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

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/cm² 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/cm², with exposure times of 50, 100, and 150 seconds for 5, 10, and 15 J/cm², respectively.

This study evaluates the efficacy of Allura Red-mediated aPDT against P. aeruginosa at different energy doses (5, 10, 15 J/cm²), 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 blue¹ was prepared in sterile distilled water and stored in amber vials at 4°C to prevent photodegradation. Aminophenyl methylene diphosphonate² and XTT sodium salt³ 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

2.5 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/cm².

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.

3. Results

A full summary of all experimental endpoints is presented in Table 3, integrating AMDA, XTT, CFU, and CV data across groups.

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/cm². Specifically, CV absorbance decreased from 0.99 in the control to 0.45 and 0.24 at 10 J/cm² and 15 J/cm², 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/cm²) 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/cm², 10 J/cm², and 15 J/cm² doses, respectively. These reductions indicate strong dose-dependent bactericidal effects, although the improvement from 10 J/cm² to 15 J/cm² 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/cm², closely matching the high ROS levels detected via AMDA. The CV biofilm assay also confirmed substantial biomass inhibition of ≈55% at 10 J/cm² and ≈76% at 15 J/cm², 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/cm² 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.