In Vitro Potentiation of Antimicrobial Photodynamic Inactivation by Addition of Potassium Iodide.

The current increase in antibiotic resistance worldwide and the emergence of microbial strains that are resistant to all known antibiotics have stimulated research into novel strategies such as aPDI that are thought to be unlikely to lead to the development of resistance. Although many studies have reported in vitro aPDI killing of microorganisms by a range of different photosensitizers, there are still limitations to the effectiveness of aPDI, and recurrence of bacterial growth may occur in animal studies after completion of the illumination. In this chapter we cover a novel and relatively simple method to improve the efficacy of aPDI against Gram-positive Staphylococcus aureus, Gram-negative Escherichia coli, and fungal yeast Candida albicans by the addition of potassium iodide, a nontoxic inorganic salt. Under some circumstances up to six-logs additional killing can be obtained.

In Vivo Potentiation of Antimicrobial Photodynamic Therapy in a Mouse Model of Fungal Infection by Addition of Potassium Iodide.

Antimicrobial photodynamic inactivation (aPDI) involves the use of a nontoxic dye or photosensitizer excited with visible light to produce reactive oxygen species that can kill all classes of microorganisms. Antimicrobial photodynamic therapy (aPDT) can be used in vivo as an alternative therapeutic strategy to treat localized infections due to its ability to selectively kill microbes while preserving host mammalian cells. aPDI can be potentiated by the addition of the nontoxic inorganic salt potassium iodide (KI). KI is an approved drug for antifungal therapy. The mechanism of potentiation with iodide is likely to be singlet oxygen addition to iodide to form iodine radicals, hydrogen peroxide, and molecular iodine. A previous chapter in this volume described potentiation of aPDI in vitro by addition of KI, while in this chapter we address the ability of KI to potentiate aPDT in vivo using an animal model of localized fungal infection. We employed oral candidiasis in immunosuppressed mice caused by a bioluminescent strain of Candida albicans and monitored by bioluminescence imaging.

Postbiotics of Lactobacillus casei target virulence and biofilm formation of Pseudomonas aeruginosa by modulating quorum sensing.

Among various anti-virulence aspects, the efficacy of the bioactive constituents of probiotics, referred to as postbiotics, to affect quorum sensing (QS)-modulated signaling of pathogens, is considered as a safe natural approach. The present study investigated the potential QS-inhibitory activity of lyophilized postbiotics from Lactobacillus casei sub sp. casei PTCC 1608 on virulence phenotypes and biofilm of two strains and three clinical isolates of Pseudomonas aeruginosa. The effect of L. casei postbiotics (LCP) at sub-minimum inhibitory concentration on the expression of QS genes including lasR/I, rhlR/I, pqsA, pqsR and virulence genes including pelF (pellicle/biofilm glycosyltransferase PelF), lasB (elastase LasB) and toxA (exotoxin A) was evaluated. The viability of mouse fibroblastic NIH/3T3 cell line treated with sub-MICS of LCP was also investigated. Postbiotics were characterized using mass spectrometry-based analyses. The QS-attenuation effect of pure lactic acid as the major constituent of LCP was determined on P. aeruginosa strains. Neutralized postbiotics and crude bacteriocin did not exhibit any antibacterial activity. It was found that sub-MICS of LCP could more drastically attenuate the tested virulence phenotypes and biofilm formation than lactic acid. Biofilm inhibition was confirmed using scanning electron microscopy. The rhlI, rhlR, and pelF genes were down-regulated after treatment with LCP. No cytotoxicity effect was observed on NIH/3T3 cell line. The findings demonstrated that postbiotics of L. casei could reduce the virulence and biofilm development of P. aeruginosa and suggested a novel safe natural source for the expansion of anti-virulence treatments.

Eradication of Acinetobacter baumannii Planktonic and Biofilm Cells Through Erythrosine-Mediated Photodynamic Inactivation Augmented by Acetic Acid and Chitosan.

Photodynamic inactivation (PDI) is an attractive treatment modality for multidrug-resistant bacterial infections. The effectiveness of photosensitization by anionic photosensitizers such as erythrosine B can be further enhanced by the addition of biological or chemical molecules. This study aimed to investigate of the enhancement effect of acetic acid and chitosan on erythrosine-mediated PDI of Acinetobacter baumannii in planktonic and biofilm forms. The planktonic cell growth of three A. baumannii strains was subjected to PDI by using erythrosine B (50 µM) in 0.01% acetic acid and green laser light (530 nm) at fluence of 40 J/cm2. The phototoxic effect of erythrosine B (100 µM) in combination with chitosan (12.5 mg/ml) (in a solution of acetic acid) at fluence of 80 J/cm2 on biofilms was also evaluated. Finally, the cytotoxicity and phototoxicity of the mentioned mixture were assessed on human fibroblasts. Planktonic cells of all three studied A. baumannii strains were almost eradicated by erythrosine B-mediated PDI in the presence of acetic acid. Also, PDI combined with chitosan resulted in a marked decrease in the number of viable biofilm cells (> 3 log10 CFU). At the same experimental conditions, only 15% of the fibroblasts were photoinactivated. The results showed that PDI by using erythrosine B in acetic acid is very effective against A. baumannii planktonic cells and could eliminate them significantly. Also, chitosan enhanced the anti-biofilm efficacy of erythrosine B-mediated PDI against A. baumannii, suggesting that combination therapy may be useful in targeting biofilms.

Quorum sensing-regulated functions of Serratia marcescens are reduced by eugenol.

BACKGROUND AND OBJECTIVES: Serratia marcescens has emerged as a nosocomial pathogen responsible for human infections, where antibiotic resistance further complicates the treatments. In S. marcescens, biofilm formation and virulence factor production are controlled via quorum sensing (QS) system. QS is a signaling system that enables gene regulation to control diverse physiological functions in bacteria. Essential oils have shown to be potential in diminishing the pathogenicity and virulence of drug-resistant bacteria. This study was performed to determine whether eugenol would affect QS system, biofilm formation and virulence factor production of S. marcescens. MATERIALS AND METHODS: Biofilm formation, extracellular virulence factor production (hemolysin and protease), swarming motility and pigment formation of S. marcescens ATCC 13880 and S. marcescens Sm2 were assessed after eugenol exposure at 1.25 and 2.5 µg/ml concentrations. The expression of genes involved in motility (flhD), attachment (fimC), biofilm formation (bsmB, bsmA), and QS regulatory (swrR) were also evaluated. RESULTS: Eugenol treatment at 1.25 and 2.5 µg/ml concentrations caused a significant reduction in biofilm formation. The pigment, hemolysin and protease production of two studied S. marcescens strains, also reduced significantly by eugenol treatments (p<0.05). The bsmA, bsmB, flhD and fimC genes were down-regulated after eugenol treatment. The swrR gene expression was also reduced significantly by eugenol in both S. marcescens strains (p<0.05). CONCLUSION: Eugenol inhibited quorum sensing-regulated functions of two studied S. marcescens strains.

Photodynamic inactivation diminishes quorum sensing-mediated virulence factor production and biofilm formation of Serratia marcescens.

Serratia marcescens is an opportunistic human pathogen causing nosocomial infections and displays expanded resistance towards the conventional antibiotics. In S. marcescens, quorum sensing (QS) mechanism coordinates the population-dependent behaviors and regulates the virulence factors production. Photodynamic inactivation (PDI) is a promising alternative for the treatment of infections caused by drug resistant bacteria. Although PDI should be applied at lethal doses, it is possible that during PDI treatment, pathogens encounter sub-lethal doses of PDI (sPDI). sPDI cannot kill microorganisms, but it can considerably influence the microbial virulence. So, in this study, the effect of methylene blue (MB)-mediated PDI on QS-mediated virulence factor production and biofilm formation of S. marcescens at lethal and sub-lethal doses was evaluated. The biofilm formation and virulence factor production of S. marcescens ATCC 13,880 and S. marcescens Sm2 were assessed before and after PDI treatment. Besides, the effect of lethal and sub-lethal PDI on expression of bsmA and bsmB (Biofilm maturation), fimA and fimC (Major fimbrial protein), flhD (Regulator of flagellar mediated swarming and swimming motility) and swrR (AHL-dependent regulator) genes were evaluated by quantitative real time polymerase chain reaction. Lethal and sub-lethal PDI resulted in a significant decrease in biofilm formation, swimming/swarming motility, and pigment and hemolysin production ability of S. marcescens strains. bsmA, bsmB, flhD and swrR genes were down-regulated after PDI treatments. In conclusion, QS-mediated virulence factor production and biofilm formation ability of the two studied S. marcescens strains decreased after both lethal and sub-lethal PDI.

Sub-lethal antimicrobial photodynamic inactivation affects Pseudomonas aeruginosa PAO1 quorum sensing and cyclic di-GMP regulatory systems.

BACKGROUND: Antimicrobial photodynamic inactivation (APDI) is a new therapeutic modality which needs more precision during application due to the possibility of exposure of bacteria to sub-lethal doses (sAPDI). In this study, we aimed to evaluate the effect of sAPDI on Pseudomonas aeruginosa quorum sensing (QS) and c-di-GMP signaling which are important virulence factor regulatory systems. METHODS: Biofilm formation, pyoverdine, pyocyanin and protease production of P. aeruginosa was evaluated before and after a single sAPDI treatment with 0.8 mM methylene blue (MB) plus 1, 2, and 5-min irradiation with red laser light. Fluorescent lasB, rhlA, pqsA, and cdrA reporters of P. aeruginosa PAO1 and P. aeruginosa ΔmexAB-oprM were treated individually with sAPDI and the regulatory signals were detected. The gene expressions were also assessed after sAPDI using quantitative real-time PCR analysis. RESULTS: Morphological observations and molecular assessments indicated that sAPDI with 0.8 mM MB along with 2- and 5-min irradiation led to an increase in the expression of the Las QS system and c-di-GMP signaling, while 1 min irradiation revealed dissimilar results (increase in lasB expression and decrease in c-di-GMP levels). Expression of rhlA and pqsA did not change in response to sAPDI. Further, a severe lethal effect of sAPDI was observed in P. aeruginosa ΔmexAB-oprM as compared with the wild type strain, whilst there was no difference in QS and c-di-GMP levels as detected by reporters between treated and untreated samples. CONCLUSION: The results suggest that sAPDI affects QS and c-di-GMP signaling inP. aeruginosa in a time-dependent manner.

Quorum-sensing-regulated virulence factors in Pseudomonas aeruginosa are affected by sub-lethal photodynamic inactivation.

BACKGROUND: Photodynamic inactivation (PDI) is recognized as a new antimicrobial approach. It is likely that in human hosts receiving this therapy, pathogens may encounter sub-lethal doses of PDI (sPDI), which may affect microbial virulence. This study was aimed to evaluate the effect of sPDI using methylene blue (MB) on the expression of genes belonging to two quorum sensing (QS) operons (rhl and las systems) and two genes necessary for pyocyanin and rhamnolipid production (phzM and rhlA) under QS control in Pseudomonas aeruginosa. METHODS: Ability of pyocyanin and rhamnolipid production of P. aeruginosa ATCC 27853 and clinical isolates exposed to sPDI (MB at 0.012 mM and light dose of 23 J/cm2 was evaluated. The effect of sPDI on expression of rhlI, rhlR, lasI, lasR, phzM and rhlA were also evaluated by quantitative real time polymerase chain reaction. RESULTS: sPDI led to the down-regulation of the expression of all four QS genes (lasI, lasR, rhlI and rhlR) and rhamnolipid gene (rhlA). However, up-regulation of pyocyanin gene (phzM) was observed after sPDI. These results were consistent with phenotypic changes. CONCLUSION: This study suggests that oxidative stress induced by sPDI can affect QS-regulated virulence factors of P. aeruginosa such as pyocyanin and rhamnolipids in different ways.

Sub-lethal antimicrobial photodynamic inactivation: an in vitro study on quorum sensing-controlled gene expression of Pseudomonas aeruginosa biofilm formation.

During antimicrobial photodynamic inactivation (APDI) in the treatment of an infection, it is likely that microorganisms would be exposed to sub-lethal doses of APDI (sAPDI). Although sAPDI cannot kill microorganisms, it can significantly affect microbial virulence. In this study, we evaluated the effect of sAPDI using methylene blue (MB) on the expression of genes belonging to two quorum sensing (QS) operons (rhl and las systems) and two genes necessary for biofilm formation (pelF and pslA) under QS control in Pseudomonas aeruginosa. Biofilm formation ability of P. aeruginosa ATCC 27853 exposed to sAPDI (MB at 0.012 mM and light dose of 23 J/cm2) was evaluated using triphenyl tetrazolium chloride (TTC) assay and scanning electron microscopy (SEM). The effect of sAPDI on expression of rhlI, rhlR, lasI, lasR, pelF, and pslA were also evaluated by quantitative real-time polymerase chain reaction. Quantitative assay (TTC) results and morphological observations (SEM) indicated that a single sAPDI treatment resulted in a significant decrease in biofilm formation ability of P. aeruginosa ATCC 27853 compared to their non-treated controls (P = 0.012). These results were consistent with the expression of genes belonging to rhl and las systems and pelF and pslA genes. The results suggested that the transcriptional decreases caused by MB-sAPDI did lead to phenotypic changes.

Advances in antimicrobial photodynamic inactivation at the nanoscale.

The alarming worldwide increase in antibiotic resistance amongst microbial pathogens necessitates a search for new antimicrobial techniques, which will not be affected by, or indeed cause resistance themselves. Light-mediated photoinactivation is one such technique that takes advantage of the whole spectrum of light to destroy a broad spectrum of pathogens. Many of these photoinactivation techniques rely on the participation of a diverse range of nanoparticles and nanostructures that have dimensions very similar to the wavelength of light. Photodynamic inactivation relies on the photochemical production of singlet oxygen from photosensitizing dyes (type II pathway) that can benefit remarkably from formulation in nanoparticle-based drug delivery vehicles. Fullerenes are a closed-cage carbon allotrope nanoparticle with a high absorption coefficient and triplet yield. Their photochemistry is highly dependent on microenvironment, and can be type II in organic solvents and type I (hydroxyl radicals) in a biological milieu. Titanium dioxide nanoparticles act as a large band-gap semiconductor that can carry out photo-induced electron transfer under ultraviolet A light and can also produce reactive oxygen species that kill microbial cells. We discuss some recent studies in which quite remarkable potentiation of microbial killing (up to six logs) can be obtained by the addition of simple inorganic salts such as the non-toxic sodium/potassium iodide, bromide, nitrite, and even the toxic sodium azide. Interesting mechanistic insights were obtained to explain this increased killing.

Can microbial cells develop resistance to oxidative stress in antimicrobial photodynamic inactivation?

Infections have been a major cause of disease throughout the history of humans on earth. With the introduction of antibiotics, it was thought that infections had been conquered. However, bacteria have been able to develop resistance to antibiotics at an exponentially increasing rate. The growing threat from multi-drug resistant organisms calls for intensive action to prevent the emergence of totally resistant and untreatable infections. Novel, non-invasive, non-antibiotic strategies are needed that act more efficiently and faster than current antibiotics. One promising alternative is antimicrobial photodynamic inactivation (APDI), an approach that produces reactive oxygen species when dyes and light are combined. So far, it has been questionable if bacteria can develop resistance against APDI. This review paper gives an overview of recent studies concerning the susceptibility of bacteria towards oxidative stress, and suggests possible mechanisms of the development of APDI-resistance that should at least be addressed. Some ways to potentiate APDI and also to overcome future resistance are suggested.

In vitro Activity of Linezolid in Combination with Photodynamic Inactivation Against Staphylococcus aureus Biofilms.

BACKGROUND: Biofilm infections are a major challenge in medical practice. Bacteria that live in a biofilm phenotype are more resistant to both antimicrobial therapy and host immune responses compared to their planktonic counterparts. So, there is need for new therapeutic strategies to combat these infections. A promising approach [known as Photodynamic Inactivation (PDI)] to kill bacteria growing as biofilms uses light in combination with a photosensitizer to induce a phototoxic reaction which produces reactive oxygen species that can destroy lipids and proteins causing cell death. PDI does not always guarantee full success, so, combination of PDI with antibiotics may give increased efficiency. This study aimed to determine if PDI was effective in the eradication of Staphylococcus aureus (S. aureus) biofilms in combination with linezolid. METHODS: The susceptibility of biofilm cultures of three S. aureus strains to Methylene Blue (MB) and Toluidine Blue O (TBO)-mediated PDI was determined alone and in combination with linezolid. RESULTS: Bactericidal activity (≥3 log10 reduction in viable cell count) was not achieved with MB/TBO-PDI or antibiotic treatment alone. When antibiotic treatment was combined with TBO-PDI, a greater reduction in viable count than antibiotic alone was observed for two strains. CONCLUSION: This study showed that although TBO-PDI did not have good bactericidal activity against S. aureus biofilms; it increased the antimicrobial activity of linezolid against these bacteria.

Chitosan nanoparticles enhance the efficiency of methylene blue-mediated antimicrobial photodynamic inactivation of bacterial biofilms: An in vitro study.

BACKGROUND: Biodegradable chitosan nanoparticles (CSNPs) with an intrinsic antimicrobial activity may be a good choice to improve the effectiveness of antimicrobial photodynamic inactivation (APDI). The aim of this study was to investigate the effect of CSNPs on the efficiency of methylene blue (MB)-mediated APDI of Staphylococcus aureus and Pseudomonas aeruginosa biofilms. We also assessed the phototoxicity of MB+CSNPs towards human fibroblasts. METHODS: CSNPs were prepared using ionic gelation method and characterized by dynamic light scattering (DLS) and field-emission scanning electron microscope (FESEM). Biofilms were developed in a 96-well polystyrene plate for 24h. In vitro phototoxic effect of MB+CSNPs (at final concentrations of 50µM MB) at fluence of 22.93J/cm(2)) on biofilms were studied. Appropriate controls were included. Also, in vitro cytotoxicity and phototoxicity of the above mixture was assessed on human dermal fibroblasts. RESULTS: DLS and FESEM measurements confirmed the nanometric size of the prepared CSNPs. APDI mediated by the mixture of MB and CSNPs showed significant anti-biofilm photoinactivation (P<0.001, >3 and >2 log10 CFU reduction in S. aureus and P. aeruginosa biofilms, respectively) while MB-induced APDI led to approximately <1 log10 CFU reduction. At the same experimental conditions, only 25.1% of the fibroblasts were photoinactivated by MB+CSNPs. CONCLUSION: Our findings showed that CSNPs enhanced the efficacy of MB-APDI; it may be due to the disruption of biofilm structure by polycationic CSNPs and subsequently deeper and higher penetration of MB into the biofilms.

Phototoxic effect of hypericin alone and in combination with acetylcysteine on Staphylococcus aureus biofilms.

BACKGROUND: Resistance of bacteria against antibiotics and antimicrobials is arising worldwide and there is an urgent need for strategies that are capable of inactivating biofilm-state pathogens with less potential of developing resistance in pathogens. A promising approach could be photodynamic inactivation (PDI) which uses light in combination with a photosensitizer to induce a phototoxic reaction. In this study, we evaluated the in vitro phototoxic effect of hypericin (HYP) alone and in combination with acetylcysteine (AC) on Staphylococcus aureus biofilms. AC, a mucolytic agent, reduces the production of extracellular polysaccharide matrix while promoting the disruption of mature biofilm. METHODS: In vitro phototoxic effect of HYP alone (0.5 µg/ml, light dose: 16 J/cm(2)), and in combination with AC (10 mg/ml) on ten clinical S. aureus isolates and S. aureus (ATCC 25923) biofilms was studied. Effect of HYP concentration (0.5 µg/ml) and light dose (8 J/cm(2)) on PDI of all eleven S. aureus strains in planktonic forms was also investigated. RESULTS: HYP-PDI did not result in a reduction in viable count for each of the strains when grown in biofilms. However, HYP-PDI applied on biofilms treated with AC was able to disrupt pre-formed biofilms (viable count reduction ranging from 5.2 to 6.3 log10-unit in comparison to controls in all tested strains). A 6.5 log killing was obtained for S. aureus (ATCC 25923) planktonic cells treated with 0.5 µg/ml at 8 J/cm(2). For this set of PDI parameters, ten clinical S. aureus isolates showed 5.5-6.7 log killing. CONCLUSION: HYP-PDI in combination with AC had significant ability to eradicate the pre-formed mature biofilms of S. aureus strains.

Effect of sub-lethal photodynamic inactivation on the antibiotic susceptibility and biofilm formation of clinical Staphylococcus aureus isolates.

BACKGROUND: A promising approach to kill antibiotic-resistant bacteria uses light in combination with a photosensitizer to induce a phototoxic reaction. A major concern with the use of any non-antibiotic antimicrobial treatment is that exposure of bacteria to sub-lethal concentrations will lead to the development of resistance to antibiotics. This study aimed to determine the effect of sub-lethal photodynamic inactivation (PDI) on the antibiotic susceptibility and biofilm formation of clinical Staphylococcus aureus isolates. METHODS: Forty clinical S. aureus isolates were exposed to PDI with toluidine blue O (TBO) and methylene blue (MB). After exposure, susceptibility of surviving organisms to a range of antibiotics was determined and compared with the susceptibility of an untreated control. PDI experiments were done during three generations for assessment of biofilm formation, to determine if biofilm formation was affected by exposure to PDI. RESULTS: It was observed that the effect of sub-lethal PDI on the antibiotic sensitivity was strain-dependent. In general, exposure to sub-lethal MB/TBO-PDI increased resistance to erythromycin, amoxicillin-clavulanate and amikacin. Biofilm formation ability of studied clinical isolates increased after second sub-lethal PDI regimen compared to that before PDI. CONCLUSION: S. aureus cells may develop resistance by growing in the presence of sub-lethal MB/TBO-PDI.

Photodynamic effect of hypericin on the microorganisms and primary human fibroblasts.

BACKGROUND: Hypericin (HYP) is a natural photosensitizer considered for the new generation of photodynamic therapy (PDT) drugs. The aim of this study was to evaluate the in vitro bactericidal effect of HYP-PDT on four bacterial species, assessing its photocytotoxicity to primary human fibroblasts to determine possible side effects. METHODS: Effect of photosensitizer concentration (0.1, 0.3, 0.6, and 1 µg/ml) and light irradiation time (3, 5, 10 min) on photodynamic inactivation of microorganisms and primary human fibroblasts was investigated. RESULTS: A 6.3 log killing was obtained for Staphylococcus aureus (ATCC 25923) treated with 1 µg/ml at 48 J/cm². For this set of PDT parameters, Enterococcus faecalis (ATCC 11700) showed 6.5 log killing, Escherichia coli (ATCC 25922) 6.2 log killing, and Pseudomonas aeruginosa (ATCC 27853) 0.7 log killing. Fibroblasts can be preserved by keeping the HYP concentration below 0.6 µg/ml and the light dose below 48 J/cm². CONCLUSION: S. aureus, E. faecalis, and E. coli appear to be suitable for treatment with HYP-PDT without significant damage to fibroblasts.

Photodynamic inactivation of primary human fibroblasts by methylene blue and toluidine blue O.

BACKGROUND: An important determinant of the clinical applicability and value of antimicrobial photodynamic inactivation (PDI) is the cytotoxicity of the treatment to human cells. We evaluated the in vitro cytotoxicity of PDI to primary human fibroblasts using methylene blue (MB) and toluidine blue O (TBO) as the photosensitizers. METHODS: The primary human fibroblasts were exposed to PDI regimes that were used for the inactivation of methicillin-resistant Staphylococcus aureus (MRSA) and multi-drug resistant Escherichia coli (MDR E. coli). Mitochondrial activity subsequent to exposure was evaluated after 24h using the methylthiazoletetrazolium assay and compared to pretreatment values. RESULTS: Mitochondrial activity of primary human fibroblasts was reduced by 27% after exposure to light (163.8 J/cm(2)) and MB (50 µg/ml). At a TBO concentration previously demonstrated to induce 99.91% and 83.2% reduction in a viable count for MRSA and MDR E. coli, respectively, 39.6% of the fibroblasts were photo-inactivated. CONCLUSION: Our findings showed that MB/TBO-PDI did not induce significant cytotoxic effects on human fibroblasts in culture.

Phototoxicity of phenothiazinium dyes against methicillin-resistant Staphylococcus aureus and multi-drug resistant Escherichia coli.

BACKGROUND: Photodynamic inactivation (PDI) has been investigated to cope with the increasing incidence of multidrug-resistant (MDR) pathogens. Here we studied the PDI mediated by methylene blue (MB) and toluidine blue O (TBO) in clinical methicillin-resistant Staphylococcus aureus and MDR Escherichia coli, together with their corresponding American Type Culture Collection (ATCC) strains. METHODS: Effect of photosensitizer concentration (12.5, 25, 50 µg/ml) and laser irradiation time (10, 20 and 30 min) on lethal photosensitization was investigated. RESULTS: TBO was more effective. TBO at 50 µg/ml, 46.8 J cm⁻², exhibited 0.7 log killing for MDR E. coli and 1.7 log killing for E. coli (ATCC 25922); 3.1 log killing for MRSA, and 4.2 log killing for S. aureus (ATCC 25923). MB at 50 µg/ml, 163.8 J cm⁻², only exhibited 2.2 log killing in MRSA and 3.1 log killing in S. aureus (ATCC 25923). MB (50 µg/ml, 163.8 J cm⁻²) induced 0.2 log killing for MDR E. coli and 0.3 log killing for E. coli (ATCC 25922). After TBO-PDI, MDR isolates were more susceptible to some antibiotics than control groups. CONCLUSION: Our studied clinical isolates were more resistant to PDI-mediated killing than their ATCC reference strains. Thus, TBO/MB-mediated PDI in other MDR isolates deserves further investigation.

Application of fnbA gene as new target for the species-specific and quantitative detection of Staphylococcus aureus directly from lower respiratory tract specimens by real time PCR.

Staphylococcus aureus is a significant cause of hospital-acquired pneumonia (HAP), particularly in mechanically ventilated patients. We used the fibronectin-binding protein A gene (fnbA) for the species-specific and quantitative detection of S. aureus directly from lower respiratory tract (LRT) specimens by a Taq Man real time PCR. For this reason, a total of 269 lower respiratory tract (LRT) specimens collected from patients with hospital-acquired pneumonia were assayed. Amplification of fnbA in serial dilutions ranged from 10(9) CFU/ ml to 10(2) CFU/ml. Standard curve of triplicate every dilution had slope 3.34±0.1 and R2>0.99 with SD 0.1. Based on these data, the sensitivity and specificity of the newly developed real time PCR targeting the fnbA gene were both 100%. The Cohen's Kappa test showed the Kappa value of 1.0. The fnbA gene is a potential marker for the species-specific detection of S. aureus and can be used to detect this bacterium in any clinical specimens by real time PCR. Moreover, this method reduces the time needed for quantitative detection of Staphylococcus aureus from LRT specimens to nearly 2 hours compared to 1 to 4 days for culture and provided sensitivity equal to or greater than culture.

A randomized clinical trial on the effect of low-level laser therapy on chronic diabetic foot wound healing: a preliminary report.

BACKGROUND AND OBJECTIVES: Low-level laser therapy (LLLT) has been shown to promote chronic wound healing in conditions of reduced microcirculation. In this preliminary study, we report the results of using LLLT to heal foot ulcers in patients with diabetes mellitus. MATERIALS AND METHODS: Twenty-three patients with a diabetic foot wound for at least 3 months were included in this double-blind randomized clinical trial. Patients were randomized to receive placebo treatment (n = 10) or LLLT (n = 13) (685 nm, energy density 10 J/cm(2)) in addition to conventional therapy. Patients were followed for 20 weeks. Ulcer size reduction and the number of patients with complete healing were compared between the LLLT and placebo groups. RESULTS: There were no significant differences in baseline characteristics of patients and foot ulcers receiving LLLT and placebo treatment. At week 4, the size of ulcers decreased significantly in the LLLT group (p = 0.04). After 20 weeks, in the LLLT group, eight patients had complete healing and in the placebo group only three patients experienced complete wound healing. The mean time of complete healing in LLLT patients (11 weeks) was less than that in placebo patients (14 weeks) though the difference was not statistically significant. CONCLUSIONS: The study provides evidence that LLLT can accelerate the healing process of chronic diabetic foot ulcers, and it can be presumed that LLLT may shorten the time period needed to achieve complete healing.