Polycationic chitosan-conjugated photosensitizer for antibacterial photodynamic therapy

The complex nature of bacterial cell membrane and structure of biofilm has challenged the efficacy of antimicrobial photodynamic therapy. This study was aimed to synthesize a polycationic chitosan-conjugated rose bengal (CSRB) photosensitizer and test its antibiofilm efficacy on Enterococcus faecalis (gram positive) and Pseudomonas aeruginosa (gram negative) using photodynamic therapy. During experiments, CSRB was tested along with an anionic photosensitizer rose bengal (RB) and a cationic photosensitizer methylene blue (MB) for uptake and killing efficacy on 7-day-old E. faecalis and P. aeruginosa biofilms. Microbiological culture based analysis was used to analyze the cell viability, while laser scanning confocal microscopy (LSCM) was used to examine the structure of biofilm. The synthesized CSRB showed absorbance spectrum similar to the RB. The concentration of CSRB uptaken by both the bacterial biofilms was significantly higher than that of RB and MB (P < 0.05). Photoactivation resulted in significantly higher elimination of both bacterial biofilms sensitized with CSRB than RB and MB. The structure of biofilm under LSCM was found to be disrupted following CSRB treatment. The present study highlighted the importance of inherent cell membrane permeabilizing effect of chitosan and increased cell/biofilm uptake of conjugated photosensitizer to produce significant antibiofilm efficacy during photodynamic therapy.     

Photodynamic Inactivation of Planktonic Cultures and Biofilms of Candida albicans Mediated by Aluminum‐Chloride‐Phthalocyanine Entrapped in Nanoemulsions

New drug delivery systems, such as nanoemulsions (NE), have been developed to allow the use of hydrophobic drugs on the antimicrobial photodynamic therapy. This study evaluated the photodynamic potential of aluminum‐chloride‐phthalocyanine (ClAlPc) entrapped in cationic and anionic NE to inactivate Candida albicans planktonic cultures and biofilm compared with free ClAlPc. Fungal suspensions were treated with different delivery systems containing ClAlPc and light emitting diode. For planktonic suspensions, colonies were counted and cell metabolism was evaluated by XTT assay. Flow cytometry evaluated cell membrane damage. For biofilms, the metabolic activity was evaluated by XTT and ClAlPc distribution through biofilms was analyzed by confocal laser scanning microscopy (CLSM). Fungal viability was dependent on the delivery system, superficial charge and light dose. Free ClAlPc caused photokilling of the yeast when combined with 100 J cm^?2. Cationic NE‐ClAlPc reduced significantly both colony counts and cell metabolism (P < 0.05). In addition, cationic NE‐ClAlPc and free ClAlPc caused significant damage to the cell membrane (P < 0.05). For the biofilms, cationic NE‐ClAlPc reduced cell metabolism by 70%. Anionic NE‐ClAlPc did not present antifungal activity. CLSM showed different accumulation on biofilms between the delivery systems. Although NE system showed a lower activity for planktonic culture, cationic NE‐ClAlPc showed better results for Candida biofilms.     

Development of novel oral formulations prepared via hot melt extrusion for targeted delivery of photosensitizer to the colon

Colon‐residing bacteria, such as vancomycin‐resistant Enterococcus faecalis and Bacteroides fragilis, can cause a range of serious clinical infections. Photodynamic antimicrobial chemotherapy (PACT) may be a novel treatment option for these multidrug resistant organisms. The aim of this study was to formulate a Eudragit^®‐based drug delivery system, via hot melt extrusion (HME), for targeting colonic release of photosensitizer. The susceptibility of E. faecalis and B. fragilis to PACT mediated by methylene blue (MB), meso‐tetra(N‐methyl‐4‐pyridyl)porphine tetra‐tosylate (TMP), or 5‐aminolevulinic acid hexyl‐ester (h‐ALA) was determined, with tetrachlorodecaoxide (TCDO), an oxygen‐releasing compound, added in some studies. Results show that, for MB, an average of 30% of the total drug load was released over a 6‐h period. For TMP and h‐ALA, these values were 50% and 16% respectively. No drug was released in the acidic media. Levels of E. faecalis and B. fragilis were reduced by up to 4.67 and 7.73 logs, respectively, on PACT exposure under anaerobic conditions, with increased kill associated with TCDO. With these formulations, photosensitizer release could potentially be targeted to the colon, and colon‐residing pathogens killed by PACT. TCDO could be used in vivo to generate oxygen, which could significantly impact on the success of PACT in the clinic.     

Photodynamic Inactivation of <Emphasis Type="BoldItalic">E. coli</Emphasis> PTCC 1276 Using Light Emitting Diodes: Application of Rose Bengal and Methylene Blue as Two Simple Models

The lack of a comparative study about potential of high-power light emitting diodes (LEDs) for photodynamic inactivation (PDI) of pathogenic microorganisms has remained as a challenging issue for researchers. Therefore, the aim of this study is to fill this gap through introduction of an efficient model for in vitro PDI in an aqueous medium. For this purpose, two individual 30 mW/cm² irradiation systems were designed using suitable sets of green and red LEDs. At another work, Methylene blue (MB) and Rose bengal (RB) as two simple models in the range of 5–150 μM were used in order to compare PDI of E. coli PTCC 1276 using red and green LED systems. Our results showed that a first-order mathematical model has the strength to describe the temporal variation of survival curves. Based on our results, when concentration of photosensitizer increased, the rate of inactivation for RB increased while MB depicted a maximum rate value at 25 μM. In a comparative study, optimum inactivation of E. coli PTCC 1276 obtained during 2- and 10-min irradiation of the LED systems using RB and MB at 150 and 25 μM, respectively. With regard to lower value of inactivation time and higher rate of inactivation for RB, use of simultaneous green high-power LEDs and RB is proposed as an efficient approach for PDI of pathogenic bacteria in future industrial applications.     

Photodynamic Inactivation Using Natural Bioactive Compound Prevents and Disrupts the Biofilm Produced by Staphylococcus saprophyticus

Staphylococcus saprophyticus, the food-borne bacteria present in dairy products, ready-to-eat food and environmental sources, has been reported with antibiotic resistance, raising concerns about food microbial safety. The antimicrobial resistance of S. saprophyticus requires the development of new strategies. Light- and photosensitizer-based antimicrobial photodynamic inactivation (PDI) is a promising approach to control microbial contamination, whereas there is limited information regarding the effectiveness of PDI on S. saprophyticus biofilm control. In this study, PDI mediated by natural bioactive compound (curcumin) associated with LED was evaluated for its potential to prevent and disrupt S. saprophyticus biofilms. Biofilms were treated with curcumin (50, 100, 200 µM) and LED fluence (4.32 J/cm², 8.64 J/cm², 17.28 J/cm²). Control groups included samples treated only with curcumin or light, and samples received neither curcumin nor light. The action was examined on biofilm mass, viability, cellular metabolic activity and cytoplasmic membrane integrity. PDI using curcumin associated with LED exhibited significant antibiofilm activities, inducing biofilm prevention and removal, metabolic inactivation, intracellular membrane damage and cell death. Likewise, scanning electronic microscopy observations demonstrated obvious structural injury and morphological alteration of S. saprophyticus biofilm after PDI application. In conclusion, curcumin is an effective photosensitizer for the photodynamic control of S. saprophyticus biofilm.     

Bacterial inactivation via microfluidic electroporation device with insulating micropillars

Electroporation is a promising method to inactivate cells and it has wide applications in medical science, biology and environmental health. Here, we investigate the bacteria inactivation performance of two different microfluidic electroporation devices with rhombus and circular micropillars used for generating locally enhanced electric field strength. Experiments are carried out to characterize the inactivation performance (i.e., the log removal efficiency) of two types of bacteria: Escherichia coli (E. coli, gram-negative) and Enterococcus faecalis (E. faecalis, gram-positive) in these two microfluidic devices. We find that under the same applied electric field, the device with rhombus micropillars performs better than the device with circular micropillars for both E. coli and E. faecalis. Numerical simulations show that due to the corner-induced singularity effect, the maximum electric field enhancement is higher in the device with rhombus micropillars than that in the device with circular micropillars. We also study the effects of DC and AC electric fields and flowrate. Our experiments demonstrate that the use of the DC field achieves higher log removal efficiencies than the use of AC field.     

Advances on antimicrobial photodynamic inactivation mediated by Zn(II) porphyrins

Over the years, microorganisms have developed several resistance mechanisms against standard treatments, thus limiting the effect of drugs and rendering ineffective therapies. Considering the growing number of resistant pathogens and adverse effects of conventional therapies, new antimicrobial technologies able to provide more effective, rapid, and safer treatments to inactivate pathogens, with unlikely chances of inducing resistance, are needed. In this regard, antimicrobial photodynamic inactivation (aPDI) has emerged as an alternative modality of treatment. In particular, Zn(II) porphyrins (ZnPs) hold great potential as photosensitizers (PSs) for aPDI and have been attracting increasing attention. The chemical structure of ZnPs can be tailored to produce PSs with improved chemical stability and photophysical properties, also modulating their amphiphilic and ionic characters, bioavailability, and (sub)cellular distribution. Thus, in this review, we provide a detailed report of studies published in about the last 10 years (2010–2021) focusing on aPDI mediated by ZnPs over a variety of pathogens, including bacteria, fungi, viruses, and protozoa. Fundamentals of aPDI, and porphyrin and its derivatives, especially ZnPs, are also included herein. We hope that this review can guide and be a reference for future studies related to aPDI mediated by ZnPs, and encourages more detailed studies on ZnP photophysical and photochemical properties, aiming to improve the fight against infectious diseases.     

Antimicrobial evaluation of selected naturally occurring oxyprenylated secondary metabolites

This study tested the antimicrobial activity of eight selected naturally occurring oxyprenylated secondary metabolites against Staphylococcus aureus ATCC 29213, S. epidermidis ATCC 35984, Escherichia coli ATCC 8739, Pseudomonas aeruginosa ATCC 9027 and Candida albicans ATCC 10231. Results showed a moderate antimicrobial activity. The most active compounds were 3-(4-geranyloxyphenyl)-1-ethanol (4) and 3-(4-isopentenyloxyphenyl)-1-propanol (5) that were tested on mature and in-formation biofilms of all micro-organisms, moreover the cytotoxic activity was evaluated. Except for S. epidermidis, both compounds reduced significantly (p < 0.05) the microbial biofilm formation at 1/2 MIC and 1/4 MIC, in particular, compounds 4 and 5 at each concentration, inhibited E. coli biofilm formation to a greater extent, the biofilm formation was never more than 44% in respect to the control, moreover both compounds showed a low cytotoxic effect. Oxyprenylated derivatives may be of great interest for the development of novel antimicrobial therapeutic strategies and the synthesis of semi-synthetic analogues with anti-biofilm efficacy.     

A photo-sensitizable phage for multidrug-resistant Acinetobacter baumannii therapy and biofilm ablation

Antibiotic abuse causes the emergence of bacterial resistance. Photodynamic antibacterial chemotherapy (PACT) has great potential to solve serious bacterial resistance, but it suffers from the inefficient generation of ROS and the lack of bacterial targeting ability. Herein, a unique cationic photosensitizer (NB) and bacteriophage (ABP)-based photodynamic antimicrobial agent (APNB) is developed for precise bacterial eradication and efficient biofilm ablation. Thanks to the structural modification of the NB photosensitizer with a sulfur atom, it displays excellent reactive oxygen species (ROS)-production ability. Moreover, specific binding to pathogenic microorganisms can be provided by bacteriophages. The developed APNB has multiple functions, including bacteria targeting, near-infrared fluorescence imaging and combination therapy (PACT and phage therapy). Both in vitro and in vivo experiments prove that APNB can efficiently treat A. baumannii infection. Particularly, the recovery from A. baumannii infection after APNB treatment is faster than that with ampicillin and polymyxin B in vivo. Furthermore, the strategy of combining bacteriophages and photosensitizers is employed to eradicate bacterial biofilms for the first time, and it shows the excellent biofilm ablation effect as expected. Thus, APNB has huge potential in fighting against multidrug-resistant bacteria and biofilm ablation in practice.

APNB for multidrug-resistant A. Baumannii therapy and biofilms ablation.     

Effectiveness of the Photoactive Dye Methylene Blue versus Caspofungin on the Candida parapsilosis Biofilm in vitro and ex vivo

This research studied the effectiveness of the photoactive compound methylene blue (MB) activated with red LED light (576–672 nm) compared to that of caspofungin (CAS) on 1 Candida albicans and 3 Candida parapsilosis strains. Results were evaluated in terms of SMIC₅₀ for CAS or in PDI (photodynamic inactivation)‐SMIC₅₀ for MB (minimal inhibitory concentration inhibiting sessile biofilm to 50% in comparison to the control without CAS or after irradiation in comparison to the control without MB). While all strains were susceptible to CAS in planktonic form, the SMIC₅₀ was determined to be >16 μg mL^?1 when CAS was added to a 24 h biofilm. However, PDI‐MIC_50s (1.67 mW cm^?2, fluence 15 J cm^?2) were 0.0075–0.03 mmol L^?1. For biofilm, PDI‐SMIC_50s were in the range from 0.7 to 1.35 mmol L^?1. MB concentration of 1 mmol L^?1 prevented a biofilm being formed ex vivo on mouse tongues after irradiation regardless of the application time, in contrast to CAS, which was only effective at a concentration of 16 μg mL^?1 when it was added at the beginning of biofilm formation. PDI seems to be a promising method for the prevention of microbial biofilms that do not respond significantly to conventional drugs.     

Hydrogen Peroxide Enhances the Antibacterial Effect of Methylene Blue‐based Photodynamic Therapy on Biofilm‐forming Bacteria

Recently, increased attention has been focused on endoscopic disinfection after outbreaks of drug‐resistant infections associated with gastrointestinal endoscopy. The aims of this study were to investigate the bactericidal efficacy of methylene blue (MB)‐based photodynamic therapy (PDT) on Pseudomonas aeruginosa (P. aeruginosa), which is the major cause of drug‐resistant postendoscopy outbreak, and to assess the synergistic effects of hydrogen peroxide addition to MB‐based PDT on biofilms. In planktonic state of P. aeruginosa, the maximum decrease was 3 log₁₀ and 5.5 log₁₀ at 20 and 30 J cm^?2, respectively, following MB‐based PDT. However, the maximum reduction of colony forming unit (CFU) was decreased by 2.5 log₁₀ and 3 log₁₀ irradiation on biofilms. The biofilm formation was significantly inhibited upon irradiation with MB‐based PDT. When the biofilm state of P. aeruginosa was treated with MB‐based PDT with hydrogen peroxide, the CFU was significantly decreased by 6 log₁₀ after 20 J cm^?2, by 7 log₁₀ after 30 J cm^?2 irradiation, suggesting significantly higher efficacy than MB‐based PDT alone. The implementation of the combination of hydrogen peroxide with MB‐based PDT through working channels might be appropriate for preventing early colonization and biofilm formation in the endoscope and postendoscopy outbreak.     

An in vitro study of the use of photodynamic therapy for the treatment of natural oral plaque biofilms formed in vivo.

Seven-day oral plaque biofilms have been formed on natural enamel surfaces in vivo using a previously reported in situ device. The devices are then incubated with a cationic Zn(II) phthalocyanine photosensitizer and irradiated with white light. Confocal scanning laser microscopy (CSLM) of the biofilms shows that the photosensitizer is taken up into the biomass of the biofilm and that significant cell death is caused by photodynamic therapy (PDT). In addition, the treated biofilms are much thinner than the control samples and demonstrate a different structure from the control samples, with little evidence of channels and a less dense biomass. Transmission electron microscopy (TEM) of the in vivo-formed plaque biofilms reveals considerable damage to bacteria in the biofilm, vacuolation of the cytoplasm and membrane damage being clearly visible after PDT. These results clearly demonstrate the potential value of PDT in the management of oral biofilms.     

Photoinactivation of Moraxella catarrhalis Using 405-nm Blue Light: Implications for the Treatment of Otitis Media

Moraxella catarrhalis is one of the major otopathogens of otitis media (OM) in childhood. M. catarrhalis tends to form biofilm, which contributes to the chronicity and recurrence of infections, as well as resistance to antibiotic treatment. In this study, we aimed to investigate the effectiveness of antimicrobial blue light (aBL; 405 nm), an innovative nonpharmacological approach, for the inactivation of M. catarrhalis OM. M. catarrhalis either in planktonic suspensions or 24-h old biofilms were exposed to aBL at the irradiance of 60 mW cm⁻². Under an aBL exposure of 216 J cm⁻², a >4-log₁₀ colony-forming units (CFU) reduction in planktonic suspensions and a >3-log₁₀ CFU reduction in biofilms were observed. Both transmission electron microscopy and scanning electron microscopy revealed aBL-induced morphological damage in M. catarrhalis. Ultraperformance liquid chromatography results indicated that protoporphyrin IX and coproporphyrin were the two most abundant species of endogenous photosensitizing porphyrins. No statistically significant reduction in the viability of HaCaT cells was observed after an aBL exposure of up to 216 J cm⁻². Collectively, our results suggest that aBL is potentially an effective and safe alternative therapy for OM caused by M. catarrhalis. Further in vivo studies are warranted before this optical approach can be moved to the clinics.     

Comparative Antibacterial Efficacy of Photodynamic Therapy and Ultrasonic Irrigation Against Enterococcus faecalis In Vitro

Enterococcus faecalis poses a challenge to the efficacy of traditional root canal disinfection methods. This study was aimed to establish a synergistic root canal disinfection strategy combining ultrasonic irrigation with photodynamic therapy (PDT) together and to test its antibacterial efficacy against E. faecalis. Twenty‐seven bovine root canals infected with E. faecalis were randomly divided into three groups and treated with different disinfection methods as follows: ultrasonic irrigation with 2.5% NaOCl, methylene blue (MB)‐mediated PDT, or combined ultrasonic irrigation and PDT as described above. Quantification of E. faecalis was performed on the root canals before and immediately after the disinfection treatment. Residual bacteria were determined by counting colony‐forming units. Samples were randomly selected from the three groups, and the morphology of residual bacteria inside the dentinal tubules was studied by scanning electron microscopy. The number of surviving E. faecalis in the group treated with the combination method was significantly lower (P < 0.05) than those in the ultrasonic irrigation‐treated or PDT‐treated groups. Similar results were found in the morphological studies of the three groups. The results of our study highlighted the importance of combination of ultrasonic irrigation and PDT to produce significant antibacterial efficacy against E. faecalis during root canal disinfection.     

水溶性有机物与细胞膜的结合作用

以二氨基蓝3R (DAB)和玫瑰红B (RB)两种离子型有机物为探针, 研究了其与体外单层磷脂膜(SPM)和大肠杆菌之间的相互作用, 结合作用符合Langmuir和Temkin等温化学吸附方程. 考察了pH、离子强度和温度对吸附作用的影响, 分析了DAB, RB与SPM和细胞的结合形式、结合键和结合位置. 结果表明, DAB, RB与SPM和细胞外膜有强烈的单分子层化学吸附, 是自发的放热反应, 主要通过电荷对吸引、氢键等非共价键结合在SPM和细胞外表面, 且绝大部分DAB, RB积累、滞留在细胞膜壁上. 混合作用时, 两种有机物存在竞争吸附结合现象.     

Positively Charged Nanoaggregates Based on Zwitterionic Pillar[5]arene that Combat Planktonic Bacteria and Disrupt Biofilms

Bacterial biofilms are difficult to eradicate because they are less susceptible to antibiotics and more easily develop resistance. Therefore, there is an urgent need for new materials that can combat planktonic bacteria and disrupt established biofilms. To tackle this challenge, we design a multifunctional zwitterionic pillar[5]arene, which can self‐assemble into weakly positively charged nanoaggregates that exhibit antibacterial activity against Gram‐negative Escherichia coli (DH5α) and Gram‐positive Staphylococcus aureus (SH1000) bacterial strains in solution. In addition, the zwitterionic pillar[5]arene can efficiently disrupt pre‐existing Escherichia coli (DH5α) biofilms and kill the biofilm‐enclosed bacteria without rapid generation of resistance.     

Increased cytotoxicity and phototoxicity in the methylene blue series via chromophore methylation

The cytotoxic and photodynamic activities of the commercially-available biological stains methylene blue (MB), 1,9-dimethyl MB (Taylor's Blue) and a newly synthesised compound, 1-methyl MB, were measured against the murine mammary tumour cell line, EMT-6 Both 1-methyl MB and 1,9-dimethyl MB exhibited increased dark toxicity with concomitant higher phototoxicity compared to MB at a light dose of 7.2 J cm⁻². While increasing the light dose as a function of the fluence rate increased the photocytotoxicity of MB, this had little effect on the methylated derivatives. In vitro chemical testing proved that successive methylation rendered the phenothiazinium chromophore both more resistant to reduction to its inactive leuco form, and also led to increased levels of singlet-oxygen production, thus providing a possible explanation for the increased toxicities of the methylated derivatives. Comparisons are made with the benzo[a]phenothiazinium photosensitizer, EtNBS.     

Enhancement of the Efficacy of Photodynamic Inactivation of Candida albicans with the Use of Biogenic Gold Nanoparticles

This study reports on successful photodynamic inactivation of planktonic and biofilm cells of Candida albicans using Rose Bengal (RB) in combination with biogenic gold nanoparticles synthesized by the cell‐free filtrate of Penicillium funiculosum BL1 strain. Monodispersed colloidal gold nanoparticles coated with proteins were characterized by a number of techniques including SEM–EDS, TEM, UV–Vis absorption and fluorescence spectroscopy, as well as Fourier transform infrared spectroscopy to be 24 ± 3 nm spheres. A Xe lamp (output power of 20mW, delivering intensity of 53 mW cm^?2) was used as a light source to study the effects of RB alone, the gold nanoparticles alone and the RB‐gold nanoparticle mixture on the viability of C. albicans cells. The most effective reduction in the number of planktonic cells was found after 30 min of Xe lamp light irradiation (95.4 J cm^?2) and was 4.89 log₁₀ that is 99.99% kill for the mixture of RB with gold nanoparticles compared with 2.19 log₁₀ or 99.37% for RB alone. The biofilm cells were more resistant to photodynamic inactivation, and the highest effective reduction in the number of cells was found after 30 min of irradiation in the presence of the RB–gold nanoparticles mixture and was 1.53 log₁₀, that is 97.04% kill compared with 0.6 log₁₀ or 74.73% for RB. The probable mechanism of enhancement of RB‐mediated photodynamic fungicidal efficacy against C. albicans in the presence of biogenic gold nanoparticles is discussed leading to the conclusion that this process may have a multifaceted character.     

Photodynamic Inactivation of Bacterial and Yeast Biofilms With a Cationic Porphyrin

The efficiency of 5,10,15,20‐tetrakis(1‐methylpyridinium‐4‐yl)porphyrin tetra‐iodide (Tetra‐Py⁺‐Me) in the photodynamic inactivation of single‐species biofilms of Staphylococcus aureus, Pseudomonas aeruginosa and Candida albicans and mixed biofilms of S. aureus and C. albicans was evaluated. The effect on the extracellular matrix of P. aeruginosa was also assessed. Irradiation with white light up to an energy dose of 64.8 J cm^?2 in the presence of 20 μm of Tetra‐Py⁺‐Me caused significant inactivation in all single‐species biofilms (3–6 log reductions), although the susceptibility was attenuated in relation to planktonic cells. In mixed biofilms, the inactivation of S. aureus was as efficient as in single‐species biofilms but the susceptibility of C. albicans decreased. In P. aeruginosa biofilms, a reduction of 81% in the polysaccharide content of the matrix was observed after treatment with a 20 μm PS concentration and a total light dose of 64.8 J cm^?2. The results show that the Tetra‐Py⁺‐Me causes significant inactivation of the microorganisms, either in biofilms or in the planktonic form, and demonstrate that polysaccharides of the biofilm matrix may be a primary target of photodynamic damage.     

Photodynamic Activity on Biofilm in Endotracheal Tubes of Patients Admitted to an Intensive Care Unit

Ventilator-associated pneumonia (VAP) is an infection that arises after endotracheal intubation affecting patients under intensive care. The presence of the endotracheal tube (ETT) is a risk factor since it is colonized by multispecies biofilm. Antimicrobial photodynamic therapy (aPDT) could be a strategy to decontaminate ETTs. We verify if methylene blue (MB) associated with external illumination of the ETT could be an alternative to destroy biofilm. We performed an in vitro and ex vivo study. In vitro study was performed with P. aeruginosa biofilm grew over ETT for 7 days. After treatment, the surviving cells were cultured for 3 days and the biofilm was analyzed by crystal violet absorbance. Ex vivo study employed ETT obtained from extubated patients. aPDT was performed with MB (100 µm ) and red LED (λ = 640±20 nm). We quantified the biofilm thickness and used scanning electron microscopy and fluorescence technique to verify morphological and functional changes after aPDT. Our results showed that bacteria remain susceptible to aPDT after sequential treatments. We also attested that aPDT can reduce biofilm thickness, disrupt biofilm attachment from ETT surface and kill microbial cells. These data suggest that aPDT should be investigated to decrease VAP incidence via ETT decontamination.