Optical method for monitoring of photodynamic inactivation of bacteria

Photodynamic inactivation is a new promising approach to treat bacterial infections. Usually, the evaluation of the efficacy of this method is done through time-consuming and labor-intensive microbiological test methods. This paper describes the development and implementation of an optical method to evaluate the photodynamic inactivation of bacteria based on non-invasive diffuse reflectance measurements. Five Staphylococcus aureus cultures and 15 mice have been used in this study. A skin lesion was created on the back of all animals, and it was contaminated with S. aureus (5.16 ± 0.013 log CFU/ml). Toluidine Blue O (c = 8.67 × 10 ^− 3 M) has been used as a photosensitiser agent. The bacterial cultures and animals were exposed to laser radiation (λ = 635 nm, P = 15 mW, DE = 8.654 J/cm²) for 20 min. The photodynamic inactivation of bacteria was monitored by acquiring the wounds’ reflection spectra at different time points and by microbiological exams on the bioptical material. The good correlation between the diffuse reflectance and colony-forming units demonstrates the value of this optical method based on diffuse reflectance measurements as a rapid technique to monitor photodynamic bacterial inactivation.     

Synergistic effect of hypocrellin B and curcumin on photodynamic inactivation of Staphylococcus aureus

Antimicrobial photodynamic inactivation (aPDI) serves as a new approach to control the growth of foodborne bacteria. It remains elusive if the photodynamic efficacy of hypocrellin B (HB) can be potentiated by joint action with curcumin. In this study, we measured the survival rate of Staphylococcus aureus strains under the varying photodynamic conditions. According to our data, a maximum of 5–6 log₁₀ decrease of bacterial survival can be achieved under the tested conditions (500 nM, 9 J cm^‒2). Regarding the bactericidal mechanisms, HB-based aPDI disrupted the membrane integrity of staphylococcal cells, probably owing to the stimulated reactive oxygen species (ROS). In addition, aPDI disrupted the enzymatic activities of bacterial antioxidant proteins and caused the leakage of multiple intracellular substances. The HB-mediated photodynamic efficacy was potentiated by the addition of curcumin with a sublethal dose. This dual-photon synergy arose from unique aPDI conditions (100 nM each and 9 J cm^‒2). The synergistic action might be accounted for by the increased type I/type II ratio of ROS, as evidenced by the effect of different quenchers. Finally, the joint use of photosensitizers reduced the microbial contamination of the tested apple while maintaining its quality. In summary, photodynamic inactivation based on dual photons showed synergistic activity in controlling the growth of Staphylococcal aureus, which provided a novel approach to maintain food safety.     

Safety and delivery efficiency of a photodynamic treatment of the lungs using indocyanine green and extracorporeal near infrared illumination

Photodynamic inactivation (PDI) is a promising alternative for combating infections caused by antimicrobial resistant bacteria. Pneumonias are among the most worrisome infections because of their high‐mortality rate. Previous studies have demonstrated the feasibility of using PDI with extracorporeal light to treat pneumonia. In this study, we analyzed key parameters for the viability of this treatment, including the selectivity of the photodynamic response for pathogens over host cells. Our results showed that PDI can induce killing of Staphylococcus aureus (of up to 4.18 log for the strain Xen29 and 3.62 log for Xen36) under conditions where little or no toxicity for host cells is observed. We validated pulmonary delivery of the photosensitizer and light in mice, using photobleaching as an indicator, and demonstrated preservation of healthy tissues as evidence of the safety of the protocol. Overall, PDI displays low toxicity on host tissues, making it a promising tool for treatment of pneumonias caused by S. aureus and other important pathogens.     

Real‐time imaging of photodynamic action in bacteria

Fluorescence imaging studies of the processes leading to photodynamic inactivation of bacteria have been limited due to the small size of microorganisms as well as by the faint fluorescence of most photosensitizers. A versatile method based on highly‐sensitive fluorescence microscopy is presented which allows to study, in real time, the incorporation of photosensitizers inside S. aureus upon photodynamic action. The method takes advantage of the fluorescence enhancement of phenothiazine and porphyrin photosensitizers upon entering the bacterial cytosol after the cell wall has been compromised. In combination with typical assays, such as the addition of specific enhancers of reactive oxygen species, it is possible to extract mechanistic information about the pathway of photodynamic damage at the single‐cell level. Imaging experiments in deuterated buffer strongly support a Type‐I mechanism for methylene blue and a very minor role of singlet oxygen.

     

Near–infrared photodynamic inactivation of S. pneumoniae and its interaction with RAW 264.7 macrophages

Pneumonia is the main cause of children mortality worldwide, and its major treatment obstacle stems from the microorganisms increasing development of resistance to several antibiotics. Photodynamic therapy has been presenting, for the last decades, promising results for some subtypes of cancer and infections. In this work we aimed to develop a safe and efficient in vitro protocol for photodynamic inactivation of Streptococcus pneumoniae, one of the most commonly found bacteria in pneumonia cases, using two near‐infrared light sources and indocyanine green, a FDA approved dye. Photodynamic inactivation experiments with bacteria alone allowed to determine the best parameters for microbial inactivation. Cytotoxicity assays with RAW 264.7 macrophages evaluated the safety of the PDI. To determine if the photodynamic inactivation had a positive or negative effect on the natural killing action of macrophages, we selected and tested fewer indocyanine green concentrations and 10 J/cm² on macrophage‐S. pneumoniae co‐cultures. We concluded that ICG has potential as a photosensitizer for near‐infrared photodynamic inactivation of S. pneumoniae, producing minimum negative impact on RAW 264.7 macrophages and having a positive interaction with the immune cell's microbicidal action.

     

Rapid detection of methicillin resistance in <Emphasis Type="Italic">Staphylococcus aureus</Emphasis> isolates; evaluation of colorimetric Quicolor ES agar and determination of breakpoint inhibition zone diameters of cefoxitin

In this study, it was aimed to evaluate colorimetric Quicolor ES agar for the rapid detection of methicillin resistance and to determine susceptibility and resistance breakpoint zone diameters for cefoxitin by using 51 methicillin susceptible Staphylococcus aureus (MSSA) and 63 methicillin resistant S. aureus (MRSA) isolates. In the study, while oxacillin and cefoxitin results were obtained within 4–7 h (5.5 h in average) for MSSA isolates, the results of MRSA isolates were obtained within 5.5–9 h (6.6 h in average) for both antibiotics on QC ES agar. QC ES agar is an inexpensive medium for rapid detection (4–9 h) of methicillin resistance by disc diffusion method using oxacillin or cefoxitin. Additional studies for further evaluation of the efficiency of QC-ES agar in rapid determination of methicillin resistance in S. aureus may be beneficial.     

Efficient photodynamic inactivation of Leishmania parasites mediated by lipophilic water-soluble Zn(II) porphyrin ZnTnHex-2-PyP4+

BackgroundPhotodynamic inactivation (PDI) is emerging as a promising alternative for cutaneous leishmaniasis (CL). The chemotherapy currently used presents adverse effects and cases of drug resistance have been reported. ZnTnHex-2-PyP⁴⁺ is a porphyrin with a high potential as a photosensitizer (PS) for PDI, due to its photophysical properties, structural stability, and cationic/amphiphilic character that can enhance interaction with cells. This study aimed to investigate the photodynamic effects mediated by ZnTnHex-2-PyP⁴⁺ on Leishmania parasites.MethodsZnTnHex-2-PyP⁴⁺ stability was evaluated using accelerated solvolysis conditions. The photodynamic action on promastigotes was assessed by (i) viability assays, (ii) mitochondrial membrane potential evaluation, and (iii) morphological analysis. The PS-promastigote interaction was studied. PDI on amastigotes and the cytotoxicity on macrophages were also analyzed.ResultsZnTnHex-2-PyP⁴⁺, under submicromolar concentration, led to immediate inactivation of more than 95% of promastigotes. PDI promoted intense mitochondrial depolarization, loss of the fusiform shape, and plasma membrane wrinkling in promastigotes. Fluorescence microscopy revealed a punctate PS labeling in the parasite cytoplasm. PDI also led to reductions of ca. 64% in the number of amastigotes/macrophage and 70% in the infection index after a single treatment session. No noteworthy toxicity was observed on mammalian cells.ConclusionsZnTnHex-2-PyP⁴⁺ is stable against demetallation and more efficient as PS than the ethyl analogue ZnTE-2-PyP⁴⁺, indicating readiness for evaluation in in vivo studies as an alternative approach to CL.General significanceThis report highlighted promising photodynamic effects mediated by ZnTnHex-2-PyP⁴⁺ on Leishmania parasites, opening up perspectives for applications in CL pre-clinical assays and PDI of other microorganisms.     

The mechanism of photodynamic inactivation ofStaphylococcus aureus by deuteroporphyrin

The mechanism ofStaphylococcus aureus inactivation by deuteroporphyrin (DT) and light was studied with singlet oxygen quenchers or hydroxyl radical scavengers. The light-activated DT (10 /ml) reduced the viability of the culture to less than 1%, whereas methionine, tryptophan, and 1,4-diazabicyclo-2,2,2-octane (DBCO) used as singlet oxygen quenchers provided almost 60% protection. Propylgallate, which is a hydroxyl free radical scavenger, also provided 60% protection. The presence of a singlet oxygen quencher and propylgallate provided almost complete protection from inactivation (96%). Photoinactivation in the absence of culture media (in saline) increased the killing rate and decreased the ability of the singlet oxygen quenchers to protect. In the same conditions damage from hydroxl free radicals was well protected by propyl gallate. The present results indicate thatS. aureus photoinactivation by DT and light is mediated by both singlet oxygen and hydroxyl free radicals.     

Liquid crystal precursor system as a vehicle for curcumin-mediated photodynamic inactivation of oral biofilms

Curcumin has great potential as a photosensitizer, but it has low solubility in aqueous solutions. This study reports the antimicrobial efficacy of photodynamic inactivation (PDI) mediated by a curcumin-loaded liquid crystal precursor (LCP) on in situ dental biofilms. Thirty volunteers used intraoral devices containing enamel samples for 48 hours for biofilm formation. The samples were then removed from the device and treated either with LCP with 160 μM of curcumin plus illumination at 18 J/cm² (C + L+ group) or with LCP without curcumin in the dark (C – L − group). Following this, the biofilm from the samples was plated for quantifying the viable colonies at 37°C for 48 hours. Specific and nonspecific media were used for the presumptive isolation of Streptococcus mutans, Lactobacillus species/aciduric microorganisms, Candida species, and total microbiota. The C + L+ group showed a highly significant (P < .001) reduction in the log₁₀ (colony forming units/mL) values as compared to the C − L − group for all culture media. Hierarchical linear regression indicated that there may be predictors at individual volunteer level explaining the difference in the PDI efficacy among different individuals (P = .001). The LCP system retained curcumin and released it slowly and continuously, thus protecting the drug from photodegradation. LCP with curcumin is considered effective for the photoinactivation of dental biofilms, but the PDI efficacy may differ based on the host's individual characteristics.     

Optimization of the photodynamic inactivation of prions by a phthalocyanine photosensitizer: The crucial involvement of singlet oxygen

Prion disorders are fatal neurodegenerative diseases caused by the autocatalytic conversion of a natively occurring prion protein (PrP^C) into its misfolded infectious form (PrP^TSE). The proven resistance of PrP^TSE to common disinfection procedures increases the risk of prion transmission in medical settings. Herein, we present the effective photodynamic inactivation (PDI) of prions by disulfonated hydroxyaluminum phthalocyanine (AlPcOH(SO₃)₂) utilizing two custom‐built red light sources. The treatment eliminates PrP^TSE signal in infectious mouse brain homogenate with efficiency that depends on light intensity but has a low effect on the overall protein content. Importantly, singlet oxygen (O₂(¹Δ_g)) is the only species significantly photogenerated by AlPcOH(SO₃)₂, and it is responsible for the PDI of prions. More intensive light conditions show not only higher O₂(¹Δ_g) production but also decreases in AlPcOH(SO₃)₂ photostability. Our findings suggest that PDI by AlPcOH(SO₃)₂‐generated O₂(¹Δ_g) represents a promising approach for prion inactivation that may be useful in future decontamination strategies for delicate medical tools.     

The photodynamic effect: the comparison of chemiexcitation by luminol and phthalhydrazide

The presence of light, oxygen and photosensitizer (organic dye) is required for the photodynamic effect. Light and photosensitizer are harmless by themselves, but when combined with oxygen, reactive oxygen species (ROS) can be produced. This photodynamic effect is used in photodynamic therapy (PDT); the production of ROS as lethal cytotoxic agents can inactivate tumor cells. However, during PDT, there are many difficulties, so it is not possible to excite the photosensitizer using a laser, a source of light at the wavelengths specific to the photosensitizer (in visible region of the spectrum). Chemiluminescence is the light emission as a result of a chemical reaction. It is possible to use a chemiluminescent mixture to excite the photosensitizer even if the light emission does not conform to the absorption maximum of the photosensitizer. Luciferin and luminol have been used as chemiluminescent compounds (energizers) for the excitation of the photosensitizers. The aim of this work was to compare the chemiexcitation of some selected photosensitizers (e.g. fluorescein, eosin, methylene blue, hypericin and phthalocyanines) by chemiluminescent mixtures containing luminol (high chemiluminescent quantum yield) or phthalhydrazide (low chemiluminescent quantum yield) on some Gram‐positive (Enterococcus faecalis, Staphylococcus aureus) and Gram‐negative (Pseudomonas aeruginosa, E. coli) bacteria and some cell lines (NIH3T3 and MCF7). The efficiency of the chemiexcitation was dependent on the kind of the photosensitizer and on the type of the bacterial strain or cell line and was independent of the energizers. Copyright © 2010 John Wiley & Sons, Ltd.     

The Synergistic Antibacterial Properties of Glycinin Basic Peptide against Bacteria via Membrane Damage and Inactivation of Enzymes

This study investigated the antibacterial properties of glycinin basic peptide (GBP), a natural antibacterial component from soybean protein, against Staphylococcus aureus (S. aureus). The minimum inhibitory and bactericidal concentrations of GBP against S. aureus were 0.2 mg/mL and 0.8 mg/mL, respectively. Flow cytometry analysis manifested that GBP decreased the number of intact and normal cells. Higher concentrations of GBP induced more severe damage of the bacterial membrane; the maximal percentage of injured and dead cells was 93.8% with 0.8 mg/mL GBP. Electron microscopy imaging visually showed the morphological damage of S. aureus by GBP. Intracellular K⁺ leakage and the membrane depolarization of S. aureus further verified that GBP could destroy the bacterial membrane. Moreover, GBP decreased the activity of nonspecific esterase and ATPase of S. aureus in a concentration-dependent manner. These results demonstrated that GBP exhibited antibacterial properties against S. aureus via synergistic actions of damage to the cell membrane and inactivation of metabolic enzymes.

     

The heme sensing response regulator HssR in <Emphasis Type="Italic">Staphylococcus aureus</Emphasis> but not the homologous RR23 in <Emphasis Type="Italic">Listeria monocytogenes</Emphasis> modulates susceptibility to the antimicrobial peptide plectasin

Background  

Host defence peptides (HDPs), also known as antimicrobial peptides (AMPs), have emerged as potential new therapeutics and their antimicrobial spectrum covers a wide range of target organisms. However, the mode of action and the genetics behind the bacterial response to HDPs is incompletely understood and such knowledge is required to evaluate their potential as antimicrobial therapeutics. Plectasin is a recently discovered HDP active against Gram-positive bacteria with the human pathogen, Staphylococcus aureus (S. aureus) being highly susceptible and the food borne pathogen, Listeria monocytogenes (L. monocytogenes) being less sensitive. In the present study we aimed to use transposon mutagenesis to determine the genetic basis for S. aureus and L. monocytogenes susceptibility to plectasin.     

Aloe-emodin-mediated antimicrobial photodynamic therapy against dermatophytosis caused by Trichophyton rubrum

Trichophyton rubrum is responsible for the majority of dermatophytosis. Current systemic and topical antifungals against dermatophytosis are often tedious and sometimes unsatisfactory. Antimicrobial photodynamic therapy (aPDT) is a non-invasive alternative suitable for the treatment of superficial fungal infections. This work investigated the photodynamic inactivation efficacy and effects of aloe-emodin (AE), a natural photosensitizer (PS) against T. rubrum microconidia in vitro, and evaluated the treatment effects of AE-mediated aPDT for T. rubrum-caused tinea corporis in vivo and tinea unguium ex vivo. The photodynamic antimicrobial efficacy of AE on T. rubrum microconidia was evaluated by MTT assay. The inhibition effect of AE-mediated aPDT on growth of T. rubrum was studied. Intracellular location of AE, damage induced by AE-mediated aPDT on cellular structure and surface of microconidia and generation of intracellular ROS were investigated by microscopy and flow cytometry. The therapeutic effects of AE-mediated aPDT against dermatophytosis were assessed in T. rubrum-caused tinea corporis guinea pig model and tinea unguium ex vivo model. AE-mediated aPDT effectively inactivated T. rubrum microconidia in a light energy dose-dependent manner and exhibited strong inhibitory effect on growth of T. rubrum. Microscope images indicated that AE is mainly targeted to the organelles and caused damage to the cytoplasm of microconidia after irradiation through generation of abundant intracellular ROS. AE-mediated aPDT demonstrated effective therapeutic effects for T. rubrum-caused tinea corporis on guinea pig model and tinea unguium in ex vivo model. The results obtained suggest that AE is a potential PS for the photodynamic treatment of dermatophytosis caused by T. rubrum, but its permeability in skin and nails needs to be improved.     

Staphylococcus aureus protects its immune‐evasion proteins against degradation by neutrophil serine proteases

Neutrophils store large quantities of neutrophil serine proteases (NSPs) that contribute, via multiple mechanisms, to antibacterial immune defences. Even though neutrophils are indispensable in fighting Staphylococcus aureus infections, the importance of NSPs in anti‐staphylococcal defence is yet unknown. However, the fact that S. aureus produces three highly specific inhibitors for NSPs [the extracellular adherence proteins (EAPs) Eap, EapH1 and EapH2], suggests that these proteases are important for host defences against this bacterium. In this study we demonstrate that NSPs can inactivate secreted virulence factors of S. aureus and that EAP proteins function to prevent this degradation. Specifically, we find that a large group of S. aureus immune‐evasion proteins is vulnerable to proteolytic inactivation by NSPs. In most cases, NSP cleavage leads to functional inactivation of virulence proteins. Interestingly, proteins with similar immune‐escape functions appeared to have differential cleavage sensitivity towards NSPs. Using targeted mutagenesis and complementation analyses in S. aureus, we demonstrate that all EAP proteins can protect other virulence factors from NSP degradation in complex bacterial supernatants. These findings show that NSPs inactivate S. aureus virulence factors. Moreover, the protection by EAP proteins can explain why this antibacterial function of NSPs was masked in previous studies. Furthermore, our results indicate that therapeutic inactivation of EAP proteins can help to restore the natural host immune defences against S. aureus.     

Antibacterial effect of silver nanoparticles on <Emphasis Type="Italic">Staphylococcus aureus</Emphasis>

The antibacterial activity and mechanism of silver nanoparticles (Ag-NPs) on Staphylococcus aureus ATCC 6538P were investigated in this study. The experiment results showed the minimum bactericidal concentration (MBC) of Ag-NPs to S. aureus was 20 μg/ml. Moreover, when bacteria cells were exposed to 50 μg/ml Ag-NPs for 6 h, the cell DNA was condensed to a tension state and could have lost their replicating abilities. When S. aureus cells were exposed to 50 μg/ml Ag-NPs for 12 h, the cell wall was breakdown, resulting in the release of the cellular contents into the surrounding environments, and finally became collapsed. And Ag-NPs could reduce the enzymatic activity of respiratory chain dehydrogenase. Furthermore, the proteomic analysis showed that the expression abundance of some proteins was changed in the treated bacterial cell with Ag-NPs, formate acetyltransferase increased 5.3-fold in expression abundance, aerobic glycerol-3-phosphate dehydrogenase decreased 6.5-fold, ABC transporter ATP-binding protein decreased 6.2-fold, and recombinase A protein decreased 4.9-fold.     

Longitudinal survey of <Emphasis Type="Italic">Staphylococcus aureus</Emphasis> in cystic fibrosis patients using a multiple-locus variable-number of tandem-repeats analysis method

Background  

Staphylococcus aureus infection in patients with cystic fibrosis (CF) is frequent and may be due to colonization by a few pathogenic lineages. Systematic genotyping of all isolates, methicillin-susceptible S. aureus (MSSA) as well as methicillin-resistant S. aureus (MRSA) is necessary to identify such lineages and follow their evolution in patients. Multiple-locus variable-number tandem repeat analysis (MLVA/VNTR) was used to survey S. aureus clinical isolates in a French paediatric CF centre.