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Photodynamic Inactivation of Acinetobacter baumannii Using Phenothiazinium Dyes: In Vitro and In Vivo Studies
Abstract
Phenothiazinium dyes have been reported to be effective photosensitizers inactivating a wide range of microorganisms in vitro after illumination with red light. However, their application in vivo has not extensively been explored. This study evaluates the bactericidal activity of phenothiazinium dyes against multidrug-resistant Acinetobacter baumannii both in vitro and in vivo.
We report the investigation of toluidine blue O, methylene blue, 1,9-dimethylmethylene blue, and new methylene blue for photodynamic inactivation of multidrug-resistant A. baumannii in vitro. The most effective dye was selected to carry out in vivo studies using third-degree mouse burns infected with a bioluminescent A. baumannii strain, upon irradiation with a 652 nm noncoherent light source. The mice were imaged daily for 2 weeks to observe differences in the bioluminescence-time curve between the photodynamic therapy (PDT)-treated mice in comparison with untreated burns.
All the dyes were effective in vitro against A. baumannii after 30 J/cm(2) irradiation of 635 or 652 nm red light had been delivered, with more effective killing when the dye remained in solution. New methylene blue was the most effective of the four dyes, achieving a 3.2-log reduction of the bacterial luminescence during PDT in vivo after 360 J/cm(2) and an 800 microM dye dose. Moreover, a statistically significant reduction of the area under the bioluminescence-time curve of PDT-treated mice was observed showing that the infection did not recur after PDT.
Phenothiazinium dyes, and especially new methylene blue, are potential photosensitizers for PDT to treat burns infected with multidrug-resistant A. baumannii in vivo.
... These include ; a broad spectrum of action, efficient inactivation of antibiotic-resistant strains, low mutagenic potential, and lack of selection of photo-resistant microbial cells [32,33]. Three different classes of compounds have been used as photosensitisers to inactivate bacteria: (1) phenothiazinium salts (2) tetrapyrroles such as phthalocyanines and porphyrins with cationic charges and (3) conjugates between tetrapyrroles with cationic polymers [7]. Phenothiazinium salts possess one intrinsic quaternary nitrogen atom and have been used as photosensitisers to kill tumour cells in vitro [34]. ...
... In the present study, we evaluated the in vitro antimicrobial photodynamic effect mediated by Methylene blue and Protoporphyrin IX as photosensitizers, using a halogen lamp as a light source on A. baumannii biofilms in order to demonstrate for the first time, the in vitro photodynamic inactivation of multi-drug-resistant A. baumannii biofilm using Protoporphyrin IX and Methylene blue. A number of studies have been performed on the effect of Methylene blue and Protoporphyrin IX on A. baumannii [7]. However, to the best of our knowledge there has not been any investigation comparing the effect of these photosensitizers on isolates obtained from clinical and extra-hospital (livestock and abattoir dam) sources. ...
... The antimicrobial effect of antimicrobial photodynamic inactivation proportionally increases (P < 0.05) with Methylene blue and Protoporphyrin IX concentrations. In a similar study using phenothiazinium dyes, Ragàs et al. reported 3 log reduction of bacterial viability by Methylene blue with 10 μM concentration and 22.5 Jcm −2 light irradiation at 652 nm [7]. They also surveyed the in vivo effect of antimicrobial photodynamic inactivation against A. baumannii from a burn wound. ...
... These dyes can be activated with an optical source within the visible red spectrum (630-670 nm) or infrared range (810 nm) [28,29]. Many studies have shown the bactericidal effects of aPDT using photochemical effects as an adjunctive therapy, along with conventional treatments without any damage to the titanium implant surface [30][31][32]. Considering that none of the implant roughed surface decontamination methods have been definitely confirmed so far, the aim of this in-vivo study was to evaluate the effect of five different antimicrobial methods in reducing the bacteria sticking to SLA titanium surfaces. ...
... It was ultimately washed by sterile normal saline (5 ml) (14) 5-aPDT Group After performing mechanical debridement with plastic curette, each disc was immersed in a container containing the photosensitizer Lasers HF Paro-PDT (Hager & Werken, Gmbh&Co.KG DuiSburg, Germany), along with tolonium chloride, for 3 min, according to the manufacturer's instruction. Then, it was exposed to a 660-nm laser diode (, ASTAR Co, Bielsko-Biala, Poland) polaris for 1 min and was set perpendicular to the disk surface with a power of 40 mw [31]. The probe tip was kept at one cm from the surface of each disk in such way that the spot size of 6 mm in diameter and the energy density of 5 J/cm2 were created on the discsurface. ...
... According to pairwise comparison of the therapeutic methods in the present study, the aPDT was and there is no even significant difference between the aPDT and Er: YAG methods in terms of their OD level in the aerobic bacteria group (P = 1). This result suggests that the aPDT method used in conjunction with a mechanical method (plastic curette) is capable of removing bacteria from oral biofilm-contaminated titanium surfaces, which is consistent with some previous studies [30,31]. In the present study, we used a PS containing a tolonium chloride compound that is activated with a 660-nm diode laser. ...
Introduction:
A main challenge in treatment of peri-implant disease is the effective decontamination of the implant surface. This challenge has always been a problem, associated with the treatment of these diseases with regard to the difficulty in removing and eliminating bacterial biofilm from the surface of dental implants, especially rough surfaces. The aim of this in-vivo study was to evaluate the effect of five different antimicrobial methods in reducing bacteria adhering to titanium surfaces.
Materials and methods:
In the present in-vivo study, the contaminated discs, except for the negative control group, randomly underwent one of five treatments: Erbium: Yattrium Aluminum Garnet (Er-YAG) laser, plastic curette, 0.12% chlorhexidine, aPDT, and 810 nm diode laser. A pectrophotometer was used to measure Optical Density (OD) in case of aerobic microorganisms. Colony-Forming Units (CFUs) were used for anaerobic bacteria. Then, all the analyses were carried out at a significance level of α = 0.05 through SPSS software.
Findings:
One-way analysis of variance (ANOVA) of aerobic bacteria showed a significant difference among 6 groups in terms of OD variations during a 0-24 h time interval (P < 0.001). The results of Kruskal-Wallis test were used to investigate the effect of study methods on anaerobic bacteria after 48 h, and the results showed a significant difference among 6 groups in terms of CFUs (P < 0.001).
Conclusion:
The results of the present study showed that all five mechanicals (plastic curette), chemical (CHX), laser (810 nm diode and Er: YAG), and aPDT methods could reduce oral biofilms from roughed surfaces of titanium discs. Er: YAG laser and plastic curette had the highest and the lowest effects respectively.
... Ninety females BALB/c mice weighing (18)(19)(20)(21)(22)(23)(24)(25)(26)(27)(28)(29)(30)(31)(32) gram were enrolled in the study.Animals were kept in individual plastic cage in hygiene conditions with wood chip bedding and maintained at 22cº in day/night light cycle and fed with standard pelted laboratory diet and had water ad libidum. The study was approved by the animal house of National center for drugs control researches committee/Iraq. ...
... Swab sample were taken from wound areas of patients whose wound infection withA.baumannii suspected(using sterile swabs in transport media).These samples were collected from patients hospitalized at AL-Yermook teaching hospital in the Baghdad during period from February 2015 to march 2015.A. baumanniiis isolated and identified using microscopic, cultural characteristic, biochemical test, and API system.One isolate of A.Baumanniiwas selected according to the resistance test to several antibiotics .Standard of suspension of Bacterial growth with dilution of (10⁻⁶ viable cells/ml) was chosen from the other serial dilution from Acinetobacter Baumannii [18]. Immediately after the creation of wound, a bacterial suspension containing 10⁸ cells in 50 µl sterile normal saline was inoculated on the surface of each wound with a pipette tip and then was smeared on to the wound surface with an inoculating loop. ...
... Immediately after the creation of wound, a bacterial suspension containing 10⁸ cells in 50 µl sterile normal saline was inoculated on the surface of each wound with a pipette tip and then was smeared on to the wound surface with an inoculating loop. [18] 2.8Antibiotic susceptibility test: ...
Despite a lot of researchers were suggested that cold laser therapy can enhance healing by reducing inflammation, increase cells proliferation, enhanced collagen synthesis, its use still controversial .The optimum parameters have been still not defined .we aimed to study the effect of cold laser therapy at a constant power density (Irradiance) at different illumination time (5,15minutes) on healing of noninfectedand infected wounds. Ninety animals with cutaneous incisional wound were divided into 12 irradiation groups, six groups for each noninfected and infected animal, and 30 animals as nonirradiated controls divided into noninfected and infected groups. All groups irradiated with red Laser light 635nm with constant irradiance 9.0 mw/cm², power= 60mw, continuous wave started immediately after surgery and repeated every day for 3,5,10 days using lens expander to cover the wound area (2.7J/cm²) for 5 minutes and (8 J/cm²) for 15 minutes. Wound healing was studied by calculating the percentage of wound closure and histological evaluation. The animals were killed either at 3, 5, or 10 days after irradiation. Specimen were taken, embedded in paraffin and sectioned, stained for histological analysis. Cold laser therapy clearly stimulates wound contraction, granulation tissue, collagen formation and reduces inflammation in both irradiated groups (infected and noninfected) groups .Polymorph nuclear infiltrate was lower in both noninfected irradiated groups at (2.7J/cm², 8.0J/cm²) compared with the control. The synthesis of collagen was enhanced in both noninfected groups compared with noninfected controls. Significant difference in the newly formed granulation tissue in the irradiated groups was recorded on five days after injury compared to noninfectedcontrols. On five days, the response of non infected wounds at (8.0J/cm²) was more than the group exposed at (2.7J/ cm²). On day 10, good response was noted in both irradiated noninfected groups compared to controls. On day 5, the irradiated groups were more responsive at (8.0J/cm²) compared to infected controls. At day10, infected irradiated groups had complete healing compared to incomplete healing in infected control group. We concluded that the most important parameter in determining theoptimal light delivering regimen is irradiated or illuminated time.
... While originally developed as an antimalarial drug [96], its absorption potential at 660 nm makes it an efficient photosensitizer that can generate significant quantities of 1 O 2 . There are numerous studies that have exploited this to treat a multitude of different pathogenic microbes in vitro [97][98][99][100], in vivo [101][102][103], and even clinically in human patients [104][105][106]. In vitro, at radiant exposures as low as 10 J/cm 2 , coupled with concentrations of MB ≤ 150 uM, MB-PDT has been shown to significantly reduce the viabilities of Candida spp. ...
... Their findings suggested the potential applicability of MB-PDT in controlling localized infections. While these results may not necessarily be perfectly extrapolated towards human participants, numerous clinical studies have supported their applicability [103][104][105][106]112]. For example, MB-PDT was successfully used in two diabetic patients suffering from osteomyelitis of the phalanges of their feet, having not responded well to conventional antibiotic therapy [112]. ...
In recent years, with the increases in microorganisms that express a multitude of antimicrobial resistance (AMR) mechanisms, the threat of antimicrobial resistance in the global population has reached critical levels. The introduction of the COVID-19 pandemic has further contributed to the influx of infections caused by multidrug-resistant organisms (MDROs), which has placed significant pressure on healthcare systems. For over a century, the potential for light-based approaches targeted at combatting both cancer and infectious diseases has been proposed. They offer effective killing of microbial pathogens, regardless of AMR status, and have not typically been associated with high propensities of resistance development. To that end, the goal of this review is to describe the different mechanisms that drive AMR, including intrinsic, phenotypic, and acquired resistance mechanisms. Additionally, the different light-based approaches, including antimicrobial photodynamic therapy (aPDT), antimicrobial blue light (aBL), and ultraviolet (UV) light, will be discussed as potential alternatives or adjunct therapies with conventional antimicrobials. Lastly, we will evaluate the feasibility and requirements associated with integration of light-based approaches into the clinical pipeline.
... Chemical structures of non-porphyrinic PSs used in aPDT bioluminescence approach As mentioned before, bioluminescent pathogens were also explored to evaluate the photodynamic action of phenothiazinium dyes, which have been recognized as effective photosensitizers toward a wide range of microorganisms [ 65,66 ]. For instance, the photodynamic effect of several phenothiazinium dyes, such as toluidine blue O (TBO), methylene blue (MB), 1,9-dimethyl methylene blue (DMMB), and new methylene blue (NMB) (Fig. 4 ), was evaluated in the photoinactivation of multidrug-resistant A. baumannii, which was transformed with the lux CDABE operon (originally cloned from Photorhabdus luminescens) [ 67 ]. All phenothiazinium dyes were effective after irradiation with red light against A. baumannii, with NMB being the most effective one. ...
... The mice were imaged daily for 2 weeks to observe differences in the bioluminescence over time between the treated mice in comparison with untreated burns. The results showed a >3 log reduction in the bacterial luminescence in the aPDT-treated mice and a statistically significant difference between the bioluminescence of all the controls and of the aPDT-treated mice over time [ 67 ]. ...
The emergence of microbial resistance to antimicrobials among several common pathogenic microbial strains is an increasing problem worldwide. Thus, it is urgent to develop not only new antimicrobial therapeutics to fight microbial infections, but also new effective, rapid, and inexpensive methods to monitor the efficacy of these new therapeutics. Antimicrobial photodynamic therapy (aPDT) and antimicrobial blue light (aBL) therapy are receiving considerable attention for their antimicrobial potential and represent realistic alternatives to antibiotics. To monitor the photoinactivation process provided by aPDT and aBL, faster and more effective methods are required instead of laborious conventional plating and overnight incubation procedures. Bioluminescent microbial models are very interesting in this context. Light emission from bioluminescent microorganisms is a highly sensitive indication of their metabolic activity and can be used to monitor, in real time, the effects of antimicrobial agents and therapeutics. This chapter reviews the efforts of the scientific community concerning the development of in vitro, ex vivo, and in vivo bioluminescent bacterial models and their potential to evaluate the efficiency of aPDT and aBL in the inactivation of bacteria.
... Nevertheless, in this work despite that cationic porphyrins 1-4 have statistically similar ΦΔ, the PDI performance is quite different. It has been extensively confirmed that the generation of 1 O2 is essential for the PDI process [34,35]; however, it is not the only factor that determines the overall PDI efficiency [23]. In particular, the interaction of the PS with the cell membrane plays a major role. ...
... Nevertheless, in this work despite that cationic porphyrins 1-4 have statistically similar Φ ∆ , the PDI performance is quite different. It has been extensively confirmed that the generation of 1 O 2 is essential for the PDI process [34,35]; however, it is not the only factor that determines the overall PDI efficiency [23]. In particular, the interaction of the PS with the cell membrane plays a major role. ...
Photodynamic inactivation (PDI) is a non-antibiotic option for the treatment of infectious diseases. Although Gram-positive bacteria have been shown to be highly susceptible to PDI, the inactivation of Gram-negative bacteria has been more challenging due to the impermeability properties of the outer membrane. In the present study, a series of photosensitizers which contain one to four positive charges (1–4) were used to evaluate the charge influence on the PDI of a Gram-negative bacteria, Escherichia coli (E. coli), and their interaction with the cell membrane. The dose-response PDI results confirm the relevance of the number of positive charges on the porphyrin molecule in the PDI of E. coli. The difference between the Hill coefficients of cationic porphyrins with 1–3 positive charges and the tetra-cationic porphyrin (4) revealed potential variations in their mechanism of inactivation. Fluorescent live-cell microscopy studies showed that cationic porphyrins with 1–3 positive charges bind to the cell membrane of E. coli, but are not internalized. On the contrary, the tetra-cationic porphyrin (4) permeates through the membrane of the cells. The contrast in the interaction of cationic porphyrins with E. coli confirmed that they followed different mechanisms of inactivation. This work helps to have a better understanding of the structure-activity relationship in the efficiency of the PDI process of cationic porphyrins against Gram-negative bacteria.
... After wound excision, an inoculum of Staphylococcus aureus suspension containing 10 6 CFU/mL was immediately applied on the wound surface on each mouse in the fourth, fifth and sixth group using a sterile loop [36]. ...
Commiphora gileadensis (CG) is a small tree distributed throughout the Middle East. It
was traditionally used in perfumes in countries in this area. In Saudi Arabia, it was used to treat wounds burns and as an antidote to scorpion stings. This study aimed to evaluate the antimicrobial activity and cutaneous wound healing efficiency of the CG extracts using microbiological tests, rate of wound contraction and histopathological changes. CG pants were extracted using the methanol extraction technique; then, the methanolic extract was characterized using liquid chromatography coupled with mass spectrometry (LC–MS). Afterwards, a six-millimetre (mm) excision wound was induced in 60 male Balb/c mice. Mice were classified into two classes; each class consisted of three groups of 10 mice. In the non-infected wound class, the group I was assigned as control and received normal saline. Group II received gentamicin treatment, and group III treated with CG-methanolic extract. In the Staphylococcus aureus-infected class, group IV received normal saline, and groups V and VI were treated with gentamicin and CG-methanolic extract, respectively. The colonization of infected wounds was determined using colony-forming units (CFUs), and the percentage of wound contraction was measured in all groups. Finally, the histopathologic semi-quantitative determination of wound healing was evaluated by inflammatory cell infiltration, the presence of collagen fibres and granulation tissue, and the grade of re-epithelization. Composition analysis of the methanolic extract confirmed the presence of a high amount of ceramide (69%) and, to a lesser extent, hexosylceramide
(18%) and phosphatidylethanolamine (7%) of the total amount. Additionally, there was a statistically significant difference between the percentage of wound contraction in the CG-treated and control groups in both Staphylococcus aureus-infected and non-infected wounds (p < 0.01). The colonization of the infected wounds was lower in the group treated with CG than in the control group (p < 0.01). In both non-infected and infected wounds, the CG-treated group showed significant statistical differences in inflammatory cell infiltration, collagen fibres, re-epithelization and granulation tissue formation compared with the control group (p < 0.01). The CG extract possesses antibacterial and
anti-inflammatory properties that induce wound healing.
... Furthermore, these phenothiazinium dyes can bind to both Gram-negative and Gram-positive bacterial membrane components, conferring their ability as photosensitizers in aPDT. Ragás and coworkers demonstrated that A. baumannii is both in vitro and in vivo sensitive to phenothiazinium dyes (toluidine blue O, methylene blue, new methylene blue N, and 1, 9-dimethylmethylene blue chloride) exposed to red radiation (635 and 652 nm) [48]. Other authors demonstrated the effectiveness of aPDT by toluidine blue O exposed to red (632.8 ...
Burns are among the most debilitating and devastating forms of trauma and severity. Such injuries are influenced by infections, causing increased morbidity, mortality, and healthcare costs. Due to the emergence of multidrug-resistant infectious agents, efficient treatment of infections in burns is a challenging issue. Antimicrobial photodynamic therapy (aPDT) is a promising approach to inactivate infectious agents, including multidrug-resistant. In this review, studies on PubMed were gathered, aiming to summarize the achievements regarding the applications of antimicrobial photodynamic therapy for the treatment of infected burns. A literature search was carried out for aPDT published reports assessment on bacterial, fungal, and viral infections in burns. The collected data suggest that aPDT could be a promising new approach against multidrug-resistant infectious agents. However, despite important results being obtained against bacteria, experimental and clinical studies are necessary yet on the effectiveness of aPDT against fungal and viral infections in burns, which could reduce morbidity and mortality of burned patients, mainly those infected by multidrug-resistant strains.
... The direct photolytic inactivation with UV light allows a rather targeted damage of biological functions of the cell due to the absorption by the bacteria. 45 In contrast, photodynamic inactivation generates reactive oxygen species mainly within the bacteria (e.g., peroxides or singlet oxygen), which attack biologically important molecules in the immediate vicinity of the used photosensitizer. A well-explored photosensitizer which generates reactive oxygen species is methylene blue (MB). ...
The increasing development of antibiotic resistance in bacteria has been a major problem for years, both in human and veterinary medicine. Prophylactic measures, such as the use of vaccines, are of great importance in reducing the use of antibiotics in livestock. These vaccines are mainly produced based on formaldehyde inactivation. However, the latter damages the recognition elements of the bacterial proteins and thus could reduce the immune response in the animal. An alternative inactivation method developed in this work is based on gentle photodynamic inactivation using carbon nanodots (CNDs) at excitation wavelengths λex > 290 nm. The photodynamic inactivation was characterized on the nonvirulent laboratory strain Escherichia coli K12 using synthesized CNDs. For a gentle inactivation, the CNDs must be absorbed into the cytoplasm of the E. coli cell. Thus, the inactivation through photoinduced formation of reactive oxygen species only takes place inside the bacterium, which means that the outer membrane is neither damaged nor altered. The loading of the CNDs into E. coli was examined using fluorescence microscopy. Complete loading of the bacterial cells could be achieved in less than 10 min. These studies revealed a reversible uptake process allowing the recovery and reuse of the CNDs after irradiation and before the administration of the vaccine. The success of photodynamic inactivation was verified by viability assays on agar. In a homemade flow photoreactor, the fastest successful irradiation of the bacteria could be carried out in 34 s. Therefore, the photodynamic inactivation based on CNDs is very effective. The membrane integrity of the bacteria after irradiation was verified by slide agglutination and atomic force microscopy. The method developed for the laboratory strain E. coli K12 could then be successfully applied to the important avian pathogens Bordetella avium and Ornithobacterium rhinotracheale to aid the development of novel vaccines.
... The efficiency of MB-mediated PACT has also been confirmed on antibioticresistant polymicrobial biofilms of P. aeruginosa and MRSA in a maxillary sinus model [21]. In addition, several in vitro studies have assessed its antimicrobial efficacy against a range of bacteria commonly isolated from burn infections [22,23]. ...
Photodynamic antimicrobial chemotherapy (PACT) is a novel alternative antimicrobial therapy that elicits a broad mechanism of action and therefore has a low probability of generating resistance. Such properties make PACT ideally suited for utilization in localized applications such as burn wounds. The aim of this study was to determine the antimicrobial activity of MB and temoporfin against both a S. aureus isolate and a P. aeruginosa isolate in light (640 nm) and dark conditions at a range of time points (0–20 min). A Staphylococcus aureus isolate and a Pseudomonas aeruginosa isolate were treated in vitro with methylene blue (MB) and temoporfin under different conditions following exposure to light at 640 nm and in no-light (dark) conditions. Bacterial cell viability [colony-forming units (c.f.u.) ml ⁻¹ ] was then calculated. Against P. aeruginosa , when MB was used as the photosensitizer, no phototoxic effect was observed in either light or dark conditions. After treatment with temoporfin, a reduction of less than one log (7.00×10 ⁷ c.f.u. ml ⁻¹ ) was observed in the light after 20 min of exposure. However, temoporfin completely eradicated S. aureus in both light and dark conditions after 1 min (where a seven log reduction in c.f.u. ml ⁻¹ was observed). Methylene blue resulted in a loss of S. aureus viability, with a two log reduction in bacterial viability (c.f.u. ml ⁻¹ ) reported in both light and dark conditions after 20 min exposure time. Temoporfin demonstrated greater antimicrobial efficacy than MB against both the S. aureus and P. aeruginosa isolates tested. At 12.5 µM temoporfin resulted in complete eradication of S. aureus . In light of this study, further research into the validity of PACT, coupled with the photosensitizers (such as temoporfin), should be conducted in order to potentially develop alternative antimicrobial treatment regimes for burn wounds.
... The direct photolytic inactivation with UV light allows a rather targeted damage of biological functions of the cell due to the absorption by the bacteria. 45 In contrast, photodynamic inactivation generates reactive oxygen species mainly within the bacteria (e.g., peroxides or singlet oxygen), which attack biologically important molecules in the immediate vicinity of the used photosensitizer. A well-explored photosensitizer which generates reactive oxygen species is methylene blue (MB). ...
The increasing development of antibiotic resistance in bacteria has been a major problem for years, both in human and veterinary medicine. Prophylactic measures, such as the use of vaccines, are of great importance in reducing the use of antibiotics in livestock. These vaccines are mainly produced based on formaldehyde inactivation. However, the latter damages the recognition elements of the bacterial proteins and thus could reduce the immune response in the animal. An alternative inactivation method developed in this work is based on gentle photodynamic inactivation using carbon nanodots (CNDs) at excitation wavelengths λex > 290 nm. The photodynamic inactivation was characterized on the nonvirulent laboratory strain Escherichia coli K12 using synthesized CNDs. For a gentle inactivation, the CNDs must be absorbed into the cytoplasm of the E. coli cell. Thus, the inactivation through photoinduced formation of reactive oxygen species only takes place inside the bacterium, which means that the outer membrane is neither damaged nor altered. The loading of the CNDs into E. coli was examined using fluorescence microscopy. Complete loading of the bacterial cells could be achieved in less than 10 min. These studies revealed a reversible uptake process allowing the recovery and reuse of the CNDs after irradiation and before the administration of the vaccine. The success of photodynamic inactivation was verified by viability assays on agar. In a homemade flow photoreactor, the fastest successful irradiation of the bacteria could be carried out in 34 s. Therefore, the photodynamic inactivation based on CNDs is very effective. The membrane integrity of the bacteria after irradiation was verified by slide agglutination and atomic force microscopy. The method developed for the laboratory strain E. coli K12 could then be successfully applied to the important avian pathogens Bordetella avium and Ornithobacterium rhinotracheale to aid the development of novel vaccines.
... Thus, MB might have a potential to be used as a drug for colistin-resistant AB or to increase the colistin susceptibility of AB strains. There are several reports that have investigated the effects of MB on AB strains but these reports were not focused on colistin-resistant AB strains or they evaluated the effects of MB upon irradiation with a light source (photodynamic therapy) [44,45]. MB may be a promising agent during outbreaks of colistin-resistant AB. ...
Background Colistin is one of the last resort antibiotics used against carbapenem-resistant Acinetobacter baumannii (AB); however, colistin resistance has been reported recently. Methylene blue (MB) is used in microbiology for staining, and in medicine as an antidote drug. Here, we investigated antimicrobial effects of MB and Eosin Methylene blue (EMB) agar against colistin-resistant AB strains. Methods The AB ATCC 19606 strain and 31 AB clinical isolates were included in the study. In the first round, ATCC strain and a clinical isolate were transformed into colistin-resistant forms, using Li's method, with increasing colistin concentrations. At each step, new MICs were determined and subcultures were inoculated to EMB and sheep blood agar (SBA). The colistin MIC values of the subcultures were also determined using Mueller Hinton Agar (MHA) containing 14 µg/mL MB. In the second round, colistin resistant clones of all collected clinical isolates (n=31) were obtained and screened to investigate their susceptibility to EMB agar by inoculating on SBA and EMB agar. Results At the beginning, the MICs of two strains were 0.5 µg/mL. At the last stage, both MICs had risen to 64 µg/mL. Subpopulations with high colistin resistance (>=32 µg/mL) were inhibited by MB and EMB agar, but could grow well on SBA. In MHA plates containing MB, the MICs decreased to the 0.5 µg/mL level for colistin-susceptible or moderately resistant clones. Additionally, clones with high colistin resistance showed atypical colony morphology on SBA. In the second round, MICs of the colistin resistant clones of all clinical isolates rose to 8 µg/mL after colistin exposure and 35% of those clinical isolates were inhibited by EMB agar while they could grow on SBA. Conclusion Highly resistant strains were totally inhibited by the effect of MB and EMB agar, while the MICs of the susceptible and moderate resistant clones decreased. EMB agar and MB may have inhibitory effects against colistin-resistant AB strains and MB may have a potential to be used as an antimicrobial drug. Secondly, using only EMB agar for subculturing may cause missing of colistin-resistant strains and giving incorrect identification or antibiogram reports in clinical microbiology laboratories.
... Several PS have been studied for their ability to bind to bacteria and efficiently g e n e r a t e r e a c t i v e o x y g e n s p e c i e s ( R O S ) u p o n photostimulation [11]. ROS are formed through type I or II mechanisms and may inactivate several classes of microbial cells including Gram-negative bacteria such as A. baumannii, which are typically characterized by an impermeable outer cell membrane that contains endotoxins and blocks antibiotics, dyes, and detergents, protecting the sensitive inner membrane and cell wall [11][12][13]. Photosensitizers are light-sensitive molecules and should be biologically stable, photochemically active, and minimally toxic to tissues of the organism. The photosensitizers used include hematoporphyrin derivatives, phenothiazines (toluidine blue and methylene blue), cyanines, phytotherapeutic agents, and phthalocyanines [14]. ...
Infections caused by Acinetobacter baumannii have become a challenge for healthcare professionals because of the rapid increase in Gram-negative bacteria resistant to carbapenem antibiotics. The objective of this study was to evaluate the effect of antimicrobial photodynamic therapy (aPDT) against different strains of A. baumannii isolated from patients with infectious process and hospitalized at the intensive care unit of the hospitals of São Jose dos Campos, São Paulo. These isolates were obtained from the Valeclin Clinical Analysis Laboratory (SP, Brazil) and were tested for susceptibility to the carbapenems imipenem and meropenem by determination of the minimal inhibitory concentration (MIC) using the broth microdilution method. The strains susceptible and resistant to these antibiotics were submitted to aPDT using methylene blue and a low-level laser with a wavelength of 660 nm and fluence of 39.5 J/cm² (energy of 15 J and time of 428 s). The number of colony-forming units (CFU/mL) was analyzed by ANOVA and the Tukey test. The laboratory of origin of the clinical isolates identified 1.54% of 13,715 strains tested over a period of 8 months as A. baumannii. Among the A. baumannii isolates, 58% were resistant to carbapenems by the disk diffusion test. Susceptible isolates exhibited MIC of 0.5 to 1 μg/mL and resistant isolates of 64 to > 128 μg/mL. PDT reduced the number of A. baumannii cells for all isolates tested, with this reduction ranging from 63 to 88% for susceptible isolates and from 26 to 97% for resistant isolates. The percentage of viability was dependent on the strain analyzed. In conclusion, these data indicate that PDT could be an alternative strategy for the control of infections caused by carbapenem-resistant A. baumannii.
... There was no recurrence of bioluminescence in the five days after PDT. This can be contrasted with PDT studies in similar animal models of superficial infections using traditional type PS where substantial recurrence of bioluminescence signal in the succeeding days was definitely observed [50][51][52] . There are further experiments that should be carried out to confirm the findings of the present study. ...
Tetracyclines (including demeclocycline, DMCT, or doxycycline, DOTC) represent a class of dual-action antibacterial compounds, which can act as antibiotics in the dark, and also as photosensitizers under illumination with blue or UVA light. It is known that tetracyclines are taken up inside bacterial cells where they bind to ribosomes. In the present study, we investigated the photochemical mechanism: Type 1 (hydroxyl radicals); Type 2 (singlet oxygen); or Type 3 (oxygen independent). Moreover, we asked whether addition of potassium iodide (KI) could potentiate the aPDI activity of tetracyclines. High concentrations of KI (200–400 mM) strongly potentiated (up to 5 logs of extra killing) light-mediated killing of Gram-negative Escherichia coli or Gram-positive MRSA (although the latter was somewhat less susceptible). KI potentiation was still apparent after a washing step showing that the iodide could penetrate the E. coli cells where the tetracycline had bound. When cells were added to the tetracycline + KI mixture after light, killing was observed in the case of E. coli showing formation of free molecular iodine. Addition of azide quenched the formation of iodine but not hydrogen peroxide. DMCT but not DOTC iodinated tyrosine. Both E. coli and MRSA could be killed by tetracyclines plus light in the absence of oxygen and this killing was not quenched by azide. A mouse model of a superficial wound infection caused by bioluminescent E. coli could be treated by topical application of DMCT and blue light and bacterial regrowth did not occur owing to the continued anti biotic activity of the tetracycline.
... The results showed that the decreasing in the number of A. baumannii in plankton condition was 2-3log 10 reported after photodynamic inactivation with 2 photosensitizers of TBO and MB. 24 Kashef et al assessed the efficacy of photodynamic therapy on A. baumannii. The findings of the study showed the reducing in logarithmic growth of live cells after photodynamic inactivation with MB and TBO (TBO) for 5 strains of Acinetobacter baumannii was between (1.3log 10 ), (3.5-2.4log 10 ), (2.9-2.2log 10 ) and (2.6log 10 ). ...
... Methylene blue is a photosensitizer that has shown promising results in the treatment of tumors 32-34 and microbial infections. 35,36 It is known that methylene blue is able to interact with DNA strands and its binding modes have been well characterized by spectroscopic techniques [37][38][39] and theoretical simulations. 40,41 Lately, it has been shown that the environment has a significant influence on the intersystem crossing mechanism of methylene blue, which is 6 enhanced when methylene blue is intercalated into DNA, instead of in water solution. ...
Interactions between chromophores and biological environments may alter the electronic properties of the chromophores. A three-layered QM/QM/MM ONIOM scheme with electrostatic embedding is implemented to investigate the influence of an additional QM layer on excited-state calculations with respect to a standard QM/MM description. The implemented ONIOM scheme is employed to compute the electronic excitations of the photosensitizer methylene blue interacting with a solvated DNA double strand. It is shown that the additional quantum mechanical description of several nucleobases in the vertical energy calculations induces energy shifts in the excited states of methylene blue, compared to the energies of a traditional QM/MM scheme, where the solvated double strand is described fully classically. The energy shifts present an electrostatic component, caused by a charge redistribution of the environment, and an electronic-coupling component, originated by the mixing between the electronic bright state of methylene blue and a charge-transfer state between methylene blue and guanine. In addition, hydrogen bonding and stacking interactions are stronger when the environment is described quantum mechanically during the geometry optimizations than when it is fully described by molecular mechanics. These larger intermolecular interactions produce further energy shifts in the excitation energies of the photosensitizer.
Antimicrobial photodynamic therapy (aPDT) is one of the efficient and alternate therapeutic strategies to combat planktonic and biofilm cells of ESKAPE pathogens. This therapeutic method requires a photosensitizer, light, and oxygen. ESKAPE superbugs are responsible for most of the nosocomial infections and it needs urgent attention in healthcare system. aPDT is emerged as a safer and effective method against the elimination of resistant ESKPAE pathogens, as the therapy is not involved in the generation of antibiotic-resistant strains. Reactive oxygen species produced through type I and II photoreactions of aPDT has multiple cellular targets in the bacteria, i.e., protein, DNA, and lipids. ROS exerts oxidation of cellular targets where the photosensitizer is localized. There are several classes of photosensitizers tested and used against variety of pathogens, especially ESKAPE such as from the family of chlorins, porphyrins, phthalocyanines, and bacteriochlorins. Additionally, several photosensitizers of plant origin and nanoparticles are found to be efficient against ESKPAE pathogens. In comparison to Gram-negative pathogens, aPDT is more efficient against Gram-positive pathogens owing to their cell wall structure. Cationic and neutral photosensitizers are more efficient in the eradication of Gram-positive pathogens, while photosensitizers are modified for Gram-negative pathogens. Here, we discussed applications and effectiveness of different photosensitizers against pathogenic forms of ESKAPE group.
Opportunistic premise plumbing pathogens (OPPPs) form a group of microorganisms that normally live in water supply systems and have adapted especially well to the conditions in premise plumbing systems, and as such pose a threat to human health. Since the beginning of the 21st century, this threat has been escalating, and it is becoming increasingly evident that current water disinfection methods fall short in effectively controlling these pathogens. In researching new approaches to this emergency, phototherapy looks promising, especially one that combines photosensitizers, light, and oxygen, which is known as photodynamic inactivation (PDI). This review describes the main characteristics of the recognized (Pseudomonas aeruginosa, Legionella pneumophila, and Mycobacterium avium) and most important emerging OPPPs, and it offers a brief overview of current disinfection methods and their limitations in the fight against OPPPs. The principle and outcomes of PDI with endogenous and, in particular, exogenous photosensitizers are then explained and described through representative examples of PDI on recognized and emerging OPPPs and their biofilms. Finally, the prospects and future directions of PDI research in water disinfection and control of OPPPs are discussed.
Nanotechnology‐based phototherapies have drawn interest in the fight against cancer because of its noninvasiveness, high flexibility, and precision in terms of cancer targeting and drug delivery based on its surface properties and size. Phototherapy has made remarkable development in recent decades. Approaches to phototherapy, which utilize nanomaterials or nanotechnology have emerged to contribute to advances around nanotechnologies in medicine, particularly for cancers. A brief overviews of the development of photodynamic therapy as well as its mechanism in cancer treatment is provided. We emphasize the design of novel nanoparticles utilized in photodynamic therapy while summarizing the representative progress during the recent years. Finally, to forecast important future research in this area, we examine the viability and promise of photodynamic therapy systems based on nanoparticles in clinical anticancer treatment applications and briefly make mention of the elimination of all reactive metabolites pertaining to nano formulations inside living organisms providing insight into clinical mechanistic processes. Future developments and therapeutic prospects for photodynamic treatments are anticipated. Our viewpoints might encourage scientists to create more potent phototherapy‐based cancer therapeutic modalities.
This article is categorized under: Therapeutic Approaches and Drug Discovery > Nanomedicine for Oncologic Disease
ABSTRACT
The present study was conducted to take advantages of the experience of an environment-friendly city with a climate similar to
the climate of Baghdad, that was Masdar City, which located in the United Arab of Emirates. The study also tried to make benefit
of the sustainability applications that implemented in Masdar City and adopt them in Baghdad. It is an attempt to take the most
important environmental solutions that are used to improve the environmental situation of Masdar city by making the emissions
of CO2 and other toxic gases in the lowest levels that are accepted globally. in addition, make the dependence in electricity
production on renewable energy sources and reduce the dependence on polluting fossil fuels, that was based on a lot of
principles, such as, adoption of recycling principle by making benefit of the construction waste and try to be recycled to produce
new environment friendly construction materials, in addition, taking advantage of the sewage and waste to produce electricity
and also for collecting the dioxide carbon to reuse it for industrial purposes and finally for the production of water that used for
the purposes of cleaning and irrigation, the other principle was taking , advantage of solar energy, for several purposes, such as
production of electrical energy, filtering and sterilizing water for drinking purposes , as well as , using solar energy for heating
water for domestic purposes. The research reaches to all those principles by adopting an analyzing study for a collection of
scientific applications In pursuit of finding facts and recognizing the need to melt all sciences in one pot, especially that urban
planning since is a comprehensive science because it dealing with all matters relating to the city and its environment , that’s only
an attempt to develop solutions for the environmental problems of Baghdad, which that : the problem of air pollution , the
problem of the scarcity of drinking water , the problem of the deficit in the wastewater and waste treatment in addition to the
problem of acute shortage of generation and providing electricity , all those problems and more resulted the bad environmental
situation of Baghdad that reflected Negatively on the health of the people who live in the city and that seems clear in Statistical
data. Accordingly, the research has suggested a range of solutions to improve the environmental situation of Baghdad by utilizing
from the environmental applications used in Masdar City.
Nontuberculous mycobacterial skin and soft tissue infections are rising and are causing social concern due to the growth of cosmetic dermatology and immune-compromised populations. For the treatment of nontuberculous mycobacteria, several novel strategies have been investigated. One of them, photodynamic therapy, is a recently developed therapeutic strategy that has shown promise in managing nontuberculous mycobacterial skin and soft tissue infections. In this review, we first present an overview of the current status of the therapy and then summarize and analyze the cases of photodynamic therapy used to treat nontuberculous mycobacterial skin and soft tissue infections. We also discussed the feasibility of photodynamic therapy for treating nontuberculous mycobacterial skin soft tissue infections and the related mechanisms, providing a new option for clinical treatment.
Herein, the interaction mechanism of new methylene blue (NMB) with human serum albumin (HSA) and bovine serum albumin (BSA) was carefully investigated both experimentally and conceptually, employing experimental and insilico analysis. The steady-state emission spectral studies showed that the emission intensity of HSA and BSA was quenched significantly by NMB. The findings of the Stern-Volmer and double logarithmic plot revealed that the observed emission quenching process was through a static quenching mechanism and the measured binding constant values (Kb) for HSA-NMB and BSA-NMB are 2.766 and 1.187 × 105 dm3 mol-1 respectively. The time-resolved fluorescence lifetime measurement and UV-vis absorption investigation further verify the complex formation between NMB and HSA/BSA. The assessment of thermodynamic parameters disclosed the binding process was spontaneous driven by hydrogen bonds (H-bond) and van der Waals interactions, which contributed a significant role in the complexation. Moreover, the secondary structural conformation and microenvironment of HSA/BSA were modified in the presence of NMB, as evidenced by circular dichroism and synchronous fluorescence data. Molecular docking study predicted a plausible binding mode of NMB inside the binding pocket of HSA and BSA. These results demonstrated that the stabilized NMB is found at the Subdomain IIA (site I) of both the proteins and the results were correlated well with the competitive binding assay. Additionally, the principal components analysis revealed less variation of docked poses for HSA, while, more dispersed docked poses were observed for the BSA model. This also highlights the effects of docking towards a modeled protein (BSA). Molecular dynamic (MD) simulation based binding free energy (ΔGmmgbsa) estimation obtained at 298, 303, 308 and 313 K, were in good agreement with our experimental (ΔGbind) values.
Chromoblastomycosis is a fungal disease presented with local warty papule, plaque, and verrucous nodules. In addition, the incidence and drug resistance of chromoblastomycosis are increasing each year worldwide. Photodynamic therapy is a promising method to treat mycoses. The purpose of this study was to evaluate the effect of new methylene blue (NMB)-induced PDT on multidrug-resistant chromoblastomycosis in vitro. We isolated one wild-type strain pathogen from one clinical patient diagnosed with chromoblastomycosis for over 27 years. The pathogen was identified by histopathology, the morphology of fungal culture, and genetic testing. Drug susceptibility testing was performed on the isolate. It was cultured with logarithmic growth phase spore in vitro and incubated with different concentrations of NMB for 30 min, and received illumination by red light-emitted diode with different light doses. After photodynamic treatment, the scanning electron microscopy (SEM) and transmission electron microscopy (TEM) were conducted. The pathogen was Fonsecaea nubica, and it was resistant to itraconazole, terbinafine, amphotericin B, voriconazole andcaspofungin. At the same NMB concentration, the sterilization efficiency of NMB-photodynamic therapy (PDT) on F. nubica increased with increasing light intensity; F. nubica was completely killed at 25 µmol/L NMB with a light dose of 40 J/cm2 or 50 µmol/L NMB and light doses of ≥ 30 J/cm2. SEM and TEM observed ultrastructural changes after PDT. NMB-PDT inactivates the survival of multidrug-resistant F. nubica in vitro; it therefore has the potential to become an alternative or adjuvant treatment for refractory chromoblastomycosis.
Antimicrobial photodynamic therapy (APDT) is a promising approach to fight the growing problem of antimicrobial resistance that threatens health care, food security and agriculture. APDT uses light to excite a light-activated chemical (photosensitiser), leading to the generation of reactive oxygen species (ROS). Many APDT studies confirm its efficacy in vitro and in vivo against bacteria, fungi, viruses and parasites. However, the development of the field is focused on exploring potential targets and developing new photosensitisers. The role of light, a crucial element for ROS production, has been neglected. What are the main parameters essential for effective photosensitiser activation? Does an optimal light radiant exposure exist? And finally, which light source is best? Many reports have described the promising antibacterial effects of APDT in vitro, however, its application in vivo, especially in clinical settings remains very limited. The restricted availability may partially be due to a lack of standard conditions or protocols, arising from the diversity of selected photosensitising agents (PS), variable testing conditions including light sources used for PS activation and methods of measuring anti-bacterial activity and their effectiveness in treating bacterial infections. We thus sought to systematically review and examine the evidence from existing studies on APDT associated with the light source used. We show how the reduction of pathogens depends on the light source applied, radiant exposure and irradiance of light used, and type of pathogen, and so critically appraise the current state of development of APDT and areas to be addressed in future studies. We anticipate that further standardisation of the experimental conditions will help the field advance, and suggest key optical and biological parameters that should be reported in all APDT studies. More in vivo and clinical studies are needed and are expected to be facilitated by advances in light sources, leading to APDT becoming a sustainable, alternative therapeutic option for bacterial and other microbial infections in the future.
Many studies show that photodynamic inactivation (PDI) is a powerful tool for the fight against pathogenic, multi‐resistant bacteria and the closing of hygiene gaps. However, PDI studies have been frequently performed under standardized in vitro conditions comprising artificial laboratory settings. Under real life conditions, however, PDI encounters substances like ions, proteins, amino acids, and fatty acids, potentially hampering the efficacy PDI to an unpredictable extent. Thus, we investigated PDI with the phenalene‐1‐one based photosensitizer SAPYR against Escherichia coli and Staphylococcus aureus in the presence of calcium or magnesium ions, which are ubiquitous in potential fields of PDI applications like in tap water or on tissue surfaces. The addition of citrate should elucidate the potential as a chelator. The results indicate that PDI is clearly affected by such ubiquitous ions depending on its concentration and the type of bacteria. The application of citrate enhanced PDI especially for Gram‐negative bacteria at certain ionic concentrations (e.g. CaCl2 or MgCl2: 7.5 to 75 mmol l‐1). Citrate also improved PDI efficacy in tap water (especially for Gram‐negative bacteria) and synthetic sweat solution (especially for Gram‐positive bacteria). In conclusion, the use of chelating agents like citrate may facilitate the application of PDI under real life conditions.
Photodynamic inactivation of microorganisms (PDI) finds use in a variety of applications. Several studies report on substances enhancing or inhibiting PDI. In this study, we analyzed the inhibitory potential of ubiquitous salts like CaCl2 and MgCl2 on PDI against Staphylococcus aureus and Pseudomonas aeruginosa cells using five cationic photosensitizers methylene blue, TMPyP, SAPYR, FLASH-02a and FLASH-06a.
TMPyP changed its molecular structure when exposed to MgCl2, most likely due to complexation. CaCl2 substantially affected singlet oxygen generation by MB at small concentrations. Elevated concentrations of CaCl2 and MgCl2 impaired PDI up to a total loss of bacterial reduction, whereas CaCl2 is more detrimental for PDI than MgCl2. Binding assays cannot not explain the differences of PDI efficacy. It is assumed that divalent ions tightly bind to bacterial cells hindering close binding of the photosensitizers to the membranes. Consequently, photosensitizer binding might be shifted to outer compartments like teichoic acids in Gram-positives or outer sugar moieties of the LPS in Gram-negatives, attenuating the oxidative damage of susceptible cellular structures.
In conclusion, CaCl2 and MgCl2 have an inhibitory potential at different phases in PDI. These effects should be considered when using PDI in an environment that contains such salts like in tap water or different fields of food industry.
Antimicrobial photodynamic therapy (APDT) has been studied as a noninvasive therapy for treating cutaneous leishmaniasis to overcome challenges with current treatment, such as toxicity, resistance, and need for in‐patient hospital treatment. Organic light‐emitting diodes (OLEDs) have emerged as an attractive technology that can provide wearable light‐emitting materials that are conformable to human skin. This makes OLEDs ideal candidates for APDT by light‐bandages for ambulatory care. In this work, suitable OLEDs are successfully developed to match the absorbance of three photosensitizers: methylene blue, new methylene blue, and 1,9‐dimethyl‐methylene blue to inactivate two Leishmania species in vitro: Leishmania major and Leishmania amazonensis. Parasites are treated either by LED (20 mW cm⁻²) or OLED (6.5 mW cm⁻²) at increasing photosensitizer concentrations at a radiant exposure of 50 J cm⁻². 1,9‐Dimethyl‐methylene blue is the most potent photosensitizer, killing both strains at nanomolar concentrations. The effect of different intensities from the OLEDs (0.7, 1.5, and 6.5 mW cm⁻²) are also explored and it is shown that effective killing of Leishmania occurs even at a very low intensity. These findings demonstrate the great potential of OLEDs as a new approach for ambulatory treatment of cutaneous leishmaniasis by APDT.
Acinetobacter baumannii (A. baumannii) has emerged as a pathogen of global importance able to cause opportunistic infections on the skin, urinary tract, lungs, and bloodstream, being frequently involved in hospital outbreaks. Such bacterium can resist a variety of environmental conditions and develop resistance to different classes of antibiotics. Antimicrobial photodynamic therapy (aPDT) has been considered a promising approach to overcome bacterial resistance once it does not cause selective environmental pressure on bacteria. In this review, studies on aPDT were accessed on PubMed, and their findings were summarized regarding its efficacy against A. baumannii. The data obtained from the literature show that exogenous photosensitizers belonging to different chemical classes are effective against multidrug-resistant A. baumannii strains. However, most of such data is from in vitro studies, and additional studies are necessary to evaluate if the exogenous photosensitizers may induce selective pressure on A. baumannii and the effectiveness of such photosensitizers in clinical practice.
Background and aim
Antimicrobial photodynamic therapy (aPDT) has shown great potential for treatment of superficial or localized multidrug-resistant (MDR) Acinetobacter baumannii infections. The purpose of this study was to investigate the cytotoxicity and in vivo safety of aloe-emodin (AE), and its photodynamic treatment efficacy against MDR A. baumannii infections.
Methods
The cytotoxicity (dark toxicity) and phototoxicity of AE to human immortalized keratinocytes and mice fibroblasts were detected by CCK-8 kit. Low and high doses of AE were intravenously injected into mice to evaluate the safety of AE in vivo. Bioluminescent MDR A. baumannii strain was employed to establish the infection model on BALB/c mice after skin scald, and infection status and therapeutic effect of AE-mediated aPDT were assessed by animal imaging system. The peripheral blood of mice was analyzed by flow cytometer.
Results
AE had low cytotoxicity to human immortalized keratinocytes and mice fibroblasts, and had certain phototoxicity to these cells under light irradiation. The in vivo experiments demonstrated that AE caused no obvious effects on the weight and pathological changes of mice. AE-mediated aPDT was effective in the treatment of MDR A. baumannii caused infections in mice after skin scald.
Conclusions
AE has potential to be used in the photodynamic treatment of MDR A. baumannii caused superficial infections after scald.
Background:
Antimicrobial photodynamic therapy (aPDT) using methylene blue (MB) plus potassium iodide (KI) has been shown to be effective in killingCandida albicans in many in vitro and in vivo studies, however, there are limited reports of clinical investigations. This study aimed to explore the clinical application of aPDT with MB plus KI for the treatment of oral infection caused by C. albicans in adult acquired immune deficiency syndrome (AIDS) patients.
Methods:
A total of 21 adult AIDS patients with C. albicans oral candidiasis were divided into two groups according to MB concentration and received two consecutive aPDT treatments. Immediately after the aPDT treatments, C. albicans yeast isolates were recovered to measure the colony-forming units per mL (CFU/mL), biofilm formation, and to analyze the 25S rDNA genotype. Patients were assessed for the clinical recovery of oral lesions and improvement of symptoms.
Results:
The Log10 CFU/mL of C. albicans decreased significantly after the second aPDT but not the first aPDT. There was no significant difference between the two MB concentrations. Both aPDT protocols decreased the oral lesions and clinical symptoms with no significant difference after 2-time aPDT. The biofilm formation of C. albicans isolates did not change before and after aPDT. The killing efficiency of 2-time-aPDT was not associated with either biofilm formation or 25S rDNA genotype.
Conclusions:
Two-time-aPDT with MB plus KI could reduce the number of fungal colonies and improve the clinical symptoms of oral candidiasis in adult AIDS patients, regardless of the biofilm formation or 25S rDNA genotype.
According to the World Health Organization, up to 30% of cancer diseases may be related to nutritional factors. Although many dietary ingredients with protective (anti-cancer) effects are known, the health-promoting impact of spices remains underestimated. Rich in the bioactive compounds, they are essential ingredients in the diet of people with cancer disease around the world. Spices have antioxidant, anti-cancer, anti-inflammatory, antifungal, antibacterial action. They inhibit the bioactivation of carcinogens in the body. Attention should be paid to natural antioxidants –cloves, oregano, thyme, cinnamon, marjoram, cumin, and basil. Their chemopreventive properties have been extensively investigated and well documented. Curcumin, rosemary, and bay leaf show anti-inflammatory effects. Ginger, infusions of mint, chamomile, sage, and lemon balm help with ailments associated with cancer therapy. Herbs increase appetite and dishes' attractiveness in patients with a lack of appetite or impaired sense of taste caused by cancer therapy. Researchers suggest that phenolic acids in spices and herbs can increase the antioxidant capacity, affecting the bioactivity of the food consumed. The purpose of the article was to show the role of spices in the diet of cancer patients generally. At first, the role of oxidative stress and chronic inflammation in cancer formation was described. Then, recommendations for dietotherapy and the role of dietary counselling were presented, focusing on selected health problems arising as a result of cancer treatment. In the following, the chemopreventive properties of bioactive ingredients of selected spices were shown. Their impact on patients' health has been described through multidirectional mechanisms of influence, paying attention to practical (culinary) use.
The next book in the series Advances in Biomedical Research edited by I. Mlynarczuk-Bialy and L. Bialy. This book contains chapters dealing with cancer research.
TABLE OF CONTENTS
Assessment of dietary intake of polyphenols in the prevention of cancer; Is regular physical activity an effective method of cancer prevention?; Psychiatric disorders in cancer patients ; Female internet blogs as (self) help in the fight against malignant cancer ; Personalized Medicine: from molecular methods to targeted therapy in cancer; Spices – bioactive compounds in the diet of patients with cancer; Computational analysis of AP-2γ role in bladder cancer; Burkitt-like lymphoma with 11q aberration a provisional entity in the 2016 WHO classification of lymphoid malignancies;. Relationship between redox status in blood of patients with chronic myeloid leukemia (CML) and values of selected inflammatory markers - preliminary studies; Patient eligibility for chemotherapy in the geriatric population – how to best balance benefits and risks?; Hormonotherapy in gynecological cancers; Sinonasal malignancies- clinicopathological characteristics and difficulties in diagnostics; Antiemetics in oncology – current knowledge and problems with effectiveness; Contemporary sonography and ultrasound-based systems used for the discrimination of the benign and malignant endometrial lesions; Antimicrobial peptides and their bactericidal, immunomodulating and anticancer activities – literature mini-review; A short guide on the selection of melanocytes and melanoma cells’ isolation procedures for cancer research; Application of Clostridium spp. and their toxins/enzymes in treatment of oncologic and other pathologies; Tumor angiogenesis – therapeutic approach; Photodynamic therapy as alternative therapy for prostate cancer and colorectal carcinoma as well as an antimicrobial treatment – a systematic review
Hormones play a significant role in the functioning of the human organism, by regulating a number of processes, including tissue activity, cell proliferation and division. Disorders in hormonal balance may promote the development of so-called hormone-dependent or hormone-sensitive cancers.
Many gynecological malignancies are dependent on estrogen and/or progesterone (e.g. epithelial and stromal ovarian carcinomas or endometrial carcinomas and sarcomas). The discovery of the relationship between hormonal stimulation and tumour progression has led to the development of new therapeutic strategy, known as the antihormone therapy (AHT) for cancer. AHT is one of the type of hormone therapy (HT) and it is based on the supressing the activity of selected hormones and/or lowering these hormones levels in the human body. The use of the phrase 'hormonal therapy' in this case seems to be incorrect as the term is associated with hormone replacement therapy, which encourages hormone activity.
Three main courses of action can be distinguished in antihormone therapy:
1.) the reduction in secretion of sex hormones, as a result of the agonism of pituitary gonadotropin-releasing hormone (GnRH) receptors,
2.) the inhibition of the aromatase enzyme activity, based on conversion of androgens into estrogens,
3.) the inhibition of proliferations of cells containing estrogen receptors (ERs) by the prevention of estrogen binding to ERs located on the cancer cells surface. Main advantage of antihormone therapy is the fact that it is better tolerated by patients compared to conventional chemotherapy. However, the limitations of this approach should be remembered, e.g. interactions with other drugs and late therapeutic response. In this article authors present a review of achievements to date of antihormone therapy in the context of some oncological gynaecology and describe the AHT strategies listed above.
Drug delivery systems (DDS) can be designed to enrich the pharmacological and therapeutic properties of several drugs. Many of the initial obstacles that impeded the clinical applications of conventional DDS have been overcome with nanotechnology-based DDS, especially those formed by chitosan (CS). CS is a linear polysaccharide obtained by the deacetylation of chitin, which has potential properties such as biocompatibility, hydrophilicity, biodegradability, non-toxicity, high bioavailability, simplicity of modification, aqueous solubility, and excellent chemical resistance. Furthermore, CS can prepare several DDS as films, gels, nanoparticles, and microparticles to improve delivery of drugs, such as photosensitizers (PS). Thus, CS-based DDS are broadly investigated for photodynamic therapy (PDT) of cancer and fungal and bacterial diseases. In PDT, a PS is activated by light of a specific wavelength, which provokes selective damage to the target tissue and its surrounding vasculature, but most PS have low water solubility and cutaneous photosensitivity impairing the clinical use of PDT. Based on this, the application of nanotechnology using chitosan-based DDS in PDT may offer great possibilities in the treatment of diseases. Therefore, this review presents numerous applications of chitosan-based DDS in order to improve the PDT for cancer and fungal and bacterial diseases.
Aim:
The purpose of this study was to investigate how Enterococcus faecalis and Streptococcus mutans behave in mono and dual species biofilm after photodynamic treatment.
Background:
Antimicrobial photodynamic therapy (aPDT) leads to the generation of reactive oxygen species (ROS) that destroys bacterial cells in presence of a photosensitizer, visible light, and oxygen.
Material and methods:
We have taken Enterococcus faecalis and Streptococcus mutans as monospecies culture and their dualspecies culture biofilm. Antibacterial effect was evaluated by colony forming unit while antibiofilm action by crystal violet and congored binding assays.
Results:
We found that dual species biofilm are more resistant than monospecies biofilm and S. mutans shows dominance over E. faecalis in dual species biofilm, it inhibited the growth of E. faecalis in dual species biofilm. Antibiofilm efficacy of TBO also validated that dualspecies show less inhibition than monospecies biofilm this may be due to different EPS constitution in dualspecies biofilm, hence less inhibition was observed in EPS production of dualspecies biofilm than monospecies biofilm. Reactive oxygen species and singlet oxygen yield was found to be light dose dependent and antimicrobial photodynamic efficiency is directly related to the ROS production.
Conclusion:
We conclude that dual species biofilm shows resistance over monospecies biofilm and S. mutans in dual species inhibits the growth of E. faecalis.
Antibacterial Photodynamic therapy (APDT) is a process utilizing light and light sensitive agents (named photosensitizer (PS)) and is usually applied in an oxygen-rich environment. The energy of the photons is absorbed by the photosensitizer and subsequently transferred to surrounding molecules. Consequently, reactive oxygen species and free radicals are formed. These oxidative molecules can damage bacterial macromolecules such as proteins, lipids and nucleic acids and may result in bacterial killing. Unlike antibiotics, APDT as a novel technique does not lead to the selection of mutant resistant strains, hence it has appealed to researchers in this field. The type of PS used in APDT is a major determinant regarding outcome. In this review, various types of PS that are used in antimicrobial Photodynamic therapy will be discussed. PSs are classified based on their chemical structure and origin. Synthetic dyes such as methylene blue and toluidine blue are the most commonly used photosensitizers in Antibacterial Photodynamic therapy (APDT). Other photosensitizers including natural PSs (e.g. curcumin and hypericin) and tetra-pyrrole structures like phthalocyanines and porphyrins have also been studied. Furthermore, nanostructures and their probable contribution to APDT will be discussed.
effect of plant extracts on the biology of insects
The study included 2328 samples of patients who attended to Specialized Chest and Respiratory Disease Center/
National Reference Laboratory for Tuberculosis (NRL) in Baghdad in the period from (February 2015 to February
2016). These samples included (1806) of pulmonary TB patients and (522) Extra-pulmonary samples. Methods
included direct examination by Zeihl-Nelsen stain, then cultures were examined traditionally and been followed by
phenotypic-based identification methods.
Out of a total sample 2328 of different specimens, 351(15.08%) were positive by direct examination (smear
microscopy) and 433(18.6%) specimens were able to grow on solid media, and it was found that cultures had
detected 82 negative specimens by smear microscopy. Results showed that males were more affected significantly
1356 (58.25%) than females 972 (41.75%), and the higher percentage of positive culture was in Baghdad (12.07%)
than other governorates (6.53 %).
The most sites of specimens samples were sputum 1462 (62.8%) with high significant difference than other
samples 866 (37.2 %) and the total pulmonary TB was 1806 (77.58%) than total extra pulmonary TB 522
(22.42%). The ages of patients were ranged from <1year to > 60 years. Age range of 30-39 showed the highest
percentage of M. tuberculosis infection (28.47%), while the lowest recorded age group was observed in (0-9) years
with percentage (5.36%). More incidence was a previously treated Pulmonary Tuberculosis patients (old case)
(25.44%) with highly significant than new patients (new cases) (10.19%), but the higher percentage of positive
culture was in multidrug resistant (MDR) (32.51%).
As antibiotic resistance increases and antimicrobial options diminish, there is a pressing need to identify and develop new and/or alternative (non-antimicrobial-based) wound therapies. The authors describe the implications of antibiotic resistance on their current wound treatment paradigms and review the most promising non-antibiotic-based antimicrobial agents currently in research and development, with a focus on preclinical and human studies of therapeutic bacteriophages, antimicrobial peptides, cold plasma treatment, photodynamic therapy, honey, silver, and bioelectric dressings.
Abstract
Introduction: The purpose of this review was to evaluate the available literature for in vitro and in vivo effectiveness of antimicrobial Photodynamic therapy (aPDT) in the field of bacteriology.
Methods: A review of the relevant articles carried out in PubMed and Scopus to determine the efficiency of aPDT used in the reduction of microbial infection. Thirty-one relevant documents retrieved from PubMed, Scopus by inserting “antimicrobial photodynamic therapy” and “bacterial infection” and “photodynamic therapy” keywords.
Results: According to different results, aPDT can be used as an adjuvant for the treatment of infectious diseases. The use of photosensitizer methylene blue, toluidine blue O (TBO), indocyanine green with light diode laser centered at (630±10 nm) and (650±10 nm) wavelengths have been shown to have significant results for the treatment of infectious diseases and bactericidal properties
Conclusion: These findings suggest that, aPDT can be an efficient method in the treatment of localized and superficial infections.
Keywords: Antimicrobial photodynamic therapy; Photosensitizer; Skin infections.
Development of antimicrobial photodynamic therapy (aPDT) is highly dependent on the development of suitable photosensitizers (PS): ideally affinity of PS towards bacterial cells should be much higher than towards mammalian cells. Cationic charge of PS may lead to selective binding of PS to bacteria mediated by electrostatic interaction; however, the photodynamic outcome is highly dependent on the lipophilicity of PS. Herein we report the aPDT effect of silicon(IV)phthalocyanine derivatives bearing four positive charges and methyl, phenyl or naphthyl substituents on the periphery of the macrocycle. We show that via modulation of lipophilicity it is possible to find a therapeutic window where bacteria but not mammalian cells are effectively killed. The photobiological activity of these PSs dropped significantly when host-guest complexes of PSs with cucurbit[7]uril (CB[7]) were used. CB[7] blocks the hydrophobic part of the molecule and reduces lipophilicity of the PS, indicating that a hydrophobic interaction with the outer membrane of bacterial cells is essential for aPDT activity. Efficiencies of obtained PSs were evaluated using different uropathogenic E. coli isolates and human kidney epithelial carcinoma cells.
Traditional methods of localizing and quantifying the presence of pathogenic microorganisms in living experimental animal models of infections have mostly relied on sacrificing the animals, dissociating the tissue and counting the number of colony forming units. However the discovery of several varieties of the light producing enzyme, luciferase, and the genetic engineering of bacteria, fungi, parasites and mice to make them emit light, either after administration of the luciferase substrate, or in the case of the bacterial lux operon without any exogenous substrate, has provided a new alternative. Dedicated bioluminescence imaging (BLI) cameras can record the light emitted from living animals in real time allowing non-invasive, longitudinal monitoring of the anatomical location and growth of infectious microorganisms as measured by strength of the BLI signal. BLI technology has been used to follow bacterial infections in traumatic skin wounds and burns, osteomyelitis, infections in intestines, Mycobacterial infections, otitis media, lung infections, biofilm and endodontic infections and meningitis. Fungi that have been engineered to be bioluminescent have been used to study infections caused by yeasts (Candida) and by filamentous fungi. Parasitic infections caused by malaria, Leishmania, trypanosomes and toxoplasma have all been monitored by BLI. Viruses such as vaccinia, herpes simplex, hepatitis B and C and influenza, have been studied using BLI. This rapidly growing technology is expected to continue to provide much useful information, while drastically reducing the numbers of animals needed in experimental studies.
Objectives: We have shown previously that a polycationic conjugate between poly-L-lysine and the photosensitizer chlorin e6 was effective in photodynamic inactivation (PDI) of both Gram- positive and Gram-negative bacteria. In this report we explore the relationship between the size of the polylysine chain and its effectiveness for mediating the killing of Gram-negative and Gram-positive bacteria. Methods: Conjugates were prepared by attaching precisely one chlorin e6 molecule to the α-amino group of poly-(e-benzyloxycarbonyl)lysines of average length eight and 37 lysine residues, followed by deprotection of the e-amino groups, and were characterized by iso-electric focusing. The uptake of these conjugates and free chlorin e6 by Gram-positive Staphylococcus aureus (ATCC 27659) and Gram-negative Escherichia coli (ATCC 29181) after washing was measured as a function of photosensitizer concentration (0-4 µM chlorin e6 equivalent) and incubation time. After incubation the bacteria were exposed to low fluences (10-40 J/cm 2 ) of 660 nm light delivered from a diode laser, and viability was assessed after serial dilutions by a colony- forming assay. Results: S. aureus and E. coli took up comparable amounts of the two conjugates, but free chlorin e6 was only taken up by S. aureus. After illumination S. aureus was killed in a fluence- dependent fashion when loaded with the 8-lysine conjugate and free chlorin e6 but somewhat less so with the 37-lysine conjugate. In contrast, PDI of E. coli was only effective with the 37-lysine conjugate at concentrations up to 4 µM. PDI using the 8-lysine conjugate and free chlorin e6 on E. coli was observed at a concentration of 100 µM. Transmission electron micrographs showed internal electron-lucent areas consistent with chromosomal damage. Conclusion: These results can be explained by the necessity of a large polycation to penetrate the impermeable outer membrane of Gram-negative E. coli, while Gram-positive S. aureus is more easily penetrated by small molecules. However, because S. aureus is more sensitive overall than E. coli the 37-lysine conjugate can effectively kill both bacteria.
Background and Objectives
Photodynamic therapy (PDT) appears to be endowed with several favorable features for the treatment of infections originated by microbial pathogens, including a broad spectrum of action, the efficient inactivation of antibiotic-resistant strains, the low mutagenic potential, and the lack of selection of photoresistant microbial cells. Therefore, intensive studies are being pursued in order to define the scope and field of application of this approach.ResultsOptimal cytocidal activity against a large variety of bacterial, fungal, and protozoan pathogens has been found to be typical of photosensitizers that are positively charged at physiological pH values (e.g., for the presence of quaternarized amino groups or the association with polylysine moieties) and are characterized by a moderate hydrophobicity (n-octanol/water partition coefficient around 10). These photosensitizers in a micromolar concentration can induce a >4–5 log decrease in the microbial population after incubation times as short as 5–10 minutes and irradiation under mild experimental conditions, such as fluence-rates around 50 mW/cm2 and irradiation times shorter than 15 minutes.ConclusionsPDT appears to represent an efficacious alternative modality for the treatment of localized microbial infections through the in situ application of the photosensitizer followed by irradiation of the photosensitizer-loaded infected area. Proposed clinical fields of interest of antimicrobial PDT include the treatment of chronic ulcers, infected burns, acne vulgaris, and a variety of oral infections. Lasers Surg. Med. 38:468–481, 2006. © 2006 Wiley-Liss, Inc.
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.
The widespread problem of antibiotic resistance in pathogens such as Staphylococcus aureus has prompted the search for new antimicrobial approaches. In this study we report for the first time the use of a light-activated antimicrobial agent, methylene blue, to kill an epidemic methicillin-resistant Staphylococcus aureus (EMRSA-16) strain in two mouse wound models.
Following irradiation of wounds with 360 J/cm(2) of laser light (670 nm) in the presence of 100 microg/ml of methylene blue, a 25-fold reduction in the number of viable EMRSA was seen. This was independent of the increase in temperature of the wounds associated with the treatment. Histological examination of the wounds revealed no difference between the photodynamic therapy (PDT)-treated wounds and the untreated wounds, all of which showed the same degree of inflammatory infiltration at 24 hours.
The results of this study demonstrate that PDT is effective at reducing the total number of viable EMRSA in a wound. This approach has promise as a means of treating wound infections caused by antibiotic-resistant microbes as well as for the elimination of such organisms from carriage sites.
We have shown previously that a polycationic conjugate between poly-L-lysine and the photosensitizer chlorin(e6) was effective in photodynamic inactivation (PDI) of both Gram-positive and Gram-negative bacteria. In this report we explore the relationship between the size of the polylysine chain and its effectiveness for mediating the killing of Gram-negative and Gram-positive bacteria.
Conjugates were prepared by attaching precisely one chlorin(e6) molecule to the alpha-amino group of poly-(epsilon-benzyloxycarbonyl)lysines of average length eight and 37 lysine residues, followed by deprotection of the epsilon-amino groups, and were characterized by iso-electric focusing. The uptake of these conjugates and free chlorin(e6) by Gram-positive Staphylococcus aureus (ATCC 27659) and Gram-negative Escherichia coli (ATCC 29181) after washing was measured as a function of photosensitizer concentration (0-4 microM chlorin(e6) equivalent) and incubation time. After incubation the bacteria were exposed to low fluences (10-40 J/cm(2)) of 660 nm light delivered from a diode laser, and viability was assessed after serial dilutions by a colony-forming assay.
S. aureus and E. coli took up comparable amounts of the two conjugates, but free chlorin(e6) was only taken up by S. aureus. After illumination S. aureus was killed in a fluence-dependent fashion when loaded with the 8-lysine conjugate and free chlorin(e6) but somewhat less so with the 37-lysine conjugate. In contrast, PDI of E. coli was only effective with the 37-lysine conjugate at concentrations up to 4 microM. PDI using the 8-lysine conjugate and free chlorin(e6) on E. coli was observed at a concentration of 100 microM. Transmission electron micrographs showed internal electron-lucent areas consistent with chromosomal damage.
These results can be explained by the necessity of a large polycation to penetrate the impermeable outer membrane of Gram-negative E. coli, while Gram-positive S. aureus is more easily penetrated by small molecules. However, because S. aureus is more sensitive overall than E. coli the 37-lysine conjugate can effectively kill both bacteria.
Porphyromonas gingivalis is one of the major causative organisms of periodontitis and has been shown to be susceptible to toluidine blue-mediated photosensitization in vitro. The aims of the present study were to determine whether this technique could be used to kill the organism in the oral cavities of rats and whether this would result in a reduction in the alveolar bone loss characteristic of periodontitis. The maxillary molars of rats were inoculated with P. gingivalis and exposed to up to 48 J of 630-nm laser light in the presence of toluidine blue. The number of surviving bacteria was then determined, and the periodontal structures were examined for evidence of any damage. When toluidine blue was used together with laser light there was a significant reduction in the number of viable P. gingivalis organisms. No viable bacteria could be detected when 1 mg of toluidine blue per ml was used in conjunction with all light doses used. On histological examination, no adverse effect of photosensitization on the adjacent tissues was observed. In a further group of animals, after time was allowed for the disease to develop in controls, the rats were killed and the level of maxillary molar alveolar bone was assessed. The bone loss in the animals treated with light and toluidine blue was found to be significantly less than that in the control groups. The results of this study show that toluidine blue-mediated lethal photosensitization of P. gingivalis is possible in vivo and that this results in decreased bone loss. These findings suggest that photodynamic therapy may be useful as an alternative approach for the antimicrobial treatment of periodontitis.
Antimicrobial photodynamic therapy was shown to be effective against a wide range of bacterial cells, as well as for fungi,
yeasts, and viruses. It was shown previously that photodestruction of yeast cells treated with photosensitizers resulted in
cell destruction and leakage of ATP. Three photosensitizers were used in this study: tetra(N-methyl-4-pyridyl)porphine tetratosylate salt (TMPyP), toluidine blue O (TBO), and methylene blue trihydrate (MB). A microdilution
method was used to determine MICs of the photosensitizers against both Escherichia coli O157:H7 and Listeria monocytogenes. To evaluate the effects of photodestruction on E. coli and L. monocytogenes cells, a bioluminescence method for detection of ATP leakage and a colony-forming assay were used. All tested photosensitizers
were effective for photodynamic destruction of both bacteria. The effectiveness of photosensitizers (in microgram-per-milliliter
equivalents) decreased in the order TBO > MB > TMPyP for both organisms. The MICs were two- to fourfold higher for E. coli O157:H7 than for L. monocytogenes. The primary effects of all of the photosensitizers tested on live bacterial cells were a decrease in intracellular ATP and
an increase in extracellular ATP, accompanied by elimination of viable cells from the sample. The time courses of photodestruction
and intracellular ATP leakage were different for E. coli and L. monocytogenes. These results show that bioluminescent ATP-metry can be used for investigation of the first stages of bacterial photodestruction.
Photodynamic therapy (PDT) employs a non-toxic dye, termed a photosensitizer (PS), and low intensity visible light which, in the presence of oxygen, combine to produce cytotoxic species. PDT has the advantage of dual selectivity, in that the PS can be targeted to its destination cell or tissue and, in addition, the illumination can be spatially directed to the lesion. PDT has previously been used to kill pathogenic microorganisms in vitro, but its use to treat infections in animal models or patients has not, as yet, been much developed. It is known that Gram-(-) bacteria are resistant to PDT with many commonly used PS that will readily lead to phototoxicity in Gram-(+) species, and that PS bearing a cationic charge or the use of agents that increase the permeability of the outer membrane will increase the efficacy of killing Gram-(-) organisms. All the available evidence suggests that multi-antibiotic resistant strains are as easily killed by PDT as naive strains, and that bacteria will not readily develop resistance to PDT. Treatment of localized infections with PDT requires selectivity of the PS for microbes over host cells, delivery of the PS into the infected area and the ability to effectively illuminate the lesion. Recently, there have been reports of PDT used to treat infections in selected animal models and some clinical trials: mainly for viral lesions, but also for acne, gastric infection by Helicobacter pylori and brain abcesses. Possible future clinical applications include infections in wounds and burns, rapidly spreading and intractable soft-tissue infections and abscesses, infections in body cavities such as the mouth, ear, nasal sinus, bladder and stomach, and surface infections of the cornea and skin.
Photodynamic therapy (PDT) employs a non-toxic dye termed a photosensitizer (PS) together with low intensity visible light, which, in the presence of oxygen, produce cytotoxic species. PS can be targeted to its destination cell or tissue and, in addition, the irradiation can be spatially confined to the lesion giving PDT the advantage of dual selectivity. This promising approach can be used for various applications including microbial inactivation and the treatment of infections. Resistance to PDT has not been shown and multiantibiotic-resistant strains are as easily killed as naive strains. It is known that Gram (+) bacteria are more sensitive to PDT as compared to Gram (-) species. However, the use of cationic PS or agents that increase the permeability of the outer membrane allows for the effective killing of Gram (-) organisms. Some PS have an innate positive charge, but our approach is to link PS to a cationic molecular vehicle such as poly-L-lysine. This modification dramatically increases PS binding to and penetrating through the negatively charged bacterial permeability barrier. Due to focused light delivery the use of PDT is possible only for localized infections. Nonetheless numerous diseases can be treated. Selectivity of the PS for microbes over host cells, accurate delivery of the PS into the infected area, and PDT dose adjustment help minimize side effects and give PDT an advantage over conventional therapy. There are only a few reports about the use of antimicrobial PDT in animal models and clinical trials. We have used genetically modified bioluminescent bacteria to follow the effect of PDT in infected wounds, burns, and soft tissue infections in mice. Not only were bacteria infecting wounds, burns, and abscesses killed, but mice were saved from death due to sepsis and wound healing was improved.
Vibrio vulnificus is a gram-negative, highly invasive bacterium responsible for human opportunistic infections. We studied the antibacterial
effects of toluidine blue O (TBO)-mediated photodynamic therapy (PDT) for V. vulnificus wound infections in mice. Fifty-three percent (10 of 19) of mice treated with 100 μg of TBO per ml and exposed to broad-spectrum
red light (150 J/cm2 at 80 mW/cm2) survived, even though systemic septicemia had been established with a bacterial inoculum 100 times the 50% lethal dose.
In vitro, the bacteria were killed after exposure to a lower light dose (100 J/cm2 at 80 mW/cm2) in the presence of low-dose TBO (0.1 μg/ml). PDT severely damaged the cell wall and reduced cell motility and virulence.
Cell-killing effects were dependent on the TBO concentration and light doses and were mediated partly through the reactive
oxygen species generated during the photodynamic reaction. Our study has demonstrated that PDT can cure mice with otherwise
fatal V. vulnificus wound infections. These promising results suggest the potential of this regimen as a possible alternative to antibiotics
in future clinical applications.
The worldwide rise in the rates of antibiotic resistance of bacteria underlines the need for alternative antibacterial agents. A promising approach to the killing of gram-positive antibiotic-resistant bacteria of the skin uses light in combination with a photosensitizer to induce a phototoxic reaction. Different concentrations (0 to 100 microM) of porphyrin-based photosensitizers (CTP1, XF70, and XF73) and different incubation times (5 min, 1 h, and 4 h) were used to determine phototoxicity against two methicillin-resistant Staphylococcus aureus strains, one methicillin-sensitive S. aureus strain, one methicillin-resistant Staphylococcus epidermidis strain, one Escherichia coli strain, and human keratinocytes and fibroblasts. Incubation with 0.005 microM XF70 or XF73, followed by illumination, yielded a 3-log10 (> or = 99.9%) decrease in the viable cell numbers of all staphylococcal strains, indicating that the XF drugs have high degrees of potency against gram-positive bacteria and also that the activities of these novel drugs are independent of the antibiotic resistance pattern of the staphylococci examined. CTP1 was less potent against the staphylococci under the same conditions. At 0.005 microM, XF70 and XF73 demonstrated no toxicity toward fibroblasts or keratinocytes. No inactivation of E. coli was detected at this concentration. XF73 was confirmed to act via a reactive oxygen species from the results of studies with sodium azide (a quencher of singlet oxygen), which reduced the killing of both eukaryotic and prokaryotic cells. When a quencher of superoxide anion and the hydroxyl radical was used, cell killing was not inhibited. These results demonstrate that the porphyrin-based photosensitizers had concentration-dependent differences in their efficacies of killing of methicillin-resistant staphylococcal strains via reactive oxygen species without harming eukaryotic cells at the same concentrations.
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.
We previously showed that covalent conjugates between poly-l-lysine and chlorin(e6) were efficient photosensitizers (PS) of both gram-positive and gram-negative bacteria. The polycationic
molecular constructs increased binding and penetration of the PS into impermeable gram-negative cells. We have now prepared
a novel set of second-generation polycationic conjugates between chlorin(e6) and three molecular forms of polyethyleneimine
(PEI): a small linear, a small cross-linked, and a large cross-linked molecule. The conjugates were characterized by high-pressure
liquid chromatography and tested for their ability to kill a panel of pathogenic microorganisms, the gram-positive Staphylococcus aureus and Streptococcus pyogenes, the gram-negative Escherichia coli and Pseudomonas aeruginosa, and the yeast Candida albicans, after exposure to low levels of red light. The large cross-linked molecule efficiently killed all organisms, while the linear
conjugate killed gram-positive bacteria and C. albicans. The small cross-linked conjugate was the least efficient antimicrobial PS and its remarkably low activity could not be explained
by reduced photochemical quantum yield or reduced cellular uptake. In contrast to polylysine conjugates, the PEI conjugates
were resistant to degradation by proteases such as trypsin that hydrolyze lysine-lysine peptide bonds, The advantage of protease
stability combined with the ready availability of PEI suggests these molecules may be superior to polylysine-PS conjugates
for photodynamic therapy of localized infections.
Multidrug-resistant (MDR) Acinetobacter baumannii, defined as resistant to three or more different antibiotic classes, is rapidly becoming a focus of attention of the medical and scientific communities. [1] The Infectious Diseases Society of America (IDSA) identified A. baumannii among the top seven pathogens threatening our healthcare-delivery system and as a prime example of unmet medical need. [2] The clinical impact and molecular basis of the ‘MDR phenotype’ have also made A. baumannii a study of ‘bug and drug’ interactions at every level, as summarized elegantly herein by Gootz et al. [3] We hope to address the following questions in this editorial: why are we so afraid of MDR A. baumannii, and is A. baumannii becoming a very ‘successful pathogen’?
Photodynamic therapy could provide an alternative to antibiotics for the treatment of local infections since a wide range of microorganisms have been shown to be susceptible to killing by photodynamic action (PDA) in vitro. The purpose of this study was to determine whether PDA was also able to affect the potency of two key bacterial virulence factors—lipopolysaccharide (LPS) and proteases. Suspensions of LPS from Escherichia coli and culture supernatants containing proteases of Pseudomonas aeruginosa were exposed to red light in the presence of toluidine blue O (TBO). The activity of each virulence factor was determined before and after irradiation. The limulus amoebocyte lysate (LAL) assay and the induction of proinflammatory cytokine (interleukin-8 and -6) release from human peripheral blood mononuclear cells (PBMC) were used for assessing the biological activity of LPS. Protease activity was quantified by azocasein hydrolysis. The biological activities of the LPS (both the LAL activity and its ability to induce cytokine release from PBMC) and the proteases were reduced significantly by irradiation with red light in the presence of TBO in a dose-dependent manner with respect to both the light energy dose and the TBO concentration. The ability of TBO-mediated PDA to reduce the activities of key virulence factors may be an additional benefit of using light-activated antimicrobial agents in the treatment of infectious diseases.
The photodynamic antibacterial properties of a closely related series of phenothiazinium dyes were tested against several pathogenic strains of Staphylococcus aureus, four of which were methicillin-resistant. Illumination of the photosensitisers at a fluence rate of 1.75 mW cm−2 generally resulted in the enhancement of antibacterial activity in liquid culture and in greater efficacy than the methicillin analogue flucloxacillin. For methylene blue, dimethyl methylene blue and new methylene blue illumination led to increases in bactericidal activity ≤16-fold, typically 4-fold. In addition, dimethyl methylene blue and new methylene blue were active against epidemic strains of methicillin-resistant Staphylococcus aureus at concentrations lower than that of vancomycin (≥0.5 μM).
Phenothiazinium dyes, and derivatives, were tested for toxicity to Escherichia coli and Staphylococcus aureus. The dyes were generally lipophilic (log P>1) and showed inherent dark toxicity (minimum lethal concentrations: 3.1–1000 μM). Dye illumination (total light dose of 3.15 J cm−1 over 30 min) led to up to eight-fold reductions in minimum lethal concentrations. Most of the illuminated dyes showed significant relative singlet oxygen yields (Φ′Δ: 0.18–1.35) suggesting a type II mechanism of generating a phototoxic response. Although generally up to six-fold more effective against S. aureus, the dyes tested efficiently killed E. coli and may be of particular use in combating Gram-negative pathogens.
5-(Ethylamino)-9-diethylaminobenzo[a]phenothiazinium chloride (EtNBS) is a photosensitizer (PS) with broad antimicrobial photodynamic activity. The objective of this study was to determine the antimicrobial photodynamic effect of side chain/end group modifications of EtNBS on two representative bacterial Gram-type-specific strains. Two EtNBS derivatives were synthesized, each functionalized with a different side-chain end-group, alcohol or carboxylic acid. In solution, both exhibited photochemical properties consistent with those of the EtNBS parent molecule. In vitro photodynamic therapy experiments revealed an initial Gram-type-specificity with two representative strains; both derivatives were phototoxic to Staphylococcus aureus 29,213 but the carboxylic acid derivative was nontoxic to Escherichia coli 25,922. This difference in photodynamic efficacy was not due to a difference in the binding of the two molecules to the bacteria as the amount of both derivatives bound by bacteria was identical. Interestingly, the carboxylic acid derivative produced no fluorescence emission when observed in cultures of E. coli via fluorescence microscopy. These early findings suggest that the addition of small functional groups could achieve Gram-type-specific phototoxicity through altering the photodynamic activity of PSs and deserve further exploration in a larger number of representative strains of each Gram type.
The photodynamic antibacterial properties of a closely related series of phenothiazinium dyes were tested against several pathogenic strains of Staphylococcus aureus, four of which were methicillin-resistant. Illumination of the photosensitisers at a fluence rate of 1.75 mW cm-2 generally resulted in the enhancement of antibacterial activity in liquid culture and in greater efficacy than the methicillin analogue flucloxacillin. For methylene blue, dimethyl methylene blue and new methylene blue illumination led to increases in bactericidal activity < or = 16-fold, typically 4-fold. In addition dimethyl methylene blue and new methylene blue were active against epidemic strains of methicillin-resistant Staphylococcus aureus at concentrations lower than that of vancomycin (> or = 0.5 microM).
The photosensitizing activity of lipophilic zinc-phthalocyanine (Zn-Pc) and its water-soluble sulphonated derivative (Zn-PcS) towards Streptococcus faecium and Candida albicans was studied and correlated with the amount of cell-bound photosensitizer. With both micro-organisms Zn-PcS was more tightly bound in larger amounts than Zn-Pc in the protoplasts of the cytoplasmic membrane. As a consequence, the photoinduced damage in S. faecium initially involved membrane proteins, while DNA was modified only upon prolonged irradiation. For C. albicans only Zn-PcS showed a preferential affinity for the spheroplasts and the decrease in cell survival was not accompanied by detectable modifications of the electrophoretic pattern of membrane proteins. The photoinduced ultrastructural alteration of both micro-organisms suggests damage at membrane level. This would indicate the involvement of different targets in bacteria and yeast for phthalocyanine photosensitization.
Photosensitization of Escherichia coli and Pseudomonas aeruginosa cells by deuteroporphyrin (DP) is shown to be possible in the presence of the polycationic agent polymyxin nonapeptide (PMNP). Previous studies established complete resistance of Gram-negative bacteria to the photodynamic effects of porphyrins. The present results show that combined treatment of E. coli or P. aeruginosa cultures with DP and PMNP inhibit cell growth and viability. No antibacterial activity of PMNP alone could be demonstrated and cell viability remained unchanged. Spectroscopically, PMNP was found to bind DP, a mechanism which probably assists its penetration into the cell's membranes. Insertion of DP into the cells was monitored by the characteristic fluorescence band of bound DP at 622 nm. Binding times were 5-40 min and the extent of binding increased with decreasing the pH from 8.5 to 6.5. DP binding constants, as well as the concentrations of PMNP which were required for maximal effect on the various Gram-negative bacteria, were determined fluorometrically. By the treatment of DP, PMNP and light the growth of E. coli and P. aeruginosa cultures was stopped and the viability of the culture was dramatically reduced. Within 60 min of treatment the survival fraction of E. coli culture was 9 x 10(-6) and that of P. aeruginosa was 5.2 x 10(-4). Electron microscopy depicted ultrastructural alterations in the Gram-negative cells treated by DP and PMNP. The completion of cell division was inhibited and the chromosomal domain was altered markedly.
A metachromatic dye, 1,9-dimethylmethylene blue (DMB) reacts with bacterial endotoxins. This interaction results in a shift of the absorption maximum of DMB to shorter wavelengths. The findings indicate that the negatively charged lipid moiety of the endotoxic lipopolysaccharide reacts with DMB. The lowest amount of endotoxin detectable by the procedure described here is approximately one microgram. The dye could not be used in the presence of serum components. DMB mixed to column chromatographic effluent showed good resolution in continuous monitoring of endotoxin components leaving the column.
To evaluate newer therapies for wound infections, it becomes necessary to quantify bacteria that invade from the infected wounds into the adjacent tissues. For example, antibody-targeted photolysis targets the invasive Pseudomonas with antibodies carrying photochemical dyes. A full-thickness burn wound was infected with Pseudomonas aeruginosa with a modification of previous methods. In mice, a skin fold was elevated, and two preheated brass blocks at 92 degrees to 95 degrees C were applied for 5 seconds, producing a 5% total body surface area injury with discrete margins. The eschars were immediately inoculated with Pseudomonas. Survival at 10 days was 100% with burn injury alone and 60% with infected burns. Pseudomonas (10(8)/gm) were recovered from the unburned muscle by 24 hours. The method produced uniform and reproducible quantitative bacteriology within the muscle immediately beneath the burn injury (SL < 0.05). Quantitative comparisons can be used to determine the effectiveness of newer modalities to control Pseudomonas burn wound infections.
The photosensitization of microorganisms is potentially useful for sterilization and for the treatment of certain bacterial diseases. Until now, any broad spectrum approach has been inhibited because, although Gram-positive bacteria can be photoinactivated by a range of photosensitizers, Gram-negative bacteria have not usually been susceptible to photosensitized destruction. In the present work, it has been shown that the Gram-negative bacteria Escherichia coli and Pseudomonas aeruginosa, as well as the Gram-positive bacterium Enterococcus seriolicida, can be photoinactivated when illuminated in the presence of a cationic water-soluble zinc pyridinium phthalocyanine (PPC). The degree of photoinactivation is dependent on both the concentration of PPC and the illumination time. In contrast, the three bacteria are not photoinactivated by illumination in the presence of a neutral tetra-diethanolamine phthalocyanine (TDEPC) or negatively charged tetra-sulphonated phthalocyanine (TSPC). Uptake studies have revealed that the lack of activity of TSPC is due to the fact that it has very little affinity for any of the organisms. However, the issue appears to be more complex than simply the gross levels of cellular uptake, since TDEPC and PPC are both taken up by the organisms but only PPC shows activity. This indicates that the localization and subcellular distribution of the phthalocyanines may be a crucial factor in determining their cell killing potential. Further analysis of the uptake data has revealed a cell-bound photosensitizer fraction, which remains tightly associated after several washings, and another weakly bound fraction, which is removed by successive washings. Analysis of the cell killing curves, carried out after successive washings of E. coli exposed to PPC, has revealed that it is the tightly associated fraction that is involved in the photosensitization. Taken together with other data, these results suggest that cationic photosensitizers may have a broader application in the photoinactivation of bacterial cells than the anionic or neutral photosensitizers commonly used in photodynamic therapy.
Previous studies on the photosensitization of bacterial cells with different neutral or negatively charged porphyrins and phthalocyanines have demonstrated that, although Gram-positive bacteria are efficiently photoinactivated, Gram-negative bacteria become photosensitive only after modification of the permeability of their outer membrane. The results described in this paper show that two meso-substituted cationic porphyrins, namely tetra(4N-methyl-pyridyl)porphine tetraiodide and tetra(4N,N,N-trimethyl-anilinium)porphine, efficiently photosensitize the inactivation of Gram-negative bacteria, such as Vibrio anguillarum and Escherichia coli. A negatively charged meso-substituted porphyrin, tetra(4-sulphonatophenyl)porphine, has no appreciable photosensitizing activity towards Gram-negative bacteria, although all three porphyrins exhibit a similar subcellular distribution pattern, being mainly localized in the protoplasts or spheroplasts. Moreover, the three porphyrins show similar efficiency in the photoinactivation of the Gram-positive bacterium Entorecoccus seriolicida.
With advances in research techniques involving molecular biology, the need for precision in the determination of surface area is even greater than previously. This study sets out to determine the most accurate Meeh constant, k, for a commonly used weight range of experimental rats. The importance of a narrow weight range is appreciated and planometric data is presented from a series of adolescent rats that allow a new k value to be calculated. The figures presented here represent the largest number of animals, of a commonly used rat strain, in the most widely used weight range yet described. Pelts were removed from 16 anaesthetized male Sprague-Dawley rats after cervical dislocation. For each animal total body surface area (TBSA) was determined planometrically, along with premarked areas on the dorsal and ventral surfaces. Entering the planometrically measured data along with the animal's weight into the Meeh formula, a k value was obtained for each rat. Surface areas were then calculated for each animal using the mean k, 9.46, and compared against figures obtained by using historical values for the Meeh constant. Using the new k value a method is described for the accurate determination of total body surface area and premarked surface areas, in experimental rats of a specific weight range.
The photodynamic antibacterial properties of a closely related series of commercially available phenothiazinium dyes were tested against a range of pathogenic strains of Gram-positive (Staphylococcus aureus, Enterococcus faecalis, Bacillus cereus) and Gram-negative organisms (Escherichia coli, Pseudomonas aeruginosa). The photosensitisers were illuminated using a non-laser light source at a fluence of 1.75 mW cm-2 and this resulted in the enhancement of antibacterial activity in liquid culture. In several cases, illumination resulted in considerable decreases in the minimum lethal concentrations required, giving up to 100-fold increases in bactericidal activity.
The toxicities and phototoxicities of methylene blue and its two methylated derivatives were measured against one standard and three vancomycin-resistant pathogenic strains of Enterococcus spp. Each of the compounds was bactericidal and the derivatives exhibited photobactericidal activity on illumination at a 'light' dose of 6.3 J/cm(2) against one or more of the strains. Increased bactericidal and photobactericidal activity in the methylated derivatives is thought to be due to their higher hydrophobicities allowing greater interaction with the bacterial cell wall. In addition, the derivatives exhibited higher inherent photosensitizing efficacies.
Photodynamic therapy could provide an alternative to antibiotics for the treatment of local infections since a wide range of microorganisms have been shown to be susceptible to killing by photodynamic action (PDA) in vitro. The purpose of this study was to determine whether PDA was also able to affect the potency of two key bacterial virulence factors--lipopolysaccharide (LPS) and proteases. Suspensions of LPS from Escherichia coli and culture supernatants containing proteases of Pseudomonas aeruginosa were exposed to red light in the presence of toluidine blue O (TBO). The activity of each virulence factor was determined before and after irradiation. The limulus amoebocyte lysate (LAL) assay and the induction of proinflammatory cytokine (interleukin-8 and -6) release from human peripheral blood mononuclear cells (PBMC) were used for assessing the biological activity of LPS. Protease activity was quantified by azocasein hydrolysis. The biological activities of the LPS (both the LAL activity and its ability to induce cytokine release from PBMC) and the proteases were reduced significantly by irradiation with red light in the presence of TBO in a dose-dependent manner with respect to both the light energy dose and the TBO concentration. The ability of TBO-mediated PDA to reduce the activities of key virulence factors may be an additional benefit of using light-activated antimicrobial agents in the treatment of infectious diseases.
Porphycenes are electronic isomers of porphyrins which, when neutral, display no appreciable photosensitizing action towards Gram-negative bacteria. The covalent binding of oligomeric polylysine moieties, which are cationic at physiological pH values, endows porphycenes with a significant phototoxic activity against Gram-negative bacteria while retaining their photoefficiency against a variety of microbial pathogens, including Gram-positive bacteria, fungi and mycoplasmas. The effect of the polylysine moiety is dependent on both the polylysine concentration and the degree of oligomerization. A suitable interplay among the various parameters opens the possibility to obtain either a broad spectrum of antimicrobial activity or a selective action toward a specific pathogen while minimizing the damage to human fibroblasts.
The worldwide rise in antibiotic resistance necessitates the development of novel antimicrobial strategies. In this study we report on the first use of a photochemical approach to destroy bacteria infecting a wound in an animal model. Following topical application, a targeted polycationic photosensitizer conjugate between poly-L-lysine and chlorin(e6) penetrated the gram (-) outer bacterial membrane, and subsequent activation with 660 nm laser light rapidly killed Escherichia coli infecting excisional wounds in mice. To facilitate real-time monitoring of infection, we used bacteria that expressed the lux operon from Photorhabdus luminescens; these cells emitted a bioluminescent signal that allowed the infection to be rapidly quantified, using a low-light imaging system. There was a light-dose dependent loss of luminescence in the wound treated with conjugate and light, not seen in untreated wounds. Treated wounds healed as well as control wounds, showing that the photodynamic treatment did not damage the host tissue. Our study points to the possible use of this methodology in the rapid control of wounds and other localized infections.
The purpose of this study was to evaluate the efficacy of using methylene blue (MB)-mediated photodynamic therapy to treat oral candidiasis in an immunosuppressed murine model, mimicking what is found in human patients.
Seventy-five experimental mice with severe combined immunodeficiency disease were inoculated orally with Candida albicans by swab 3 times a week for a 4-week period. On treatment day, mice were cultured for baseline fungal growth and received a topical oral cavity administration of 0.05 mL MB solution at one of the following concentrations: 250, 275, 300, 350, 400, 450, or 500 microgram/mL. After 10 minutes the mice were recultured and underwent light activation with 664 nm of diode laser light with a cylindrical diffuser. After photodynamic therapy the mice were cultured again for colony-forming units per milliliter and then killed, their tissue harvested for histopathology.
The results indicate an MB dose-dependent effect. Concentrations from 250 to 400 microgram/mL reduced fungal growth but did not eliminate Candida albicans. MB concentrations of 450 and 500 microgram/mL totally eradicated Candida albicans from the oral cavity, resulting in reductions from 2.5 log(10) and 2.74 log(10) to 0, respectively. These results suggest that MB-mediated photodynamic therapy can potentially be used to treat oral candidiasis in immunodeficient patients.
Phenothiazinium-based molecules exhibit phototoxicity against a broad range of bacteria. In general, these photosensitizers use several cellular uptake pathways, coupled to type II mechanisms of photo-oxidation, to inflict bacterial damage. These molecules show potential to act as novel alternatives to conventional antibiotics.
The difference in the photobactericidal efficacy of methylene blue and toluidine blue against gram-negative bacteria may result from their primary reaction with lipopolysaccharides (LPS) of the outer bacterial membrane. The aim of the present study was to compare the reactivity of these dyes with LPS extracted from different gram-negative bacteria.
The interactions of methylene blue and toluidine blue with LPS from Escherichia coli (E. coli), Pseudomonas aeruginosa (P. aeruginosa), Klebsiella pneumoniae (K. pneumoniae), and Serratia marcescens (S. marcescens) were studied spectrophotometrically in 0.45% saline. The dyes were used at the concentration of 10 microM. The concentrations of LPS ranged from 5-100 microg/ml.
Methylene blue and toluidine blue enter into a metachromatic reaction with the LPS resulting the in generation of dimers of methylene blue and higher aggregates of toluidine blue. The more significant hypochromic and hypsochromic effects in the reaction of the latter with LPS indicate a greater metachromatic efficacy of toluidine blue than methylene blue. The equilibrium constants of the metachromatic complex between toluidine blue and different LPS were calculated. The spectrophotometric titration of LPS with the dyes was used to estimate the equivalent weight of LPS.
Toluidine blue interacts with LPS more significantly than methylene blue in vitro. This may be one of the main factors determining its greater photobactericidal efficacy against gram-negative bacteria.
Methylene blue (MB) is a phenothiazinium photosensitizer with promising applications in the photodynamic therapy (PDT) for anticancer treatment. The binding properties of MB to herring sperm DNA have been investigated by the measurements of absorption spectra, quenching experiments and the elucidation of the photobleaching processes. Remarkable hypochromic and bathochromic effects of MB in the presence of increasing amounts of DNA have been observed in the absorption spectra. The quenching of MB by the DNA bases obeys the Stern-Volmer equation and ferrocyanide quenching of MB in the absence and presence of DNA is also measured as extended experiments. Results from the above spectral measurements are all consistent with the intercalative binding mode of MB to DNA with the Kb value of 1.89 x 10(4) M(-1). The photobleaching processes of MB and its DNA complex have also been studies, which indicate that the photobleaching of MB and its DNA complex proceeds with different mechanisms and the reactive oxygen species are responsible for the self-sensitized photooxidation of MB.
Methylene blue (MB) and toluidine blue (TB) form metachromatic complexes with lipopolysaccharides (LPS). The greater photobactericidal efficacy of TB may be explained by its affinity for LPS. This study aims to elucidate the difference in photobactericidal efficacies between the dyes using Ca(2+) as a competitor for dye-binding sites on the bacterial outer membrane.
Fixed dye concentration solutions with gram-negative bacteria and increasing concentrations of CaCl(2) were exposed to red laser light. Bacterial survival and spectrophotometry were used to describe the effect of Ca(2+) on dye interaction with bacteria and LPS.
MB-mediated photokilling was inhibited more significantly than that of TB. CaCl(2) inhibited dye photobleaching and suppressed the metachromatic reaction between the dyes and LPS, in particular TB.
CaCl(2) inhibits bacterial photokilling by binding with LPS, as well as other anionic polymers including outer membrane proteins. LPS is chiefly involved in TB-mediated photokilling, whereas outer membrane proteins probably are more involved in MB-mediated photokilling.
The evolving problem of antimicrobial resistance in Pseudomonas aeruginosa, Acinetobacter baumannii and Klebsiella pneumoniae has led to the emergence of clinical isolates susceptible to only one class of antimicrobial agents and eventually to pandrug-resistant (PDR) isolates, i.e. resistant to all available antibiotics. We reviewed the available evidence from laboratory and clinical studies that reported on polymyxin-resistant and/or PDR P. aeruginosa, A. baumannii or K. pneumoniae clinical isolates. Eleven laboratory studies reported on isolates with resistance to polymyxins, three of which (including two surveillance studies) also included data regarding PDR isolates. In addition, two clinical studies (from Central and Southern Europe) reported on the clinical characteristics and outcomes of patients infected with PDR isolates. These data suggest that polymyxin-resistant or PDR P. aeruginosa, A. baumannii and K. pneumoniae clinical isolates are currently relatively rare. However, they have important global public health implications because of the therapeutic problems they pose. The fears for the dawn of a post-antibiotic era appear to be justified, at least for these three Gram-negative bacteria. We must increase our efforts to preserve the activity of available antibiotics, or at least expand as much as possible the period of their use, whilst intense research efforts should be focused on the development and introduction into clinical practice of new antimicrobial agents.
Acinetobacter baumannii is a highly resilient, gram-negative coccobacillus that thrives within the unique and complex ecological setting of an intensive care unit. This evolving pathogen has now surpassed human capacity to create new antimicrobials, and has led physicians into a concerning era for hospital-acquired infections. This review presents the available evidence on the therapeutic strategies for A. baumannii infection, with a particular focus on clinical human data. The utility of existing and older antimicrobials such as sulbactam and the polymyxins are explored, as well as, the potential role of newer agents such as tigecycline. Other important adjunctive strategies such as pharmacodynamic target attainment and infection control implementation are briefly discussed. It is now clear that new antimicrobials with unique mechanisms of action are urgently required to combat the rising trends seen globally with drug-resistant A. baumannii.
Periodontitis is a disease caused by bacterial infection accompanied with the inflammation of connected tissues and resorption of alveolar bone. The aim of this study was to investigate the in vivo photosensitization of periodontal bacteria in rats and to compare its efficacy with that of routine scaling and root planing.
Periodontitis was developed by submerging ligatures at the subgingival region of maxillary molars in 16 rats. Six weeks later, the infection sites were treated either with 1 mg/mL of toluidine blue plus 12 J/cm2 red laser irradiation, or by routine scaling and root planing. The therapeutic efficacy was assessed by evaluating the reduction of total bacterial flora and histological changes of periodontal tissues.
Significant reduction of total bacterial flora was achieved by both photodynamic therapy and conventional therapy. The signs of inflammation that accompanied periodontitis, such as redness, increased plaque index and gingival index values, bleeding on probing and inflammatory cell infiltration, were greatly reduced without any obvious detectable injury to host tissues. Both photodynamic therapy and conventional therapy showed similar therapeutic results.
Toluidine blue-mediated photodynamic therapy could effectively treat periodontitis in vivo and has high potential in clinical application.
Amid the recent attention focused on the growing impact of methicillin-resistant Staphylococcus aureus and multidrug-resistant Pseudomonas aeruginosa infections, the pathogen Acinetobacter baumannii has been stealthily gaining ground as an agent of serious nosocomial and community-acquired infection. Historically, Acinetobacter spp. have been associated with opportunistic infections that were rare and of modest severity; the last two decades have seen an increase in both the incidence and seriousness of A. baumannii infection, with the main targets being patients in intensive-care units. Although this organism appears to have a predilection for the most vulnerable patients, community-acquired A. baumannii infection is an increasing cause for concern. The increase in A. baumannii infections has paralleled the alarming development of resistance it has demonstrated. The persistence of this organism in healthcare facilities, its inherent hardiness and its resistance to antibiotics results in it being a formidable emerging pathogen. This review aims to put into perspective the threat posed by this organism in hospital and community settings, describes new information that is changing our view of Acinetobacter virulence and resistance, and calls for greater understanding of how this multifaceted organism came to be a major pathogen.
Rapid control of wound infections by targeted photodynamic therapy monitored by in vivo bioluminescence imaging
- Hamblin