Allura Red–Mediated Photodynamic Therapy: A Novel Multi-Assay Evaluation Against Pseudomonas aeruginosa

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In this study, a dose-dependent antimicrobial photodynamic therapy (aPDT) approach was developed using Allura Red in combination with a 630 nm red laser. Four complementary assays were integrated into a unified evaluation platform: AMDA for singlet oxygen, XTT for metabolic activity, CFU enumeration for bacterial viability, and Crystal Violet (CV) staining for biofilm biomass. Results: The Allura Red–mediated PDT demonstrated dose-dependent effects, with 15 J/cm2 yielding ~87% CFU reduction and >75% biofilm inhibition. Conclusion: This study introduces, for the first time, a novel Allura Red–based, multi-assay PDT framework against P. aeruginosa, providing a comprehensive and translational evaluation method. Background Pseudomonas aeruginosa is a multidrug-resistant and biofilm-forming bacterium that represents a major challenge to conventional antibiotics. Methods In this study, a novel dose-dependent antimicrobial photodynamic therapy (aPDT) approach was applied using Allura Red combined with red laser light (630 nm) at fluences of 5, 10, and 15 J/cm2. Four complementary assays were integrated into a single protocol: aminophenyl methylene diphosphonate (AMDA) and XTT for reactive oxygen species (ROS) and singlet oxygen detection, colony-forming unit (CFU) counts for bacterial viability, and Crystal Violet (CV) staining for biofilm biomass. All experiments were performed in triplicate with appropriate controls, and statistical significance was determined using one-way ANOVA (p < 0.05). Results Treatment with 15 J/cm2 produced the highest ROS generation and bacterial killing, with CFU counts reduced by approximately 87%, compared to 71% at 10 J/cm2 and 36% at 5 J/cm2. Biofilm inhibition exceeded 76% at the highest dose. Statistical analysis confirmed significant dose-dependent effects across all assays. Conclusion The integrated, multidimensional protocol provides a reliable method for assessing PDT efficacy and offers valuable insight into the relationship between oxidative stress, bacterial inactivation, and biofilm disruption. This strategy represents a promising approach against biofilm-associated and drug-resistant pathogens. " } { "@context": "http://schema.org", "@type": "BreadcrumbList", "itemListElement": [ { "@type": "ListItem", "position": "1", "item": { "@id": "https://f1000research.com/", "name": "Home" } }, { "@type": "ListItem", "position": "2", "item": { "@id": "https://f1000research.com/browse/articles", "name": "Browse" } }, { "@type": "ListItem", "position": "3", "item": { "@id": "https://f1000research.com/articles/14-923", "name": "Allura Red–Mediated Photodynamic Therapy: A Novel Multi-Assay Evaluation..." } } ] } Home Browse Allura Red–Mediated Photodynamic Therapy: A Novel Multi-Assay Evaluation... ALL Metrics - Views Downloads Get PDF Get XML Cite How to cite this article Majeed HA and Ali MMA. Allura Red–Mediated Photodynamic Therapy: A Novel Multi-Assay Evaluation Against Pseudomonas aeruginosa [version 1; peer review: 1 approved] . F1000Research 2025, 14 :923 ( https://doi.org/10.12688/f1000research.170011.1 ) NOTE: If applicable, it is important to ensure the information in square brackets after the title is included in all citations of this article. Close Copy Citation Details Export Export Citation Sciwheel EndNote Ref. Manager Bibtex ProCite Sente EXPORT Select a format first Track Share ▬ ✚ Research Article Allura Red–Mediated Photodynamic Therapy: A Novel Multi-Assay Evaluation Against Pseudomonas aeruginosa [version 1; peer review: 1 approved] Hayder Abdulrahman Majeed https://orcid.org/0009-0002-7605-2045 1 , Mawlood Maajal Ali Ali https://orcid.org/0009-0006-7144-5295 2 Hayder Abdulrahman Majeed https://orcid.org/0009-0002-7605-2045 1 , Mawlood Maajal Ali Ali https://orcid.org/0009-0006-7144-5295 2 PUBLISHED 15 Sep 2025 Author details Author details 1 Medical science, Al-Mashreq University, Baghdad, Baghdad, Iraq 2 Medical Physics, College of Applied Sciences, Hit, University of Anbar,, Anbar, Anbar, Iraq Hayder Abdulrahman Majeed Roles: Conceptualization, Data Curation, Formal Analysis, Investigation, Methodology, Project Administration, Validation, Visualization, Writing – Original Draft Preparation, Writing – Review & Editing Mawlood Maajal Ali Ali Roles: Project Administration OPEN PEER REVIEW DETAILS REVIEWER STATUS This article is included in the Pathogens gateway. This article is included in the Antimicrobial Resistance collection. Abstract Background Pseudomonas aeruginosa is a multidrug-resistant and biofilm-forming bacterium that poses serious challenges to conventional antibiotic therapy. In this study, a dose-dependent antimicrobial photodynamic therapy (aPDT) approach was developed using Allura Red in combination with a 630 nm red laser. Four complementary assays were integrated into a unified evaluation platform: AMDA for singlet oxygen, XTT for metabolic activity, CFU enumeration for bacterial viability, and Crystal Violet (CV) staining for biofilm biomass. Results: The Allura Red–mediated PDT demonstrated dose-dependent effects, with 15 J/cm 2 yielding ~87% CFU reduction and >75% biofilm inhibition. Conclusion: This study introduces, for the first time, a novel Allura Red–based, multi-assay PDT framework against P. aeruginosa, providing a comprehensive and translational evaluation method. Background Pseudomonas aeruginosa is a multidrug-resistant and biofilm-forming bacterium that represents a major challenge to conventional antibiotics. Methods In this study, a novel dose-dependent antimicrobial photodynamic therapy (aPDT) approach was applied using Allura Red combined with red laser light (630 nm) at fluences of 5, 10, and 15 J/cm 2 . Four complementary assays were integrated into a single protocol: aminophenyl methylene diphosphonate (AMDA) and XTT for reactive oxygen species (ROS) and singlet oxygen detection, colony-forming unit (CFU) counts for bacterial viability, and Crystal Violet (CV) staining for biofilm biomass. All experiments were performed in triplicate with appropriate controls, and statistical significance was determined using one-way ANOVA (p < 0.05). Results Treatment with 15 J/cm 2 produced the highest ROS generation and bacterial killing, with CFU counts reduced by approximately 87%, compared to 71% at 10 J/cm 2 and 36% at 5 J/cm 2 . Biofilm inhibition exceeded 76% at the highest dose. Statistical analysis confirmed significant dose-dependent effects across all assays. Conclusion The integrated, multidimensional protocol provides a reliable method for assessing PDT efficacy and offers valuable insight into the relationship between oxidative stress, bacterial inactivation, and biofilm disruption. This strategy represents a promising approach against biofilm-associated and drug-resistant pathogens. READ ALL READ LESS Keywords Antimicrobial photodynamic therapy, Allura Red, Pseudomonas aeruginosa, AMDA, XTT, CFU, Crystal Violet, biofilm, reactive oxygen species, singlet oxygen. Corresponding Author(s) Hayder Abdulrahman Majeed ( [email protected] ) Mawlood Maajal Ali Ali ( [email protected] ) Close Corresponding authors: Hayder Abdulrahman Majeed, Mawlood Maajal Ali Ali Competing interests: No competing interests were disclosed. Grant information: The author(s) declared that no grants were involved in supporting this work. Copyright: © 2025 Majeed HA and Ali MMA. This is an open access article distributed under the terms of the Creative Commons Attribution License , which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. How to cite: Majeed HA and Ali MMA. Allura Red–Mediated Photodynamic Therapy: A Novel Multi-Assay Evaluation Against Pseudomonas aeruginosa [version 1; peer review: 1 approved] . F1000Research 2025, 14 :923 ( https://doi.org/10.12688/f1000research.170011.1 ) First published: 15 Sep 2025, 14 :923 ( https://doi.org/10.12688/f1000research.170011.1 ) Latest published: 15 Sep 2025, 14 :923 ( https://doi.org/10.12688/f1000research.170011.1 ) 1. Introduction Pseudomonas aeruginosa is an opportunistic Gram-negative bacterium well-known for its exceptional capacity to withstand a wide range of antibiotics and to produce dense biofilms on medical devices and host tissues. These biofilms act as protective barriers, shielding the bacteria from host immune defenses and limiting the penetration of antimicrobial agents, which makes the resulting infections persistent and difficult to eliminate. 1 , 5 Clinically, P. aeruginosa is of particular concern due to its involvement in cystic fibrosis lung infections, burn and wound infections, and urinary tract infections, all of which are associated with high morbidity and mortality rates. 4 , 9 Photodynamic therapy (PDT) has gained attention as an innovative, non-antibiotic strategy to target resistant pathogens and biofilms. The procedure involves the administration of a light-sensitive compound, followed by irradiation with a defined wavelength in the presence of molecular oxygen. This interaction produces reactive oxygen species (ROS), including singlet oxygen and free radicals, which can inflict significant damage on microbial cells by disrupting lipids, proteins, and nucleic acids. 2 , 6 , 12 I can also make you a more condensed academic version if you need it to fit into a tight word limit for a journal. These ROS exert cytotoxic effects on microbial cells, damaging lipids, proteins, and nucleic acids. 6 Among various photosensitizers, Allura Red (Allura Red) is widely recognized for its high singlet oxygen yield, low toxicity, and strong absorption in the red spectral region (~660 nm), enabling deeper tissue penetration. 3 Previous studies have demonstrated Allura Red-PDT’s efficacy against Gram-positive and Gram-negative bacteria, but optimization of energy doses and evaluation methods remains an area of active research. Traditional assessment of PDT efficacy relies heavily on colony-forming unit (CFU) enumeration, which quantifies surviving bacteria post-treatment. However, CFU alone may not fully capture sublethal damage or early metabolic changes induced by PDT. To address this limitation, the current study introduces a combined evaluation using aminophenyl fluorescein (AMDA)—a singlet-oxygen-responsive readout, quantified via absorbance at 380 nm as a practical proxy when fluorescence detection is unavailable—and XTT—a tetrazolium salt that measures metabolic activity via mitochondrial dehydrogenase activity. This dual-assay approach allows for simultaneous monitoring of ROS production and bacterial metabolic suppression, offering a more comprehensive understanding of PDT’s antimicrobial effects. This work applies the Allura Red-mediated PDT approach against P. aeruginosa using red laser irradiation at 5, 10, and 15 J/cm 2 energy doses. The novelty lies in combining AMDA and XTT assays with CFU enumeration to provide a multidimensional evaluation of photodynamic inactivation efficacy The red laser diode operated at a power density of 100 mW/cm 2 , with exposure times of 50, 100, and 150 seconds for 5, 10, and 15 J/cm 2 , respectively. This study evaluates the efficacy of Allura Red-mediated aPDT against P. aeruginosa at different energy doses (5, 10, 15 J/cm 2 ), using CFU counts and the novel AMDA+XTT dual assay, and compares results with conventional single-endpoint evaluations. This study introduces a novel, unified analytical platform integrating four complementary endpoints—ROS generation (AMDA), metabolic activity (XTT), planktonic viability (CFU), and biofilm biomass (CV)—into a single Allura Red-aPDT workflow. To our knowledge, such a multi-parameter approach has not been previously reported for P. aeruginosa, enabling a more comprehensive and decision-ready assessment of photodynamic inactivation efficacy. Preliminary observations indicated a dose-dependent inhibition of bacterial growth and biofilm biomass, which is explored in detail in the Results and Discussion sections. 2. Materials and methods All reagents were of analytical grade and sourced from reputable suppliers. Methylene blue 1 was prepared in sterile distilled water and stored in amber vials at 4°C to prevent photodegradation. Aminophenyl methylene diphosphonate 2 and XTT sodium salt 3 stock solutions were freshly prepared before each experiment. The red laser diode (model XYZ, Thorlabs, USA) was calibrated using a digital optical power meter before every run to ensure precise fluence delivery. A BioTek Synergy HT microplate reader (BioTek Instruments, USA) was used for all absorbance measurements, with wavelength calibration verified weekly. All experiments were carried out under aseptic conditions inside a Class II laminar flow biosafety cabinet (Thermo Scientific, USA) to prevent contamination. 5 , 7 , 8 , 11 2.1 Bacterial strain and culture Clinical P. aeruginosa was grown overnight (37°C, 150 rpm) and adjusted to OD600 ≈ 0.10 (~1×10^8 CFU/mL). 13 2.2 Photosensitizer (Allura Red) preparation Allura Red stock (5 mM) in sterile water, 0.22 μm-filtered, stored at 4°C (amber vials); working 25–50 μM in PBS, 10 min dark pre-incubation. 15 2.3 Experimental groups Six groups: Control (no Allura Red/no light), Light only (630–660 nm, 15 J/cm 2 ), Allura Red only (25–50 μM, dark), Allura Red + 5 J/cm 2 , Allura Red + 10 J/cm 2 , Allura Red + 15 J/cm 2 . 2.4 Light source and dosimetry Collimated 630–660 nm diode at 100 mW/cm 2 ; fluences by time: 5 J/cm 2 (50 s), 10 J/cm 2 (100 s), 15 J/cm 2 (150 s); 3 cm distance; plate temperature ≤ 28°C. 2.5 Plate setup and reagent volumes Per well: 100 μL bacterial suspension, then 50 μL Allura Red/PBS (dark pre-incubation 10 min), followed by 25 μL AMDA and 25 μL XTT/menadione (added immediately before irradiation to capture early ROS/metabolic changes). The total volume per well was 200 μL; edge wells were filled with PBS to minimize edge effects. Results for AMDA and XTT are presented in Figure 2 and Figure 3 , respectively. Figure 1. Experimental workflow for the integrated AMDA–XTT–CFU–CV evaluation of Allura Red-mediated aPDT against Pseudomonas aeruginosa. Figure 2. AMDA (A380) across treatment groups indicating dose-dependent ROS generation. Figure 3. XTT (A450–470) across treatment groups showing metabolic suppression with increasing fluence. Per well: 100 μL bacteria + 50 μL Allura Red/PBS + 25 μL AMDA (50 μM) + 25 μL XTT/menadione (500 μg/mL XTT + 50 μM menadione); 200 μL total; edge wells filled with PBS. 2.6 AMDA and XTT readouts AMDA absorbance was measured at 380 nm using a standard microplate reader (common and accessible in Iraq), as fluorescence detection systems are not widely available. XTT absorbance was measured at 450–470 nm. Readings were recorded immediately post-irradiation and normalized to the Control group (see Figure 2 and Figure 3 ). Dose–response relationships are summarized in Figure 7 . Figure 4. Absolute CFU counts (CFU/mL) across groups demonstrating progressive killing. Figure 5. CFU log10 values across groups showing the magnitude of bacterial reduction. Figure 6. Crystal Violet biofilm biomass (A590) quantification with inhibition at 10–15 J/cm 2 . Figure 7. Dose–response curves for AMDA (ROS) and XTT (metabolic activity) in Allura Red + Light groups. AMDA at A380 and XTT at A450–470 were recorded immediately post-irradiation and normalized to Control. 2.7 CFU counts Serial dilutions (10^-1–10^-6) were plated on LB agar (100 μL) and incubated 24 h at 37°C. Absolute CFU/mL and log10 reductions versus Control were calculated (see Figure 4 and Figure 5 ). Serial dilutions (10^-1–10^-6) plated on LB agar (100 μL); 24 h at 37°C. Reported as absolute CFU/mL and log10 reduction versus Control. 2.8 Crystal violet biofilm assay Biofilms were formed for 24 h, washed, stained with 0.1% CV (15 min), rinsed, and destained (30% acetic acid, 15 min); absorbance was read at 590 nm. Biofilm inhibition (%) was calculated relative to Control (see Figure 6 ). Biofilms formed 24 h; washed; stained with 0.1% CV (15 min); rinsed; destained (30% acetic acid, 15 min); read at A590; inhibition (%) vs Control. 2.9 Statistics Data are presented as mean ± SD from n = 3 independent biological replicates; within each biological replicate, plate wells were recorded in technical duplicates. One-way ANOVA with Tukey’s post-hoc was used (p < 0.05). Mean ± SD (n = 3 independent experiments). One-way ANOVA + Tukey’s post-hoc (p < 0.05). Pearson correlations optionally evaluated. 3. Results A full summary of all experimental endpoints is presented in Table 3 , integrating AMDA, XTT, CFU, and CV data across groups. Table 1. Summary of endpoints (mean ± SD) with p-values versus Control for each group. Data are expressed as mean ± standard deviation (SD) from three independent replicates (n = 3). All experiments were conducted under controlled photodynamic therapy (PDT) conditions using Allura Red as the photosensitizer and red laser irradiation (630 nm) at specified doses. Statistical analysis was performed using one-way ANOVA with significance set at p < 0.05. Group AMDA (Mean ± SD) XTT (Mean ± SD) CFU log10 (Mean ± SD) Biofilm CV A590 (Mean ± SD) p-value vs Control Control 0.12 ± 0.01 0.85 ± 0.00 8.02 ± 0.03 0.99 ± 0.03 - Light only 0.19 ± 0.04 0.80 ± 0.01 8.02 ± 0.04 0.93 ± 0.02 NS Allura Red only 0.34 ± 0.03 0.65 ± 0.02 7.93 ± 0.08 0.73 ± 0.05 <0.05 Allura Red + 5 J/cm 2 0.55 ± 0.07 0.49 ± 0.03 7.75 ± 0.01 0.61 ± 0.04 <0.01 Allura Red + 10 J/cm 2 0.75 ± 0.06 0.33 ± 0.02 7.53 ± 0.05 0.45 ± 0.04 <0.001 Allura Red + 15 J/cm 2 0.95 ± 0.04 0.20 ± 0.01 7.01 ± 0.05 0.24 ± 0.02 <0.001 Table 2. Group-wise data (mean ± SD) from n = 3 independent experiments under Allura Red PDT at specified doses. Data represent mean ± standard deviation (SD) from three independent experiments (n = 3). Samples were analyzed under specified photodynamic therapy (PDT) conditions using Allura Red as a photosensitizer and red laser irradiation at 630 nm with indicated energy doses. Statistical significance was determined using one-way ANOVA with p < 0.05 considered significant. Number Title Page Figure 1 Experimental workflow for the integrated AMDA–XTT–CFU–CV evaluation of Allura Red-mediated aPDT against Pseudomonas aeruginosa. 5 Table 1 Summary of endpoints (mean ± SD) with p-values versus Control for each group. 6 Figure 2 AMDA fluorescence intensity across treatment groups, showing dose-dependent ROS generation. 7 Figure 3 XTT assay absorbance at 450 nm across treatment groups, indicating reduced metabolic activity with increasing light dose. 8 Figure 4 Absolute CFU counts (CFU/mL) across treatment groups, demonstrating progressive bacterial killing. 9 Figure 5 CFU log10 values across treatment groups, showing the magnitude of bacterial reduction. 10 Figure 6 Crystal Violet biofilm biomass quantification, demonstrating inhibition at higher fluences. 11 Figure 7 Dose–response curves for AMDA (ROS) and XTT (metabolic activity) in Allura Red + Light groups. 12 Table 3. Comprehensive summary of AMDA, XTT, absolute CFU, CFU log10, and CV (A590) across all experimental groups. Summary of experimental endpoints and related data. Group AMDA Mean AMDA SD XTT Mean XTT SD CFU abs Mean CFU abs SD CFU log10 Mean CFU log10 SD CV A590 Mean ± SD Control 0.12 0.01 0.85 0.00 95944650 11920751 8.02 0.03 0.99 ± 0.03 Light only 0.19 0.04 0.80 0.01 87345277 11439975 8.02 0.04 0.93 ± 0.02 Allura Red only 0.34 0.03 0.65 0.02 81010565 12822788 7.93 0.08 0.73 ± 0.05 Allura Red + 5J 0.55 0.07 0.49 0.03 61651524 7988680 7.75 0.01 0.61 ± 0.04 Allura Red + 10J 0.75 0.06 0.33 0.02 28265034 3088384 7.53 0.05 0.45 ± 0.04 Allura Red + 15J 0.95 0.04 0.20 0.01 12447414 1338627 7.01 0.05 0.24 ± 0.02 Oxidative signals (AMDA, XTT) AMDA increased with light dose in Allura Red-treated arms (indicating more ROS), while XTT decreased (indicating reduced metabolic activity); Light-only and Allura Red-only groups remained near baseline. These trends were mirrored by microbiological outcomes: absolute CFU/mL decreased progressively and CFU log10 levels reflected stronger killing at higher fluence. Biofilm biomass (CV) was substantially reduced at 10–15 J/cm 2 . Specifically, CV absorbance decreased from 0.99 in the control to 0.45 and 0.24 at 10 J/cm 2 and 15 J/cm 2 , corresponding to ≈55% and ≈76% biofilm inhibition, respectively (see Table 3 ). 4. Discussion Moreover, previous work has highlighted the role of antimicrobial photodynamic inactivation as a promising strategy to overcome resistance mechanisms in microbes. 14 The present study demonstrates the effectiveness of Allura Red–mediated photodynamic therapy (PDT) against Pseudomonas aeruginosa, highlighting the integration of multiple evaluation assays as a novel methodological advancement. The findings revealed a clear dose-dependent antibacterial effect, with the highest fluence (15 J/cm 2 ) producing significant reductions in colony-forming units (CFU), marked increases in reactive oxygen species (ROS), strong inhibition of metabolic activity, and substantial biofilm disruption. Previous research has extensively explored Allura Red and other conventional photosensitizers in antimicrobial PDT. However, the current work differs by employing Allura Red, a food-grade dye with established safety but underexplored potential as a photosensitizer. This provides a dual advantage: enhancing the therapeutic scope of PDT and offering a cost-effective, biocompatible alternative. To our knowledge, this is the first study to systematically integrate AMDA, XTT, CFU, and CV within a single Allura Red–based PDT workflow. The strong correlation between ROS generation and reductions in CFU and metabolic activity underscores the central role of oxidative stress in the bactericidal mechanism of PDT. Despite promising outcomes, limitations include the in vitro nature of the study, which does not fully replicate the complexity of host environments. Future investigations should explore the efficacy of Allura Red–PDT in vivo. Taken together, this study establishes Allura Red–mediated PDT as a promising strategy for managing multidrug-resistant, biofilm-associated infections. All presented data were obtained from n = 3 independent biological replicates with technical duplicates, and statistical analysis was performed using one-way ANOVA followed by Tukey’s post-hoc test (p < 0.05). The present study demonstrates the potent antibacterial efficacy of Allura Red (Allura Red)-mediated photodynamic therapy (PDT) against Pseudomonas aeruginosa, highlighting a novel combination of AMDA and XTT assays for a more comprehensive evaluation of treatment effects. The results showed that Allura Red alone or red laser irradiation alone induced minimal bactericidal effects, consistent with the known low intrinsic toxicity of Allura Red in the absence of light activation and the inability of red light at the applied wavelength to damage bacterial cells without a photosensitizer. 6 , 11 , 13 Significant CFU reductions were observed in the Allura Red + Laser groups, with ≈36%, ≈71%, and ≈87% reduction for the 5 J/cm 2 , 10 J/cm 2 , and 15 J/cm 2 doses, respectively. These reductions indicate strong dose-dependent bactericidal effects, although the improvement from 10 J/cm 2 to 15 J/cm 2 was modest, suggesting a photodynamic saturation threshold. 1 , 5 The AMDA absorbance at 380 nm assay confirmed that the enhanced bactericidal activity in the Allura Red + Laser groups was due to a significant increase in intracellular ROS, especially singlet oxygen (^1O_2), which is the primary cytotoxic agent in Allura Red-mediated PDT. The high ROS levels detected at both 10 J and 15 J doses align with the observed CFU reductions, reinforcing the ROS-mediated killing mechanism. 3 , 6 The XTT assay further validated these findings by demonstrating decreased metabolic activity, with the greatest suppression (≈76%) observed at 15 J/cm 2 , closely matching the high ROS levels detected via AMDA. The CV biofilm assay also confirmed substantial biomass inhibition of ≈55% at 10 J/cm 2 and ≈76% at 15 J/cm 2 , highlighting the potential of Allura Red-PDT to disrupt established biofilms. 11 , 12 From a translational perspective, these findings are significant because P. aeruginosa is known for its multidrug resistance and biofilm-forming capabilities, making it a challenging pathogen in clinical settings, especially in immunocompromised patients and in chronic wound infections. The demonstrated effectiveness of Allura Red-PDT in reducing bacterial load and metabolic activity suggests potential for application in topical and wound-care formulations, particularly where conventional antibiotics fail. 10 , 15 Several previous studies have examined Allura Red-PDT against Gram-negative bacteria, but few have incorporated both ROS quantification (AMDA) and metabolic activity assessment (XTT) in the same experimental framework, making this study novel. The integration of these two assays allows for a more nuanced understanding of how PDT parameters influence both oxidative stress induction and functional bacterial viability. 13 , 15 Despite the promising findings, certain limitations should be acknowledged. The study was performed under in vitro conditions, which cannot fully mimic the complexity of in vivo infections, where factors such as tissue penetration, immune responses, and oxygen availability may influence PDT outcomes. Further investigations are recommended to assess the performance of Allura Red-PDT with this dual-assay strategy in biofilm models and animal infection systems to confirm its clinical applicability. 6 , 11 , 13 In conclusion, the results strongly indicate that integrating AMDA and XTT assays provides a valuable approach for PDT studies, supporting more accurate optimization of treatment parameters and offering deeper insights into the underlying antimicrobial mechanisms. 1 , 5 5. Conclusion Allura Red–mediated PDT demonstrated strong dose-dependent antibacterial activity against P. aeruginosa. By unifying AMDA, XTT, CFU, and CV assays, this study provides a novel multi-assay framework for PDT evaluation. The approach highlights Allura Red as a promising photosensitizer for future antimicrobial applications. This study presents a novel multi-assay framework integrating AMDA, XTT, CFU, and CV to evaluate Allura Red-mediated PDT against Pseudomonas aeruginosa. The approach captured oxidative stress, metabolic suppression, viable cell reduction, and biofilm disruption in parallel, overcoming the limitations of single-endpoint methods. A fluence of 15 J/cm 2 showed the highest antimicrobial effect, with ~87% CFU reduction, ~76% metabolic decline, and ~76% biofilm decrease, alongside significant ROS generation. The consistent agreement across assays highlights ROS as a key driver of bacterial killing and biofilm destruction. This reproducible and scalable methodology offers strong potential for optimizing PDT protocols, particularly for multidrug-resistant, biofilm-associated infections, and could be extended to in vivo models for clinical validation. 5.1 Highlights • First unified AMDA–XTT–CFU–CV workflow for Allura Red-aPDT against P. aeruginosa. All findings are based on n = 3 independent biological replicates with technical duplicates, analyzed using one-way ANOVA and Tukey’s post-hoc test (p < 0.05). • Dose-dependent AMDA/XTT aligns with CFU killing and CV biofilm inhibition. All findings are based on n = 3 independent biological replicates with technical duplicates, analyzed using one-way ANOVA and Tukey’s post-hoc test (p < 0.05). • Dual CFU reporting (absolute and log-reduction) improves interpretability. All findings are based on n = 3 independent biological replicates with technical duplicates, analyzed using one-way ANOVA and Tukey’s post-hoc test (p < 0.05). • Reduced sample burden and assay variability versus fragmented workflows. All findings are based on n = 3 independent biological replicates with technical duplicates, analyzed using one-way ANOVA and Tukey’s post-hoc test (p < 0.05). • Transferable to other photosensitizers and preclinical models. All findings are based on n = 3 independent biological replicates with technical duplicates, analyzed using one-way ANOVA and Tukey’s post-hoc test (p < 0.05). List of Figures and Tables All findings are based on n = 3 independent biological replicates with technical duplicates, analyzed using one-way ANOVA and Tukey’s post-hoc test (p < 0.05). Data availability The datasets generated and analyzed during this study are openly available in Zenodo at DOI: https://doi.org/10.5281/zenodo.16988108 under a CC-BY license. All raw values behind means, standard deviations, figures, and tables are included. References 1. Misba L, Zaidi S, Khan AU: Efficacy of photodynamic therapy against Pseudomonas aeruginosa biofilm: Role of extracellular polymeric substances. Photodiagn. Photodyn. Ther. 2019; 26 : 383–388. PubMed Abstract | Publisher Full Text 2. Khan AU, Metgud R, Hamblin MR: Reconsidering photodynamic therapy against multidrug-resistant pathogens: A new light in the dark. Photodiagn. Photodyn. Ther. 2020; 30 : 101645. 3. Hamblin MR, Hasan T: Photodynamic therapy: A new antimicrobial approach to infectious disease? Photochem. Photobiol. Sci. 2004; 3 (5): 436–450. PubMed Abstract | Publisher Full Text | Free Full Text 4. Wainwright M, Maisch T, Nonell S, et al. : Photoantimicrobials—are we afraid of the light? Lancet Infect. Dis. 2017; 17 (2): e49–e55. PubMed Abstract | Publisher Full Text | Free Full Text 5. Pourhajibagher M, Chiniforush N, Bahador A: Antimicrobial photodynamic therapy with phenothiazinium dyes against multidrug-resistant bacteria. J. Lasers Med. Sci. 2018; 9 (3): 139–146. 6. Cieplik F, Tabenski L, Buchalla W, et al. : Antimicrobial photodynamic therapy for inactivation of biofilms formed by oral key pathogens. Front. Microbiol. 2014; 5 : 405. 7. Diogo P, Faustino MA, Neves MG, et al. : Photodynamic antimicrobial chemotherapy for root canal system asepsis: A narrative literature review. Int J Dent. 2015; 2015 : 1–26. Publisher Full Text 8. Tegos GP, Hamblin MR: Phenothiazinium antimicrobial photosensitizers are substrates of bacterial multidrug resistance pumps. Antimicrob. Agents Chemother. 2006; 50 (1): 196–203. PubMed Abstract | Publisher Full Text | Free Full Text 9. Almeida A, Faustino MA, Neves MG: Antimicrobial photodynamic therapy in the control of multidrug-resistant bacteria: A new approach to antimicrobial resistance. Antibiotics. 2019; 8 (3): 122. 10. Mesbah L, Misba L, Khan AU: Light-based approaches to inactivate multidrug-resistant pathogens. Lasers Med. Sci. 2020; 35 (2): 275–283. 11. Dai T, Gupta A, Murray CK, et al. : Blue light for infectious diseases: Propionibacterium acnes, Helicobacter pylori, and beyond. Drug Resist. Updat. 2012; 15 (4): 223–236. PubMed Abstract | Publisher Full Text | Free Full Text 12. Kashef N, Hamblin MR: Can microbial cells develop resistance to oxidative stress in antimicrobial photodynamic inactivation? Drug Resist. Updat. 2017; 31 : 31–42. PubMed Abstract | Publisher Full Text | Free Full Text 13. Misba L, Abdulrahman H, Khan AU: Photodynamic strategies to tackle multidrug-resistant biofilms. Photodiagn. Photodyn. Ther. 2019; 28 : 219–227. 14. Hamblin MR: Antimicrobial photodynamic inactivation: A bright new technique to kill resistant microbes. Curr. Opin. Microbiol. 2016; 33 : 67–73. PubMed Abstract | Publisher Full Text | Free Full Text 15. Khan AU, Mesbah L: Innovative applications of photodynamic therapy against resistant pathogens. Photodiagn. Photodyn. Ther. 2021; 34 : 102233. Comments on this article Comments (0) Version 1 VERSION 1 PUBLISHED 15 Sep 2025 ADD YOUR COMMENT Comment Author details Author details 1 Medical science, Al-Mashreq University, Baghdad, Baghdad, Iraq 2 Medical Physics, College of Applied Sciences, Hit, University of Anbar,, Anbar, Anbar, Iraq Hayder Abdulrahman Majeed Roles: Conceptualization, Data Curation, Formal Analysis, Investigation, Methodology, Project Administration, Validation, Visualization, Writing – Original Draft Preparation, Writing – Review & Editing Mawlood Maajal Ali Ali Roles: Project Administration Competing interests No competing interests were disclosed. Grant information The author(s) declared that no grants were involved in supporting this work. Article Versions (1) version 1 Published: 15 Sep 2025, 14:923 https://doi.org/10.12688/f1000research.170011.1 Copyright © 2025 Majeed HA and Ali MMA. This is an open access article distributed under the terms of the Creative Commons Attribution License , which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Download Export To Sciwheel Bibtex EndNote ProCite Ref. Manager (RIS) Sente metrics Views Downloads F1000Research - - PubMed Central info_outline Data from PMC are received and updated monthly. - - Citations open_in_new 0 open_in_new 0 open_in_new SEE MORE DETAILS CITE how to cite this article Majeed HA and Ali MMA. Allura Red–Mediated Photodynamic Therapy: A Novel Multi-Assay Evaluation Against Pseudomonas aeruginosa [version 1; peer review: 1 approved] . F1000Research 2025, 14 :923 ( https://doi.org/10.12688/f1000research.170011.1 ) NOTE: If applicable, it is important to ensure the information in square brackets after the title is included in all citations of this article. COPY CITATION DETAILS track receive updates on this article Track an article to receive email alerts on any updates to this article. TRACK THIS ARTICLE Share Open Peer Review Current Reviewer Status: ? Key to Reviewer Statuses VIEW HIDE Approved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested Approved with reservations A number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit. Not approved Fundamental flaws in the paper seriously undermine the findings and conclusions Version 1 VERSION 1 PUBLISHED 15 Sep 2025 Views 0 Cite How to cite this report: Das S. Reviewer Report For: Allura Red–Mediated Photodynamic Therapy: A Novel Multi-Assay Evaluation Against Pseudomonas aeruginosa [version 1; peer review: 1 approved] . F1000Research 2025, 14 :923 ( https://doi.org/10.5256/f1000research.187421.r422754 ) The direct URL for this report is: https://f1000research.com/articles/14-923/v1#referee-response-422754 NOTE: it is important to ensure the information in square brackets after the title is included in this citation. Close Copy Citation Details Reviewer Report 12 Nov 2025 Sharmistha Das , The Neotia University, Sarisha, West Bengal, India Approved VIEWS 0 https://doi.org/10.5256/f1000research.187421.r422754 Major Comments The use of a food-grade dye (Allura Red) as a photosensitizer is innovative and practical, but the rationale for its selection should be strengthened with physicochemical justification (e.g., absorption coefficient, singlet oxygen quantum ... Continue reading READ ALL Major Comments The use of a food-grade dye (Allura Red) as a photosensitizer is innovative and practical, but the rationale for its selection should be strengthened with physicochemical justification (e.g., absorption coefficient, singlet oxygen quantum yield, molecular structure relevant to PDT efficiency) compared to established sensitizers like methylene blue or toluidine blue. The manuscript would benefit from clarifying whether Allura Red acts primarily through Type I or Type II photochemical pathways and how its ROS profile compares with conventional photosensitizers. While ROS generation is quantified through AMDA absorbance, there is limited mechanistic discussion linking oxidative stress to cellular damage. Transmission electron microscopy (TEM) or confocal imaging data could substantiate claims of membrane disruption or intracellular oxidation. The potential role of efflux pump inhibition or alterations in extracellular polymeric substances (EPS) post-PDT could be hypothesized based on the observed biofilm reduction. The inclusion of “light only” and “dye only” controls is commendable; however, a dark toxicity control over time (to rule out dye degradation or spontaneous ROS formation) should be described. The AMDA assay was measured at 380 nm (absorbance) instead of fluorescence, which is acceptable given equipment constraints, but the authors should acknowledge that this might underestimate singlet oxygen yield and discuss the limitation accordingly. The results demonstrate statistical significance, but the description could be strengthened by including exact p -values rather than categorical ranges (e.g., < 0.05, < 0.001). Correlation analysis between AMDA and CFU/XTT outcomes (Pearson r ) should be included in the main text or supplementary data to quantify the strength of association between oxidative stress and viability suppression. Is the work clearly and accurately presented and does it cite the current literature? Yes Is the study design appropriate and is the work technically sound? Yes Are sufficient details of methods and analysis provided to allow replication by others? Partly If applicable, is the statistical analysis and its interpretation appropriate? Partly Are all the source data underlying the results available to ensure full reproducibility? Yes Are the conclusions drawn adequately supported by the results? Yes Competing Interests: No competing interests were disclosed. Reviewer Expertise: Microbiology, biotchnology, biofilm biology, antimicrobial resistance, AI-ML in drug dose optimization I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard. Close READ LESS CITE CITE HOW TO CITE THIS REPORT Das S. Reviewer Report For: Allura Red–Mediated Photodynamic Therapy: A Novel Multi-Assay Evaluation Against Pseudomonas aeruginosa [version 1; peer review: 1 approved] . F1000Research 2025, 14 :923 ( https://doi.org/10.5256/f1000research.187421.r422754 ) The direct URL for this report is: https://f1000research.com/articles/14-923/v1#referee-response-422754 NOTE: it is important to ensure the information in square brackets after the title is included in all citations of this article. COPY CITATION DETAILS Report a concern Respond or Comment COMMENT ON THIS REPORT Comments on this article Comments (0) Version 1 VERSION 1 PUBLISHED 15 Sep 2025 ADD YOUR COMMENT Comment keyboard_arrow_left keyboard_arrow_right Open Peer Review Reviewer Status info_outline Alongside their report, reviewers assign a status to the article: Approved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested Approved with reservations A number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit. Not approved Fundamental flaws in the paper seriously undermine the findings and conclusions Reviewer Reports Invited Reviewers 1 Version 1 15 Sep 25 read Sharmistha Das , The Neotia University, Sarisha, India Comments on this article All Comments (0) Add a comment Sign up for content alerts Sign Up You are now signed up to receive this alert Browse by related subjects keyboard_arrow_left Back to all reports Reviewer Report 0 Views copyright © 2025 Das S. This is an open access peer review report distributed under the terms of the Creative Commons Attribution License , which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. 12 Nov 2025 | for Version 1 Sharmistha Das , The Neotia University, Sarisha, West Bengal, India 0 Views copyright © 2025 Das S. This is an open access peer review report distributed under the terms of the Creative Commons Attribution License , which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. format_quote Cite this report speaker_notes Responses (0) Approved info_outline Alongside their report, reviewers assign a status to the article: Approved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested Approved with reservations A number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit. Not approved Fundamental flaws in the paper seriously undermine the findings and conclusions Major Comments The use of a food-grade dye (Allura Red) as a photosensitizer is innovative and practical, but the rationale for its selection should be strengthened with physicochemical justification (e.g., absorption coefficient, singlet oxygen quantum yield, molecular structure relevant to PDT efficiency) compared to established sensitizers like methylene blue or toluidine blue. The manuscript would benefit from clarifying whether Allura Red acts primarily through Type I or Type II photochemical pathways and how its ROS profile compares with conventional photosensitizers. While ROS generation is quantified through AMDA absorbance, there is limited mechanistic discussion linking oxidative stress to cellular damage. Transmission electron microscopy (TEM) or confocal imaging data could substantiate claims of membrane disruption or intracellular oxidation. The potential role of efflux pump inhibition or alterations in extracellular polymeric substances (EPS) post-PDT could be hypothesized based on the observed biofilm reduction. The inclusion of “light only” and “dye only” controls is commendable; however, a dark toxicity control over time (to rule out dye degradation or spontaneous ROS formation) should be described. The AMDA assay was measured at 380 nm (absorbance) instead of fluorescence, which is acceptable given equipment constraints, but the authors should acknowledge that this might underestimate singlet oxygen yield and discuss the limitation accordingly. The results demonstrate statistical significance, but the description could be strengthened by including exact p -values rather than categorical ranges (e.g., < 0.05, < 0.001). Correlation analysis between AMDA and CFU/XTT outcomes (Pearson r ) should be included in the main text or supplementary data to quantify the strength of association between oxidative stress and viability suppression. Is the work clearly and accurately presented and does it cite the current literature? Yes Is the study design appropriate and is the work technically sound? Yes Are sufficient details of methods and analysis provided to allow replication by others? Partly If applicable, is the statistical analysis and its interpretation appropriate? Partly Are all the source data underlying the results available to ensure full reproducibility? Yes Are the conclusions drawn adequately supported by the results? Yes Competing Interests No competing interests were disclosed. Reviewer Expertise Microbiology, biotchnology, biofilm biology, antimicrobial resistance, AI-ML in drug dose optimization I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard. reply Respond to this report Responses (0) Das S. Peer Review Report For: Allura Red–Mediated Photodynamic Therapy: A Novel Multi-Assay Evaluation Against Pseudomonas aeruginosa [version 1; peer review: 1 approved] . F1000Research 2025, 14 :923 ( https://doi.org/10.5256/f1000research.187421.r422754) NOTE: it is important to ensure the information in square brackets after the title is included in this citation. The direct URL for this report is: https://f1000research.com/articles/14-923/v1#referee-response-422754 Alongside their report, reviewers assign a status to the article: Approved - the paper is scientifically sound in its current form and only minor, if any, improvements are suggested Approved with reservations - A number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit. Not approved - fundamental flaws in the paper seriously undermine the findings and conclusions Adjust parameters to alter display View on desktop for interactive features Includes Interactive Elements View on desktop for interactive features Competing Interests Policy Provide sufficient details of any financial or non-financial competing interests to enable users to assess whether your comments might lead a reasonable person to question your impartiality. 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