure to antibacterial MMs is not accompanied by resistance development. Finally, therapeutic doses of MMs mitigate mortality associated with bacterial infection in an in vivo model of burn wound infection. Visible light-activated MMs represent an unconventional antibacterial mode of action by mechanical disruption at the molecular scale, not existent in nature and to which resistance development is unlikely.
RESUMO
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.
Assuntos
Acetatos/química , Infecções Bacterianas/tratamento farmacológico , Queimaduras/microbiologia , Quitosana/química , Quitosana/uso terapêutico , Nanopartículas Metálicas/química , Nanopartículas Metálicas/uso terapêutico , Prata/química , Animais , Feminino , Camundongos , Camundongos Endogâmicos BALB CRESUMO
The objective of this study was to investigate photodynamic therapy (PDT), using blue dye and red light, for prophylaxis and treatment of cutaneous Candida albicans infections in mice. A mouse model of skin abrasion infected with C. albicans was developed by inoculating wounds measuring 1.2 cm by 1.2 cm with 10(6) or 10(7) CFU. The use of a luciferase-expressing strain of C. albicans allowed real-time monitoring of the extent of infection in mice noninvasively through bioluminescence imaging. The phenothiazinium salts toluidine blue O (TBO), methylene blue (MB), and new methylene blue (NMB) were compared as photosensitizers (PS) for the photodynamic inactivation of C. albicans in vitro. PDT in vivo was initiated either at 30 min or at 24 h after fungal inoculation to investigate the efficacies of PDT for both prophylaxis and treatment of infections. Light at 635 ± 15 nm or 660 ± 15 nm was delivered with a light dose of 78 J/cm(2) (for PDT at 30 min postinfection) or 120 J/cm(2) (for PDT at 24 h postinfection) in multiple exposures with bioluminescence imaging taking place after each exposure of light. In vitro studies showed that NMB was superior to TBO and MB as the PS in the photodynamic inactivation of C. albicans. The efficacy of PDT was related to the ratio of PS concentration to fungal cell density. PDT in vivo initiated either at 30 min or at 24 h postinfection significantly reduced C. albicans burden in the infected mouse skin abrasion wounds. These data suggest that PDT is a viable approach for prophylaxis and treatment of cutaneous C. albicans infections.
Assuntos
Candida albicans/efeitos dos fármacos , Candidíase Cutânea/tratamento farmacológico , Corantes/uso terapêutico , Luz , Fotoquimioterapia , Fármacos Fotossensibilizantes/uso terapêutico , Animais , Candida albicans/crescimento & desenvolvimento , Candidíase Cutânea/prevenção & controle , Corantes/administração & dosagem , Modelos Animais de Doenças , Feminino , Humanos , Azul de Metileno/administração & dosagem , Azul de Metileno/análogos & derivados , Azul de Metileno/uso terapêutico , Camundongos , Fármacos Fotossensibilizantes/administração & dosagem , Cloreto de Tolônio/administração & dosagem , Cloreto de Tolônio/uso terapêutico , Resultado do TratamentoRESUMO
OBJECTIVES: 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. METHODS: 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. RESULTS: 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. CONCLUSIONS: 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.
Assuntos
Transportadores de Cassetes de Ligação de ATP/metabolismo , Antifúngicos/toxicidade , Candida albicans/efeitos dos fármacos , Candida albicans/metabolismo , Azul de Metileno/toxicidade , Fármacos Fotossensibilizantes/toxicidade , Transportadores de Cassetes de Ligação de ATP/antagonistas & inibidores , Antifúngicos/metabolismo , Humanos , Azul de Metileno/metabolismo , Viabilidade Microbiana/efeitos dos fármacos , Microscopia Confocal , Fármacos Fotossensibilizantes/metabolismoRESUMO
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 microM 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.
Assuntos
Anti-Infecciosos/síntese química , Anti-Infecciosos/farmacologia , Fármacos Fotossensibilizantes/síntese química , Fármacos Fotossensibilizantes/farmacologia , Porfirinas/síntese química , Porfirinas/farmacologia , Anti-Infecciosos/efeitos adversos , Anti-Infecciosos/química , Candida albicans/efeitos dos fármacos , Candida albicans/efeitos da radiação , Sobrevivência Celular/efeitos dos fármacos , Escherichia coli/efeitos dos fármacos , Escherichia coli/efeitos da radiação , Células HeLa , Humanos , Luz , Microscopia Confocal , Estrutura Molecular , Fármacos Fotossensibilizantes/efeitos adversos , Fármacos Fotossensibilizantes/química , Porfirinas/efeitos adversos , Porfirinas/química , Staphylococcus aureus/efeitos dos fármacos , Staphylococcus aureus/efeitos da radiaçãoRESUMO
BACKGROUND AND OBJECTIVE: Methicillin-resistant Staphylococcus aureus (MRSA) skin infections are now known to be a common and important problem in the Unites States. The objective of this study was to investigate the efficacy of photodynamic therapy (PDT) for the treatment of MRSA infection in skin abrasion wounds using a mouse model. STUDY DESIGN/MATERIALS AND METHODS: A mouse model of skin abrasion wound infected with MRSA was developed. Bioluminescent strain of MRSA, a derivative of ATCC 33591, was used to allow the real-time monitoring of the extent of infection in mouse wounds. PDT was performed with the combination of a polyethylenimine (PEI)-ce6 photosensitizer (PS) and non-coherent red light. In vivo fluorescence imaging was carried out to evaluate the effect of photobleaching of PS during PDT. RESULTS: In vivo fluorescence imaging of conjugate PEI-ce6 applied in mice indicated the photobleaching effect of the PS during PDT. PDT induced on average 2.7 log(10) of inactivation of MRSA as judged by loss of bioluminescence in mouse skin abrasion wounds and accelerated the wound healing on average by 8.6 days in comparison to the untreated infected wounds. Photobleaching of PS in the wound was overcome by adding the PS solution in aliquots. CONCLUSION: PDT may represent an alternative approach for the treatment of MRSA skin infections.