Domestic LED bulb induced photodynamic effect of Toluidine Blue O-embedded silicone catheters against urinary tract infection.

BACKGROUND: Novel combination of Toluidine Blue O (TBO) embedded silicone catheter with domestic/household LED bulb has a potential in clinical infection such as prevention of multi drug resistant catheter-associated urinary tract infections (CAUTIs) through photodynamic therapy. MATERIAL AND METHODS: Preliminarily, TBO was entrapped into silicone catheter by swell-encapsulation-shrink method. Further, in vitro study was carried out to check the antimicrobial photodynamic efficacy of TBO with domestic/household LED light. Antibiofilm activity was evaluated by scanning electron microscopy. RESULTS: The results showed that these modified TBO embedded silicone catheters showed significant antimicrobial and antibiofilm activity against vancomycin resistant Staphylococcus aureus VRSA. Small piece (1 cm) of TBO-embedded silicone catheter (700 µM) showed 6 log10 reduction in the viable count when exposed for only 5 min of domestic/household LED bulb, while 1 cm piece of 500 µM and 700 µM concentration of TBO-embedded catheter eradicated all bacterial load when exposed to 15 min of light. Segment of medical grade TBO-embedded silicone catheters were used to carry out investigation of reactive oxygen species generation mainly singlet oxygen that contributes to type II phototoxicity. CONCLUSION: These modified catheter provides cost effective, easy to manage and less time consuming therapy to eliminate CAUTIs.

Antimicrobial and antibiofilm photodynamic therapy against vancomycin resistant Staphylococcus aureus (VRSA) induced infection in vitro and in vivo.

Biofilm mediated infection caused by multi-drug resistant bacteria are difficult to treat since it protects the microorganisms by host defense system, making them resistant to antibiotics and other antimicrobial agents. Combating such type of nosocomial infection, especially in immunocompromised patients, is an urgent need and foremost challenge faced by clinicians. Therefore, antimicrobial photodynamic therapy (aPDT) has been intensely pursued as an alternative therapy for bacterial infections. aPDT leads to the generation of reactive oxygen species (ROS) that destroy bacterial cells in the presence of a photosensitizer, visible light and oxygen. Here, we elucidated a possibility of its clinical application by reducing the treatment time and exposing curcumin to 20 J/cm2 of blue laser light, which corresponds to only 52 s to counteract vancomycin resistant Staphylococcus aureus (VRSA) both in vitro and in vivo. To understand the mechanism of action, the generation of total reactive oxygen species (ROS) was quantified by 2'-7'-dichlorofluorescein diacetate (DCFH-DA) and the type of phototoxicity was confirmed by fluorescence spectroscopic analysis. The data showed more production of singlet oxygen, indicating type-II phototoxicity. Different anti-biofilm assays (crystal violet and congo red assays) and microscopic studies were performed at sub-MIC concentration of curcumin followed by treatment with laser light against preformed biofilm of VRSA. The result showed significant reduction in the preformed biofilm formation. Finally, its therapeutic potential was validated in skin abrasion wistar rat model. The result showed significant inhibition of bacterial growth. Furthermore, immunomodulatory analysis with rat serum was performed. A significant reduction in expression of proinflammatory cytokines TNF-α and IL-6 were observed. Hence, we conclude that curcumin mediated aPDT with 20 J/cm2 of blue laser treatment (for 52 s) could be used against multi-drug resistant bacterial infections and preformed biofilm formation as a potential therapeutic approach.

Inhibition of multi-drug resistant Klebsiella pneumoniae: Nanoparticles induced photoinactivation in presence of efflux pump inhibitor.

In the current scenario, frontline antibiotics are losing effectiveness against multidrug-resistant (MDR) bacteria because of the single mode of action. The accumulation of mutations and spread of antibiotic resistance markers among the bacteria results into the severe threat to community health. Now, there is an urgent requirement for the development of an alternate and as well as multiple-targeted action of drugs to stop the spread of resistance in bacteria. Here, we showed an alternative nanoparticle based photodynamic therapy (PDT) targeting the bacterial efflux pumps and its cell wall. The dextran capped gold nanoparticles (GNPDEX) were localized to the bacterial surface by nanoparticle attached Concanavalin-A (ConA), where GNPDEX attached methylene blue (MB) photosensitizer as an MB@GNPDEX-ConA formulation induced the killing of MDR Klebsiella pneumoniae clinical isolates in no time. The intervention of efflux pump inhibitor (EPI) further improved the MB@GNPDEX-ConA treatment modality and displayed the maximum bactericidal cytoplasmic phototoxicity. The CCCP EPI (carbonyl cyanide m-chlorophenylhydrazone) with the PDT increased the bacterial killing by>3 log10 as compared with or without EPI intervention. Further, the fractionated (two light treatment after long dark phase) PDT treatment modality decreased the bacterial biofilm growth up to ~90%. The microscopic as well as ROS fluorescent probes showed the singlet oxygen mediated cytotoxicity. The mode of interactions and genomic DNA photo-toxicity confirmed that EPI enhanced the killing mediated by singlet oxygen generation. The multi-targeted (Cell wall, DNA and efflux pump) modality of MB@GNPDEX-ConA in presence of EPI is an effective and alternative therapeutic approach against most potent Klebsiella MDR infections.

Curcumin induced photodynamic therapy mediated suppression of quorum sensing pathway of Pseudomonas aeruginosa: An approach to inhibit biofilm in vitro.

OBJECTIVE: The objective of this study was to inhibit the Pseudomonas aeruginosa biofilm through curcumin-mediated antimicrobial photodynamic therapy (A-PDT). BACKGROUND: The mechanism behind A-PDT mediated photoinactivation depend upon reactive oxygen species (ROS) production, like singlet oxygen and free radicals. METHODS: To evaluate the antibacterial efficacy of curcumin induced A-PDT on P. aeruginosa by colony forming unit (CFU) while antibiofilm action was determined by the use of crystal violet, XTT, congored binding assay and confocal laser scanning microscope (CLSM). RESULTS: We found that curcumin with 10 J/cm2 of light reduces P. aeruginosa biofilm more efficiently than without light. Extracellular polymeric substances (EPS) production was also reduced by approx 94 % with 10 J/cm2 of light dose. CLSM images showed that the thickness of biofilms were reduced from >30 µm to <5 µm after treatment with curcumin followed by 10 J/cm2 of light irradiation. Curcumin showed better bacteriostatic activity than bactericidal activity. Singlet oxygen is primarily responsible for photodamage and cytotoxic reactions caused by curcumin-mediated APDT. Genes involved in quorum sensing (QS) pathway was also found to be inhibited after APDT. Curcumin with 5 J/cm2 light inhibits QS genes and on increasing light dose i.e10 J/cm2, we found a drastic reduction in gene expression. CONCLUSION: We conclude that the curcumin mediated A-PDT inhibits biofilm formation ofP. aeruginosa through QS pathway by the action of singlet oxygen generation which in turn reduced EPS of the biofilm.

Synergistic fungicidal activity with low doses of eugenol and amphotericin B against Candida albicans.

Candida albicans frequently causes variety of superficial and invasive disseminated infections in HIV infected patients. Further, the emergence of non albicans species causing candidiasis predominantly in patients with advanced immune-suppression and drug resistance brings great apprehension. Hence, in this study we evaluate the capability of eugenol (EUG), a natural compound in combination with less toxic concentrations of amphotericin B (AmpB) for enhanced antifungal effects and reduced toxicity. Antifungal activity and time-kill assay were employed according to Clinical Laboratory Standard Institute (CLSI) guidelines with minor modifications on clinical isolates of Candida albicans. To confirm the synergistic interaction of EUG and AmpB, checkerboard experiments were employed. Interestingly, EUG-Amp B combination shows many fold higher anti-candida activity compared to single component treatment. Furthermore, our results depicts reactive oxygen species (ROS) driven killing and mitochondrial hyperpolarisation on treatment. Our data also suggests inhibition of calcium channel by EUG and predicts longer retainment of AmpB. Pronounced cellular damage was observed with combination treatment than to EUG and AmpB alone. Our finding is helpful for the removal of toxic concentrations of antifungal agents.

Photodynamic efficacy of toluidine blue O against mono species and dual species bacterial biofilm.

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.

Antibiotics versus biofilm: an emerging battleground in microbial communities.

Biofilm is a complex structure of microbiome having different bacterial colonies or single type of cells in a group; adhere to the surface. These cells are embedded in extracellular polymeric substances, a matrix which is generally composed of eDNA, proteins and polysaccharides, showed high resistance to antibiotics. It is one of the major causes of infection persistence especially in nosocomial settings through indwelling devices. Quorum sensing plays an important role in regulating the biofilm formation. There are many approaches being used to control infections by suppressing its formation but CRISPR-CAS (gene editing technique) and photo dynamic therapy (PDT) are proposed to be used as therapeutic approaches to subside bacterial biofim infections, especially caused by deadly drug resistant bad bugs.

Efficacy of photodynamic therapy against Streptococcus mutans biofilm: Role of singlet oxygen.

In photodynamic therapy (PDT), killing is entirely based on the ROS generation and among different types of ROS generated during PDT, singlet oxygen is considered as the most potential as illustrated in many studies and therefore it is predominantly responsible for photodamage and cytotoxic reactions. The aim of this study was to check whether singlet oxygen (Type II photochemistry) is more potential than free radicals (Type I photochemistry) against Streptococcus mutans biofilm. We have taken two phenothiazinium dyes i.e. toluidine blue O (TBO) and new methylene blue (NMB). TBO was found to have better antibacterial as well as antibiofilm effect than NMB. Antibacterial effect was evaluated by colony forming unit while antibiofilm action by crystal violet and congo red binding assays. We have also evaluated the disruption of preformed biofilm by biofilm reduction assay, confocal laser electron and scanning electron microscopy. More singlet oxygen production was detected in case of TBO than NMB while more Free radical (HO) was produced by NMB than TBO. TBO showed better antibacterial as well as antibiofilm effect than NMB so; we conclude that potency of a photosensitizer is correlated with the capability to produce singlet oxygen.

Enhanced photodynamic therapy using light fractionation against Streptococcus mutans biofilm: type I and type II mechanism.

AIM: The objective of the study was to look the efficacy of fractionated light against Streptococcus mutans biofilm. MATERIALS & METHODS: Antibiofilm assays (crystal violet, congo red), electron microscopic, confocal and spectroscopic studies were performed to check the effect of fractionated light. RESULTS: 6-6.5 log10 reduction of planktonic and 3.6-4.2 log10 reduction in biofilm were observed after irradiation with fractionated as compared with continuous light. Increased permeability to propidium iodide and leakage of cellular constituent validate the greater antibiofilm effect of fractionated light. Spectroscopic studies confirmed the relative contribution of type I and type II photochemistry. CONCLUSION: Phenothiazinium dyes have a potential against bacterial biofilm in combination with light fractionation and it offers new opportunities to explore its clinical implication.

Nano-therapeutics: A revolution in infection control in post antibiotic era.

With the arrival of antibiotics 70 years ago, meant a paradigm shift in overcoming infectious diseases. For decades, drugs have been used to treat different infections. However, with time bacteria have become resistant to multiple antibiotics, making some diseases difficult to fight. Nanoparticles (NPs) as antibacterial agents appear to have potential to overcome such problems and to revolutionize the diagnosis and treatment of bacterial infections. Therefore, there is significant interest in the use of NPs to treat variety of infections, particularly caused by multidrug-resistant (MDR) strains. This review begins with illustration of types of NPs followed by the literature of current research addressing mechanisms of NPs antibacterial activity, steps involved in NP mediated drug delivery as well as areas where NPs use has potential to improve the treatment, like NP enabled vaccination. Besides, recently emerged innovative NP platforms have been highlighted and their progress made in each area has been reviewed.

CRISPR Interference (CRISPRi) Inhibition of luxS Gene Expression in E. coli: An Approach to Inhibit Biofilm.

Biofilm is a sessile bacterial accretion embedded in self-producing matrix. It is the root cause of about 80% microbial infections in human. Among them, E. coli biofilms are most prevalent in medical devices associated nosocomial infections. The objective of this study was to inhibit biofilm formation by targeting gene involved in quorum sensing, one of the main mechanisms of biofilm formation. Hence we have introduced the CRISPRi, first time to target luxS gene. luxS is a synthase, involved in the synthesis of Autoinducer-2(AI-2), which in turn guides the initial stage of biofilm formation. To implement CRISPRi system for luxS gene suppression, we have synthesized complementary sgRNA to target gene sequence and co-expressed with dCas9, a mutated form of an endonuclease. Suppression of luxS expression was confirmed through qRT-PCR. The effect of luxS gene on biofilm inhibition was studied through crystal violet assay, XTT reduction assay and scanning electron microscopy. We conclude that CRISPRi system could be a potential strategy to inhibit bacterial biofilm through mechanism base approach.

A comparison of antibacterial and antibiofilm efficacy of phenothiazinium dyes between Gram positive and Gram negative bacterial biofilm.

BACKGROUND: Antimicrobial photodynamic therapy (APDT) is a process that generates reactive oxygen species (ROS) in presence of photosensitizer, visible light and oxygen which destroys the bacterial cells. We investigated the photoinactivation efficiency of phenothiazinium dyes and the effect of ROS generation on Gram positive and Gram negative bacterial cell as well as on biofilm. MATERIAL AND METHODS: Enterococcus faecalis and Klebsiella pneumonia were incubated with all the three phenothiazinium dyes and exposed to 630nm of light. After PDT, colony forming unit (CFU) were performed to estimate the cell survival fraction. Intracellular reactive oxygen species (ROS) was detected by DCFH-DA. Crystal violet (CV) assay and extracellular polysaccharides (EPS) reduction assay were performed to analyze antibiofilm effect. Confocal laser electron microscope (CLSM) scanning electron microscope (SEM) was performed to assess the disruption of biofilm. RESULTS: 8log10 reduction in bacterial count was observed in Enterococcus faecalis while 3log10 in Klebsiella pneumoniae. CV and EPS reduction assay revealed that photodynamic inhibition was more pronounced in Enterococcus faecalis. In addition to this CLSM and SEM study showed an increase in cell permeability of propidium iodide and leakage of cellular constituents in treated preformed biofilm which reflects the antibiofilm action of photodynamic therapy. CONCLUSION: We conclude that Gram-positive bacteria (Enterococcus faecalis) are more susceptible to APDT due to increased level of ROS generation inside the cell, higher photosensitizer binding efficiency and DNA degradation. Phenothiazinium dyes are proved to be highly efficient against both planktonic and biofilm state of cells.

Antibiofilm action of a toluidine blue O-silver nanoparticle conjugate on Streptococcus mutans: a mechanism of type I photodynamic therapy.

The objective of this study was to evaluate the anti-biofilm efficacy of photodynamic therapy by conjugating a photosensitizer (TBO) with silver nanoparticles (AgNP). Streptococcus mutans was exposed to laser light (630 nm) for 70 s (9.1 J cm(-2)) in the presence of a toluidine blue O-silver nanoparticle conjugate (TBO-AgNP). The results showed a reduction in the viability of bacterial cells by 4 log10. The crystal violet assay, confocal laser scanning microscopy and scanning electron microscopy revealed that the TBO-AgNP conjugates inhibited biofilm formation, increased the uptake of propidium iodide and leakage of the cellular constituents, respectively. Fluorescence spectroscopic studies confirmed the generation of OH(•) as a major reactive oxygen species, indicating type I phototoxicity. Both the conjugates down-regulated the expression of biofilm related genes compared to TBO alone. Hence TBO-AgNP conjugates were found to be more phototoxic against S. mutans biofilm than TBO alone.

Viability of Mycobacterium leprae in the environment and its role in leprosy dissemination.

BACKGROUND: Leprosy, a chronic disease caused by Mycobacterium leprae, is a public health concern in certain countries, including India. Although the prevalence of the disease has fallen drastically over time, new cases continue to occur at nearly the same rate in many regions. Several endemic pockets have been observed in India and elsewhere. The precise dynamics of leprosy transmission are still not clearly understood. Both live bacilli as well as M. leprae DNA have been detected in the soil and water of endemic areas; they possibly play an important role in disease transmission. AIMS: To study the occurrence of viable M. leprae in environmental samples collected from areas of residence of patients with active leprosy. METHODS: The study was conducted on 169 newly diagnosed leprosy patients in Ghatampur, Uttar Pradesh, India. Soil and water samples were collected from their areas of residence using a standardized protocol. An equal number of soil and water samples were also collected from non-patient areas of the same or adjoining villages. The environmental samples collected from the patients surroundings were subjected to 16S ribosomal RNA gene analysis after obtaining informed consent. RESULTS: About a quarter of the environmental samples collected from patient areas, (25.4% of soil samples and 24.2% of water samples) were found to be positive for specific 16S ribosomal RNA genes of M. leprae. Environmental samples collected from non-patient areas were all found negative for M. leprae 16S ribosomal RNA genes. LIMITATIONS: The major limitation of the study was that the sample size was small. CONCLUSION: The study demonstrated the presence of viable strains of M. leprae in skin smear samples of paucibacillary patients and multibacillary patients, as well as in the environmental samples obtained from around their houses. This could play an important role in the continued transmission of leprosy.

Calcium fluoride nanoparticles induced suppression of Streptococcus mutans biofilm: an in vitro and in vivo approach.

Biofilm formation on the tooth surface is the root cause of dental caries and periodontal diseases. Streptococcus mutans is known to produce biofilm which is one of the primary causes of dental caries. Acid production and acid tolerance along with exopolysaccharide (EPS) formation are major virulence factors of S. mutans biofilm. In the current study, calcium fluoride nanoparticles (CaF2-NPs) were evaluated for their effect on the biofilm forming ability of S. mutans in vivo and in vitro. The in vitro studies revealed 89 % and 90 % reduction in biofilm formation and EPS production, respectively. Moreover, acid production and acid tolerance abilities of S. mutans were also reduced considerably in the presence of CaF2-NPs. Confocal laser scanning microscopy and transmission electron microscopy images were in accordance with the other results indicating inhibition of biofilm without affecting bacterial viability. The qRT-PCR gene expression analysis showed significant downregulation of various virulence genes (vicR, gtfC, ftf, spaP, comDE) associated with biofilm formation. Furthermore, CaF2-NPs were found to substantially decrease the caries in treated rat groups as compared to the untreated groups in in vivo studies. Scanning electron micrographs of rat's teeth further validated our results. These findings suggest that the CaF2-NPs may be used as a potential antibiofilm applicant against S. mutans and may be applied as a topical agent to reduce dental caries.