RESUMO
In recent years, some microorganisms have shown resistance to conventional treatments. Considering this increase in resistant pathogens, treatment alternatives are needed to promote greater treatment efficiency. In this sense, antimicrobial photodynamic therapy (aPDT) has been an alternative treatment. This technique uses a photosensitizer that is activated by light with a specific wavelength producing reactive species, leading to the death of pathogenic microorganisms. In this study, bacteriochlorophyll derivatives such as bacteriochlorin metoxi (Bchl-M) and bacteriochlorin trizma (Bchl-T) obtained from purple bacterium (Rhodopseudomonas faecalis), were evaluated as photosensitizers in the aPDT. Photodynamic inactivation (PDI) of the microorganisms Staphylococcus aureus, Micrococcus luteus, Candida albicans and Pseudomonas aeruginosa was investigated with both bacteriochlorins (Bchl-M and Bchl-T) at different concentrations (1, 15 and 30 µM for S. aureus; 1, 15, 30, 45, 60 and 75 µM for M. luteus; 30, 60, 90, 105, 120 and 150 µM for C. albicans; and 200 µM for P. aeruginosa) and different doses of light (20 and 30 J/cm2 for S. aureus and M. luteus; 30 and 45 J/cm2 for C. albicans; and 45 J/cm2 for P. aeruginosa) to inactivate them. Both photosensitizers showed good activation against S. aureus and for M. luteus, we observed the inactivation of these microorganisms at approximately 3 log, showing to be a good photosensitizers for these microorganisms.
Assuntos
Candida albicans , Luz , Fotoquimioterapia , Fármacos Fotossensibilizantes , Pseudomonas aeruginosa , Staphylococcus aureus , Fármacos Fotossensibilizantes/farmacologia , Fármacos Fotossensibilizantes/química , Candida albicans/efeitos dos fármacos , Candida albicans/efeitos da radiação , Pseudomonas aeruginosa/efeitos dos fármacos , Pseudomonas aeruginosa/efeitos da radiação , Staphylococcus aureus/efeitos dos fármacos , Staphylococcus aureus/efeitos da radiação , Fotoquimioterapia/métodos , Porfirinas/farmacologia , Porfirinas/química , Viabilidade Microbiana/efeitos dos fármacos , Viabilidade Microbiana/efeitos da radiação , Micrococcus luteus/efeitos dos fármacos , Micrococcus luteus/efeitos da radiação , Bactérias/efeitos dos fármacos , Bactérias/efeitos da radiaçãoRESUMO
Photobiomodulation has been used to inactivate bacterial growth, in different laser or LED protocols. Thus, the aim of this study was to verify the inhibition of Staphylococcus aureus, Pseudomonas aeruginosa, and Escherichia coli, in ATCC strains and bacteria collected from patients with skin burns, after irradiation with LED; 300 µl of saline solution with bacterial suspension was irradiated at a concentration of 0.5-0.63, by the McFarland scale, after five serial dilutions, with evaluation of pre- and post-irradiation pH and temperature control. The cultures were placed in a bacteriological incubator at 37 °C for 24 h for later counting of colony-forming units (CFU). Data were analyzed by Shapiro-Wilk tests and single-factor ANOVA, with Tukey post hoc (p < 0.05). Both wavelengths and energy densities tested showed inhibition of bacterial growth. The comparison of the irradiated groups (ATCC) with the control group showed the following: S. aureus and P. aeruginosa 465 nm (40 J/cm2) and 630 nm (50 J/cm2) and E. coli 465 nm (40 J/cm2) and 630 nm (30 J/cm2). Among the ATCC S. aureus groups, there was a difference for 630 nm (30 J/cm2) and 465 nm (30, 40, 50 J/cm2). The bacteria from the burned patients were S. aureus (30 and 50 J/cm2) and P. aeruginosa (50 J/cm2). We conclude that different bacterial strains were reduced into colony-forming units after LED irradiation.
Assuntos
Terapia com Luz de Baixa Intensidade , Staphylococcus aureus , Escherichia coli/efeitos da radiação , Humanos , Luz , Pseudomonas aeruginosa/efeitos da radiação , Staphylococcus aureus/efeitos da radiaçãoRESUMO
Light sensing has been extensively characterized in the human pathogen Acinetobacter baumannii at environmental temperatures. However, the influence of light on the physiology and pathogenicity of human bacterial pathogens at temperatures found in warm-blooded hosts is still poorly understand. In this work, we show that Staphylococcus aureus, Acinetobacter baumannii, and Pseudomonas aeruginosa (ESKAPE) priority pathogens, which have been recognized by the WHO and the CDC as critical, can also sense and respond to light at temperatures found in human hosts. Most interestingly, in these pathogens, light modulates important pathogenicity determinants as well as virulence in an epithelial infection model, which could have implications in human infections. In fact, we found that alpha-toxin-dependent hemolysis, motility, and growth under iron-deprived conditions are modulated by light in S. aureus Light also regulates persistence, metabolism, and the ability to kill competitors in some of these microorganisms. Finally, light exerts a profound effect on the virulence of these pathogens in an epithelial infection model, although the response is not the same in the different species; virulence was enhanced by light in A. baumannii and S. aureus, while in A. nosocomialis and P. aeruginosa it was reduced. Neither the BlsA photoreceptor nor the type VI secretion system (T6SS) is involved in virulence modulation by light in A. baumannii Overall, this fundamental knowledge highlights the potential use of light to control pathogen virulence, either directly or by manipulating the light regulatory switch toward the lowest virulence/persistence configuration.IMPORTANCE Pathogenic bacteria are microorganisms capable of producing disease. Dangerous bacterial pathogens, such as Staphylococcus aureus, Pseudomonas aeruginosa, and Acinetobacter baumannii, are responsible for serious intrahospital and community infections in humans. Therapeutics is often complicated due to resistance to multiple antibiotics, rendering them ineffective. In this work, we show that these pathogens sense natural light and respond to it by modulating aspects related to their ability to cause disease; in the presence of light, some of them become more aggressive, while others show an opposite response. Overall, we provide new understanding on the behavior of these pathogens, which could contribute to the control of infections caused by them. Since the response is distributed in diverse pathogens, this notion could prove a general concept.
Assuntos
Acinetobacter baumannii/patogenicidade , Pseudomonas aeruginosa/patogenicidade , Staphylococcus aureus/patogenicidade , Fatores de Virulência/efeitos da radiação , Acinetobacter baumannii/efeitos da radiação , Infecções Bacterianas/microbiologia , Epitélio/microbiologia , Células HaCaT , Hemólise/efeitos da radiação , Humanos , Luz , Modelos Biológicos , Pseudomonas aeruginosa/efeitos da radiação , Staphylococcus aureus/efeitos da radiação , Virulência/efeitos da radiaçãoRESUMO
Pseudomonas aeruginosa, a versatile bacterium present in terrestrial and aquatic environments and a relevant opportunistic human pathogen, is largely known for the production of robust biofilms. The unique properties of these structures complicate biofilm eradication, because they make the biofilms very resistant to diverse antibacterial agents. Biofilm development and establishment is a complex process regulated by multiple regulatory genetic systems, among them is quorum sensing (QS), a mechanism employed by bacteria to regulate gene transcription in response to population density. In addition, environmental factors such as UVA radiation (400-315 nm) have been linked to biofilm formation. In this work, we further investigate the mechanism underlying the induction of biofilm formation by UVA, analysing the role of QS in this phenomenon. We demonstrate that UVA induces key genes of the Las and Rhl QS systems at the transcriptional level. We also report that pelA and pslA genes, which are essential for biofilm formation and whose transcription depends in part on QS, are significantly induced under UVA exposure. Finally, the results demonstrate that in a relA strain (impaired for ppGpp production), the UVA treatment does not induce biofilm formation or QS genes, suggesting that the increase of biofilm formation due to exposure to UVA in P. aeruginosa could rely on a ppGpp-dependent QS induction.
Assuntos
Biofilmes/efeitos da radiação , Pseudomonas aeruginosa/fisiologia , Percepção de Quorum/fisiologia , 4-Butirolactona/análogos & derivados , 4-Butirolactona/genética , 4-Butirolactona/metabolismo , Biofilmes/crescimento & desenvolvimento , Regulação Bacteriana da Expressão Gênica/efeitos da radiação , Genes Bacterianos/genética , Guanosina Tetrafosfato/genética , Guanosina Tetrafosfato/metabolismo , Mutação , Polissacarídeos Bacterianos/genética , Polissacarídeos Bacterianos/metabolismo , Pseudomonas aeruginosa/efeitos da radiação , Percepção de Quorum/genética , Percepção de Quorum/efeitos da radiação , Transcrição Gênica/efeitos da radiação , Raios UltravioletaRESUMO
The dispersion of pollutants and proliferation of antibiotic resistant bacteria in the aquatic environment are an emerging health concern worldwide. In this sense, it is essential to develop new technologies to increase the quality of wastewater treatment, which is spread throughout the environment. The present study has demonstrated evidence of the existence of antibiotic and mercury-resistant bacteria in the aquatic environment. The application of heterogeneous photocatalysis with UVA/TiO2 P25 slurry (200â¯mgâ¯L-1), UVA/TiO2-immobilized, and UVA/TiO2-immobilized/H2O2 were evaluated for the simultaneous elimination of a mixture of contaminants of emerging concern (acetamiprid (ACP), imazalil (IMZ) and bisphenol A (BPA)) and inactivation of antibiotic and mercury-resistant bacteria (Pseudomonas aeruginosa and Bacillus subtilis). UVA/TiO2-immobilized/H2O2 increased the inactivation and elimination of the contaminants. After the combined treatment, the mixture of BPA, IMZ and ACP decreased 62%, 21% and <5%, respectively, after 300â¯minâ¯at 13.10â¯kJâ¯L-1 of accumulated UV energy. The Pseudomonas aeruginosa strain was inactivated after 120â¯min using 5.24â¯kJâ¯L-1 of accumulated UV energy, whereas the Bacillus subtilis strain was shown to be extremely resistant, with a capacity to develop mechanisms to avoid the oxidation process.
Assuntos
Farmacorresistência Bacteriana , Recuperação e Remediação Ambiental/métodos , Raios Ultravioleta , Águas Residuárias , Purificação da Água/métodos , Bactérias/efeitos dos fármacos , Bactérias/efeitos da radiação , Catálise , Farmacorresistência Bacteriana/efeitos dos fármacos , Farmacorresistência Bacteriana/efeitos da radiação , Peróxido de Hidrogênio/química , Processos Fotoquímicos , Pseudomonas aeruginosa/efeitos dos fármacos , Pseudomonas aeruginosa/efeitos da radiação , Titânio/química , Águas Residuárias/química , Águas Residuárias/microbiologia , Poluentes Químicos da Água/química , Poluentes Químicos da Água/efeitos da radiaçãoRESUMO
Persister bacteria tolerate bactericidal antibiotics due to transient and reversible phenotypic changes. As these bacteria can limit the effectiveness of antibiotics to eradicate certain infections, their elimination is a relevant issue. Photodynamic therapy seems suitable for this purpose, but phenotypic tolerance to it has also been reported for Pseudomonas aeruginosa. To test whether any phenotypic feature could confer tolerance against both antibiotics and photoinactivation, survivors from exposures to light in the presence of methylene blue were treated with ofloxacin, an antibiotic effective on nongrowing bacteria. Susceptibility to ofloxacin was normal in these bacteria in spite of their increased ability to survive photodynamic inactivation, suggesting the absence of cross-tolerance. It thus seemed possible to use one of these treatments to eliminate bacteria which had phenotypic tolerance to the other. To test this strategy, persister bacteria emerging from ofloxacin treatments were submitted to the action of light and methylene blue while the antibiotic remained in the bacterial suspension. Persisters lost their clonogenic ability under these conditions and the effects of the treatments seemed to be synergistic. These observations suggest that photodynamic antimicrobial therapy could be used as a complement to antibiotic treatments to eliminate persister bacteria from localized infections.
Assuntos
Biofilmes/efeitos da radiação , Azul de Metileno/farmacologia , Viabilidade Microbiana/efeitos da radiação , Fármacos Fotossensibilizantes/farmacologia , Pseudomonas aeruginosa/efeitos dos fármacos , Antibacterianos/farmacologia , Biofilmes/efeitos dos fármacos , Farmacorresistência Bacteriana/efeitos dos fármacos , Farmacorresistência Bacteriana/efeitos da radiação , Inibidores Enzimáticos/farmacologia , Luz , Viabilidade Microbiana/efeitos dos fármacos , Ofloxacino/farmacologia , Fotoquimioterapia , Pseudomonas aeruginosa/fisiologia , Pseudomonas aeruginosa/efeitos da radiaçãoRESUMO
Planktonic Pseudomonas aeruginosa cells harvested in stationary phase were exposed to red light in the presence of methylene blue to study the potential occurrence of persistence in bacterial populations submitted to photodynamic antimicrobial therapy. Survival curves revealed the existence of small subpopulations of cells exhibiting increased ability to tolerate the treatment. These subpopulations were detected even using high concentrations of photosensitizer, whether added in a single step or following a fractionated scheme, and when the irradiation medium was modified to delay the photodecomposition of methylene blue. When cells grown from survivors to the treatment were cultured and exposed to red light and dye, their responses were similar to that of the original strain. These results exclude exhaustion of the photosensitizer and selection of resistant mutants as explanations for the features of the survival curves. Cells able to tolerate the treatment were found even when radiation was imparted at a high-dose rate. They exhibit a response typical of persisters, which tolerate antimicrobial agents due to transient and reversible changes in their phenotype, suggesting that persistence is a factor to consider upon evaluating the efficacy of photodynamic antimicrobial therapy.
Assuntos
Luz , Azul de Metileno/farmacologia , Viabilidade Microbiana/efeitos dos fármacos , Viabilidade Microbiana/efeitos da radiação , Pseudomonas aeruginosa/efeitos dos fármacos , Pseudomonas aeruginosa/efeitos da radiação , Inibidores Enzimáticos/farmacologia , LasersRESUMO
Solar UVA radiation is one of the main environmental stress factors for Pseudomonas aeruginosa. Exposure to high UVA doses produces lethal effects by the action of the reactive oxygen species (ROS) it generates. P. aeruginosa has several enzymes, including KatA and KatB catalases, which provide detoxification of ROS. We have previously demonstrated that KatA is essential in defending P. aeruginosa against high UVA doses. In order to analyse the mechanisms involved in the adaptation of this micro-organism to UVA, we investigated the effect of exposure to low UVA doses on KatA and KatB activities, and the physiological consequences. Exposure to UVA induced total catalase activity; assays with non-denaturing polyacrylamide gels showed that both KatA and KatB activities were increased by radiation. This regulation occurred at the transcriptional level and depended, at least partly, on the increase in H2O2 levels. We demonstrated that exposure to low UVA produced a protective effect against subsequent lethal doses of UVA, sodium hypochlorite and H2O2. Protection against lethal UVA depends on katA, whilst protection against sodium hypochlorite depends on katB, demonstrating that different mechanisms are involved in the defence against these oxidative agents, although both genes can be involved in the global cellular response. Conversely, protection against lethal doses of H2O2 could depend on induction of both genes and/or (an)other defensive factor(s). A better understanding of the adaptive response of P. aeruginosa to UVA is relevant from an ecological standpoint and for improving disinfection strategies that employ UVA or solar irradiation.
Assuntos
Adaptação Fisiológica/fisiologia , Catalase/metabolismo , Peróxido de Hidrogênio/farmacologia , Oxidantes/farmacologia , Estresse Oxidativo/efeitos da radiação , Pseudomonas aeruginosa/efeitos da radiação , Hipoclorito de Sódio/farmacologia , Adaptação Fisiológica/genética , Regulação Bacteriana da Expressão Gênica/efeitos da radiação , Peróxido de Hidrogênio/metabolismo , Oxirredução/efeitos da radiação , Pseudomonas aeruginosa/genética , Pseudomonas aeruginosa/metabolismo , Raios UltravioletaRESUMO
Low-level laser therapy (LLLT) is used in chronic wounds due to its healing effects. However, bacterial species may colonize these wounds and the optimal parameters for effective bacterial inhibition are not clear. The aim of this study was to analyze the effect of LLLT on bacterial growth in vitro. Bacterial strains including Staphylococcus aureus, Escherichia coli, and Pseudomonas aeruginosa were suspended in saline solution at a concentration of 10(3) cells/ml and exposed to laser irradiation at wavelengths of 660, 830, and 904 nm at fluences of 0 (control), 3, 6, 12, 18, and 24 J/cm(2). An aliquot of the irradiated suspension was spread on the surface of petri plates and incubated at 37 °C for quantification of colony-forming unit after 24, 48, and 72 h. Laser irradiation inhibited the growth of S. aureus at all wavelengths and fluences higher than 12 J/cm(2), showing a strong correlation between increase in fluence and bacterial inhibition. However, for P. aeruginosa, LLLT inhibited growth at all wavelengths only at a fluence of 24 J/cm(2). E. coli had similar growth inhibition at a wavelength of 830 nm at fluences of 3, 6, 12, and 24 J/cm(2). At wavelengths of 660 and 904 nm, growth inhibition was only observed at fluences of 12 and 18 J/cm(2), respectively. LLLT inhibited bacterial growth at all wavelengths, for a maximum of 72 h after irradiation, indicating a correlation between bacterial species, fluence, and wavelength.
Assuntos
Escherichia coli/efeitos da radiação , Terapia com Luz de Baixa Intensidade , Pseudomonas aeruginosa/efeitos da radiação , Úlcera Cutânea/microbiologia , Staphylococcus aureus/efeitos da radiação , Escherichia coli/fisiologia , Humanos , Raios Infravermelhos , Pseudomonas aeruginosa/fisiologia , Úlcera Cutânea/radioterapia , Staphylococcus aureus/fisiologia , CicatrizaçãoRESUMO
OBJECTIVE: The purpose of this study was to analyze the influence of blue laser on bacterial growth of the main species that usually colonize cutaneous ulcers, as well as its effect over time following irradiation. BACKGROUND DATA: The use of blue laser has been described as an adjuvant therapeutic method to inhibit bacterial growth, but there is no consensus about the best parameters to be used. METHODS: Strains of Staphylococcus aureus ATCC 25923, Pseudomonas aeruginosa ATCC 27853, and Escherichia coli ATCC 25922 were suspended in saline solution at a concentration of 1.5×10(3) colony forming units (CFU)/mL. Next, 300 µL of this suspension was transferred to a microtitulation plate and exposed to a single blue laser irradiation (450 nm) at fluences of 0 (control), 3, 6, 12, 18, and 24 J/cm(2). Each suspension was spread over the surface of a Petri plate before being incubated at 37°C, and counts of CFU were determined after 24 and 48 h. RESULTS: Blue laser inhibited the growth of S. aureus and P. aeruginosa at fluences >6 J/cm(2). On the other hand, E. coli was inhibited at all fluences tested, except at 24 J/cm(2). CONCLUSIONS: Blue laser light was capable of inhibiting bacterial growth at low fluences over time, thus presenting no time-dependent effect.