Enhancing antimicrobial efficacy against Pseudomonas aeruginosa biofilm through carbon dot-mediated photodynamic inactivation

Abstract

Pseudomonas aeruginosa biofilms shield the bacteria from antibiotics and the body’s defenses, often leading to chronic infections that are challenging to treat. This study aimed to assess the impact of sub-lethal doses of antimicrobial photodynamic inactivation (sAPDI) utilizing carbon dots (CDs) derived from gentamicin and imipenem on biofilm formation and the expression of genes (pelA and pslA) associated with P. aeruginosa biofilm formation.

The anti-biofilm effects of sAPDI were evaluated by exposing P. aeruginosa to sub-minimum biofilm inhibitory concentrations (sub-MBIC) of CDsGEN-NH₂, CDsIMP-NH₂, CDsGEN-IMP, and CDsIMP-GEN, combined with sub-lethal UVA light irradiation. Biofilm formation ability was assessed by crystal violet (CV) assay and enumeration method. Additionally, the impact of sAPDI on the expression of pelF and pslA genes was evaluated using real-time quantitative polymerase chain reaction (RT-qPCR).

Compared to the control group, the sAPDI treatment with CDsGEN-NH₂, CDsIMP-NH₂, CDsGEN-IMP, and CDsIMP-GEN resulted in a significant reduction in biofilm activity of P. aeruginosa ATCC 27853 (P < 0.0001). The CV assay method demonstrated reductions in optical density of 83.70%, 81.08%, 89.33%, and 75.71%, while the CFU counting method showed reductions of 4.03, 3.76, 4.39, and 3.21 Log₁₀ CFU/mL. qRT-PCR analysis revealed decreased expression of the pelA and pslA genes in P. aeruginosa ATCC 27853 following sAPDI treatment compared to the control group (P < 0.05).

The results indicate that sAPDI using CDs derived from gentamicin and imipenem can decrease the biofilm formation of P. aeruginosa and the expression of the pelA and pslA genes associated with its biofilm formation.

Introduction

Pseudomonas aeruginosa plays a crucial role as an opportunistic microorganism in healthcare-associated infections, especially in burn units, resulting in significant morbidity and mortality among affected patients (Gomersall et al. 2023). The limited penetration of antimicrobial agents through the cell wall of P. aeruginosa and its rapid development of drug resistance contribute to its high mortality rates. Furthermore, P. aeruginosa demonstrates remarkable adaptability, rapidly acquiring resistance to antimicrobial agents and producing various virulence factors and biofilms (Gomersall et al. 2023; Fakhry and Aljanabi 2024). Biofilms are recognized as structured populations of microbial cells that adhere to living or non-living surfaces and are surrounded by a matrix composed of extracellular polymeric substances (EPS) (Penesyan et al. 2021). EPS plays a crucial role in forming the structural framework of biofilms. These exopolysaccharides within the biofilm matrix have the ability to impede the penetration of antimicrobial agents and act as a protective shield, preventing host immune cells from effectively engulfing and destroying the biofilm. It appears that P. aeruginosa possesses the capacity to generate various kinds of polysaccharides, including alginate, Pel, and psl (Chung et al. 2023). Alginate, composed of monomers such as mannuronic acid and guluronic acid, contributes to the formation of bacterial microcolonies within living organisms (Moradali and Rehm 2019). The exact composition of Pel polysaccharide remains uncertain, whereas psl consists of a recurring penta-saccharide sequence containing D-mannose, L-rhamnose, and D-glucose. Both Pel and psl polysaccharides play roles in the initial phases of biofilm formation. The pelA and pslA genes are pivotal in the biofilm formation process, being the most crucial genes required for it (Hendiani et al. 2019; Soleymani-Fard et al. 2024). Considering the limited effectiveness of antibiotics and the increasing prevalence of drug resistance, alternative therapies such as antimicrobial photodynamic inactivation (APDI) have gained attention for the treatment of P. aeruginosa infections. APDI offers a unique mechanism compared to antibiotics, and research suggests that the likelihood of bacterial resistance to APDI is low, making it a promising treatment option (Dong et al. 2017; Abdelkarim-Elafifi et al. 2021). APDI employs light to activate a photosensitizer (PS) in the presence of oxygen, resulting in the generation of reactive oxygen species (ROS) that induce cell death through apoptosis or necrosis (Lin et al. 2017, 2018). APDI shows great promise, and carbon dots (CDs) are being investigated as potential nanoparticles for their role as photosensitizers in combating pathogens (Meziani et al. 2016; Anand et al. 2019). CDs possess a wide range of optical capabilities and exceptional properties, making them suitable nanomaterials for photosensitization across various light spectrums, including UV-visible, Xe lamp, blue, and NIR light (Marković et al. 2019; Nie et al. 2020; Liu et al. 2021, 2022; Yan et al. 2021; Wu et al. 2022). To date, CDs have been synthesized using diverse carbon sources. Scientists have developed nanocomposites, including CD/metal oxide combinations, modified CDs incorporating common PSs such as methylene blue (MB) and antibiotics, aiming to enhance the effectiveness of APDI against microorganisms (Sidhu et al. 2017; Liu et al. 2018; Zhang et al. 2018; Yao et al. 2019). However, there is limited research exploring the anti-biofilm effects of antibiotic-derived CDs. Antibiotics like imipenem and gentamicin are commonly used to treat burn wound infections caused by P. aeruginosa (Memar et al. 2021). In APDI for the treatment of infections, exposure to sub-lethal doses of APDI (sAPDI) may not eradicate microorganisms entirely, but it significantly impacts their virulence. Several studies have shown that sAPDI, employing various PS types, influences bacteria’s biofilm formation ability and the expression of genes associated with it (Hendiani et al. 2019; Mahmoud et al. 2023; Pires et al. 2024). Nevertheless, there is a lack of documentation regarding the effects of sAPDI using antibiotics as PS on biofilm formation ability of P. aeruginosa and the expression of genes associated with biofilm formation. Thus, this study aimed to evaluate the effects of sAPDI using CDs derived from gentamicin and imipenem on biofilm formation and the expression of key genes (pelA and pslA) associated with P. aeruginosa biofilm formation.

Materials and methods

Bacterial strains

In the investigation, the P. aeruginosa ATCC 27853 standard strain and a clinical isolate of P. aeruginosa, resistant to imipenem and gentamicin, were employed. A clinical isolate of P. aeruginosa was isolated from specimens of burn wounds. The P. aeruginosa ATCC 27853 used in this study was obtained from the microbial bank of the Microbiology Laboratory, Faculty of Medicine, Hamadan University of Medical Sciences. The burn wound samples used for isolating P. aeruginosa were collected from the burn unit of Be’sat Hospital of Hamadan University of Medical Sciences, Hamadan, Iran. The susceptibility of microorganisms to imipenem and gentamicin was assessed using disc diffusion and broth microdilution methods, following the guidelines outlined by the Clinical and Laboratory Standards Institute (CLSI) (Wayne 2018). Antibiotic discs were sourced from Condalab in Spain, while the antibiotic powders were obtained from Sigma-Aldrich in the USA.

Photosensitizer

In this study, CDs synthesized in a previous investigation by Shiralizadeh et al. were used as photosensitizers (Data not shown). Amine-functionalized CDs were synthesised from gentamicin (CDsGEN-NH₂) and imipenem (CDsIMP-NH₂) precursors using a hydrothermal method. CDsGEN-NH₂ and CDsIMP-NH₂ were conjugated with imipenem and gentamicin respectively, resulting in CDsGEN-IMP and CDsIMP-GEN.

Light source

In line with previous research by our team, this study also used the TUV6WE (Cathodeon, UK), a low-pressure mercury vapor device that primarily emits UV radiation at 320 nm.

Biofilm evaluation of bacterial strains

In the assessment of biofilm formation, a microtiter plate (MTP) assay was employed, following a previously described methodology (Stepanović et al. 2007). Briefly, the bacterial strains isolated from overnight broth cultures were diluted at a 1:100 ratio in Luria-Bertani (LB) broth and then introduced into a microtiter plate, with 200 µL per well. The strains were tested three times, and the resulting average was calculated. Sterile LB broth filled the blank wells. After incubating the microplate at 37 °C for 24 h, the contents of the wells were removed and washed with saline solution. After that, the wells were stained with 0.1% crystal violet (CV). The CV was then removed, and any residual CV in the wells was dissolved with the addition of 95% ethanol. The measurement of optical density (OD) in the wells was conducted at 570 nm, and the biofilm assay was performed in accordance with the criteria established by Stepanović et al. (Stepanović et al. 2007).

Determination of minimum biofilm inhibitory concentration (MBIC) and sub-MBIC of CDsGEN-NH2, CDsIMP-NH2, CDsGEN-IMP, and CDsIMP-GEN

In accordance with CLSI recommendations, the MBIC is described as the minimum concentration of an antibacterial drug needed to prevent biofilm formation (Wayne 2018). The sub-MBIC is the highest concentration of CDs at which bacterial biofilm formation is observed in the microtiter plate well. To determine the MBIC, 100 µL of bacterial suspension at a concentration of 1.5 × 10⁶ CFU/mL was inoculated into microtiter plate wells. Adding 100 µL of CDsGEN-NH₂, CDsIMP-NH₂, and CDsGEN-IMP at various concentrations, the plates underwent incubation at 37 °C for 24 h (with final concentrations of 48, 24, 12, 6, 3, 1.5, 0.75, and 0.37 mg/mL). Concerning CDsIMP-GEN, 100 µL of CDs was inoculated at various concentrations (with final concentration of 4, 2, 1, 0.5, 0.25, 0.12, 0.06, and 0.03 mg/mL). Following incubation, the unattached cells and any residual CDs were eliminated through PBS washing, and subsequent procedures were conducted in a manner consistent with the prior study (Pourhajibagher et al. 2016a, b).

Determination of sub-lethal dose of UVA light irradiation time

To evaluate the sub-lethal dose of UVA light exposure time against P. aeruginosa, a methodology was employed based on a previous study (Pourhajibagher et al. 2016). In summary, various durations of UVA light exposure at 320 nm were irradiated to 200 µL of a P. aeruginosa suspension containing 1.5 × 10⁵ CFU/mL (10, 20, 30, 40, 50, and 60 s with 3.12, 6.25, 9.37, 12.5, 15.62, and 18.75 J/cm²). The group designated as the control did not undergo any form of treatment. Following the treatment, the plates were incubated at 37 °C for 24 h, and CFU/mL was assessed in accordance with the method described by Miles and Misra (Miles et al. 1938).

Determination of the anti-biofilm effects of sAPDI using CDsGEN-NH2, CDsIMP-NH2, CDsGEN-IMP, and CDsIMP-GEN

The effect of sAPDI using CDsGEN-NH₂, CDsGEN-IMP and CDsIMP-GEN on P. aeruginosa biofilm was evaluated by culturing a 100 µL bacterial suspension of P. aeruginosa (1.5 × 10⁶ CFU/mL) in a 96-well microtiter plate. Afterward, 100 µL of CDsGEN-NH₂, CDsIMP-NH₂, CDsGEN-IMP, and CDsIMP-GEN, each at sub-MBIC concentrations, were individually introduced into the wells. Following this, the microtiter plate was incubated for 5 min at ambient temperature and then subjected to sub-lethal dose of UVA light irradiation time. After incubating at 37 °C for 24 h, the biofilm mass was evaluated using the CV assay method described in the earlier study, measuring the OD at 570 nm (Pourhajibagher et al. 2016a, b). Viable bacteria were quantified through colony counting of bacteria collected from the well bed and reported as Log₁₀ CFU/mL, as previously outlined (Malone and Swanson 2017). The equation below was used to calculate the percentage reduction in bacterial cells after each treatment:

P= (1–10− L) × 100

P: percent reduction.

L: log reduction.

Observation of theP. aeruginosaATCC 27853 biofilm using field emission scanning electron microscopy (FESEM).

The effects of sAPDI on the biofilm of P. aeruginosa ATCC 27853 were studied by FESEM according to the study by Lee et al. (Lee et al. 2020). Sterile plastic squares, each measuring 1 cm², were placed in a 24-well microtiter plate. These wells were then inoculated with a bacterial solution of P. aeruginosa ATCC 27853 at a concentration of 1.5 × 10⁶ CFU/mL, along with sub-MBIC concentrations of CDsGEN-NH₂, CDsIMP-NH₂, CDsGEN-IMP, and CDsIMP-GEN. The plate was allowed to incubate for 5 min at ambient temperature before being subjected to a sub-lethal dose of UVA light. Subsequent incubation occurred at 37 °C for 24 h. Post-incubation, the plastic surfaces were washed with sterile saline and fixed with 2.5% glutaraldehyde for 12 h, followed by a wash in PBS. The samples were then dehydrated in a series of ethanol solutions at concentrations of 30%, 50%, 70%, 80%, 90%, and finally 100%. The biofilms formed on the plastic surfaces were then analyzed using a FESEM (S-4800; Hitachi, Tokyo, Japan).

Quantification of gene expression using quantitative real-time-PCR (qRT-PCR)

To explore the impact of sAPDI on the genes expression associated with biofilms in P. aeruginosa ATCC 27853, total RNA was extracted from the sAPDI-treated and control groups using RNX-Plus solution (SinaClone Co, Iran). Subsequently, cDNA was synthesized using a kit from Parstous Co, Iran, following the provided guidelines. The primers utilized are listed in Table 1. qRT-PCR was conducted on a LightCycler 96 system (Roche, Germany) using RealQ Plus 2x Master Mix Green Without ROX (Ampliqon, Denmark). The protocol included an initial denaturation at 95 °C for 15 min, followed by 40 cycles of denaturation at 95 °C for 15 s, annealing at 60 °C for 20 s, and extension at 72 °C for 20 s. The relative gene expression levels of pelA and pslA were analysed using the 2^−ΔΔCt method and normalised to rpoD gene as an internal control (Schmittgen and Livak 2008).

Table 1 Sequence of used primers

Statistical analysis

Experiments were performed in triplicate, and results are presented as means ± either standard deviation (SD) or standard error of the mean (SEM). Two-way ANOVA and Dunnett’s test were used to compare group means. A significance level of P < 0.05 was considered statistically significant.

Results

The sensitivity test of bacterial strains

The standard strain of P. aeruginosa ATCC 27853 and a clinical isolate were included in the study. The clinical isolate exhibited resistance to imipenem and gentamicin, as indicated by inhibition zones of ≤ 15 mm and ≤ 12 mm, respectively. Furthermore, the clinical isolate demonstrated minimum inhibitory concentration (MIC) values of ≥ 8 µg/mL and ≥ 16 µg/mL for imipenem and gentamicin, respectively.

Biofilm evaluation of bacterial isolates

Both the P. aeruginosa ATCC 27853 standard strain and a clinical isolate exhibited strong biofilm formation ability as assessed by the microtiter plate method.

MBIC and sub-MBIC of CDsGEN-NH2, CDsIMP-NH2, CDsGEN-IMP, and CDsIMP-GEN

At concentrations ranging from 12 to 48 mg/mL, CDsGEN-NH₂ showed a statistically significant decrease in the ability of both the standard strain P. aeruginosa ATCC 27853 and a clinical isolate to form biofilms compared to the control (P < 0.05, Fig. 1a and b). However, reducing the concentration of CDsGEN-NH₂ from 12 to 3 mg/mL did not significantly affect biofilm formation (P > 0.05). CDsIMP-NH₂ and CDsGEN-IMP showed a significant reduction in biofilm formation for both the standard P. aeruginosa ATCC 27853 strain and a clinical isolate when used in the concentration range of 24–48 mg/mL compared to the control (P < 0.05, Fig. 1a and b). However, reducing the concentrations of CDsIMP-NH₂ and CDsGEN-IMP from 24 to 3 mg/mL did not significantly affect biofilm formation (P > 0.05). The MBIC values for CDsGEN-NH₂, CDsIMP-NH₂, and CDsGEN-IMP in both the standard P. aeruginosa ATCC 27853 strain and the clinical isolate were 12, 24, and 24 mg/mL, respectively (Fig. 1a and b). Additionally, the sub-MBIC values for these compounds in both strains were 6, 12, and 12 mg/mL, respectively (Fig. 1a and b). CDsIMP-GEN, when was used at concentrations of 2 to 4 mg/mL, significantly reduced the ability of both the standard P. aeruginosa ATCC 27853 strain and the clinical isolate to form biofilms compared to the control (P < 0.05, Fig. 1c). In contrast, decreasing the concentration of CDsIMP-GEN from 2 to 0.25 mg/mL had no significant effect on biofilm formation (P > 0.05). The MBIC value of CDsIMP-GEN for both strains was 2 mg/mL (Fig. 1c), while the sub-MBIC values for these strains were 1 mg/mL (Fig. 1c).

Fig. 1

The effect of CD concentrations and UVA light exposure times on the cell viability of P. aeruginosa in biofilm formation

(a) The effect of varying concentrations of CDsGEN-NH₂, CDsIMP-NH₂, and CDsGEN-IMP on the cell viability (OD 570 nm) of P. aeruginosa ATCC 27853 in biofilm formation, (b) The effect of varying concentrations of CDsGEN-NH₂, CDsIMP-NH₂, and CDsGEN-IMP on the cell viability (OD 570 nm) of a clinical isolate of P. aeruginosa in biofilm formation, (c) The effect of varying concentrations of CDsIMP-GEN on the cell viability (OD 570 nm) of both P. aeruginosa ATCC 27853 and the clinical isolate in biofilm formation. (d) The effect of different UVA light irradiation times on the cell viability (CFU/mL) of both P. aeruginosa ATCC 27853 and the clinical isolate in biofilm formation. The results are reported as mean ± SD. * P < 0.05, ** P < 0.01, **** P < 0.0001 indicates significant differences compared to the control group

Sub-lethal dose of UVA light irradiation time

Our results showed a significant reduction in viability of the standard strain P. aeruginosa ATCC 27853 and a clinical isolate after UVA light irradiation for 50 s (15.62 J/cm²) and 40 s (12.5 J/cm²), respectively, compared to the control (p < 0.05, Fig. 1d). Based on these findings, UVA light exposure durations of 40 s (12.5 J/cm²) and 30 s (9.37 J/cm²) were determined as the highest sub-lethal doses of UVA light against P. aeruginosa ATCC 27853 and the clinical isolate, respectively.

Anti-biofilm ability of sAPDI againstP. aeruginosabiofilm.

In Table 2, the OD of the control biofilm for P. aeruginosa ATCC 27853 and the clinical isolate of P. aeruginosa were 1.563 and 1.608, respectively. The reduction in biofilm activity of sAPDI-CDsGEN-NH₂, sAPDI-CDsIMP-NH₂, sAPDI-CDsIMP-GEN and sAPDI-CDsGEN-IMP using sub-MBIC alongside a sub-lethal dose of UVA light exposure time against both P. aeruginosa ATCC 27853 and clinical isolate of P. aeruginosa biofilms was significant compared to the control (P < 0.0001). The mean Log₁₀ CFU/mL of control biofilm for P. aeruginosa ATCC 27853 and clinical isolate of P. aeruginosa were 11.13 and 11.30, respectively. There was a significant difference between the mean of Log₁₀ CFU/mL in the biofilm of P. aeruginosa ATCC 27853 and the clinical isolate of P. aeruginosa treated with sAPDI-CDsGEN-NH₂, sAPDI-CDsIMP-NH₂, sAPDI-CDsIMP-GEN and sAPDI-CDsGEN-IMP using sub-MBIC plus sub-lethal dose of UVA light irradiation time and the control group (P < 0.0001). As shown in Table 3, analysis of the reduction in Log₁₀ CFU/mL demonstrated that sAPDI-CDsIMP-GEN using sub-MBIC plus sub-lethal dose of UVA light irradiation was more potent in inhibiting biofilm formation of P. aeruginosa ATCC 27853 and a clinical isolate by 99.9959% and 99.9966%, respectively.

Table 2 Anti-biofilm effects of sPDI based on different CDs against P. Aeruginosa biofilms using the crystal violet assay

Table 3 Anti-biofilm effects of sPDI based on different CDs against P. Aeruginosa biofilms using colony-forming unit (CFU) counting method

FESEM results

The impact of sAPDI-CDsGEN-NH₂, sAPDI-CDsIMP-NH₂, sAPDI-CDsGEN-IMP, and sAPDI-CDsIMP-GEN on the biofilm formation of P. aeruginosa ATCC 27853 was examined using FESEM. The control group of P. aeruginosa formed a compact, cohesive biofilm that adhered evenly across the surface. The majority of cells were found to stick together, forming a multi-layered aggregation, as shown in Fig. 2a. The treatments with sAPDI-CDsGEN-NH₂, sAPDI-CDsIMP-NH₂, sAPDI-CDsGEN-IMP, and sAPDI-CDsIMP-GEN successfully prevented the formation of biofilms. Following the treatment with sAPDI-CDsGEN-NH₂, scarcely any P. aeruginosa ATCC 27853 cells remained, and the few that were observed were dispersed, as depicted in Fig. 2b. The treatments with sAPDI-CDsIMP-NH₂ and sAPDI-CDsIMP-GEN led to the near disappearance of the biofilm’s typical structure, illustrated in Fig. 2c and d. Post-treatment with sAPDI-CDsGEN-IMP, only small clusters of cells with altered shapes were seen, indicating a clear disruption in biofilm structural development, as seen in Fig. 2e.

Fig. 2

FESEM images of the biofilm formation of P. aeruginosa

(a) Control group, (b) sAPDI-CDsGEN-NH₂ treatment, (c) sAPDI-CDsIMP-NH₂ treatment, (d) sAPDI-CDsIMP-GEN treatment, (e) sAPDI-CDsGEN-IMP treatment. Scale bar of the a–e = 10 μm

The expression of the biofilm-related genes

RT-qPCR analysis was conducted on P. aeruginosa ATCC 27853, which showed that the sAPDI groups caused a decrease in the expression of genes associated with biofilm formation. Figure 3 illustrates that the sAPDI groups, using sub-MBIC of CDsGEN-NH₂, CDsIMP-NH₂, CDsIMP-GEN, and CDsGEN-IMP, along with sub-lethal doses of UVA light irradiation, resulted in the diminished expression of pelA and pslA genes compared to the control group (P < 0.05). In contrast, sub-MBIC from CDsGEN-NH₂, CDsIMP-NH₂, CDsIMP-GEN, CDsGEN-IMP, and sub-lethal doses of UVA light irradiation alone did not significantly reduce the expression of pelA and pslA genes compared to the control group. (P > 0.05).

Fig. 3

The expression levels of the biofilm-related genes in P. aeruginosa ATCC 27853

a) pelA, and b) pslA genes in treatment with sAPDI-CDsGEN-NH₂, sAPDI-CDsIMP-NH₂, sAPDI-CDsGEN-IMP, and sAPDI-CDsIMP-GEN using sub-MBIC of CDs alongside a sub-lethal dose of UVA light irradiation time, sub- MBIC from CDsGEN-NH₂, CDsIMP-NH₂, CDsIMP-GEN, CDsGEN-IMP, and sub-lethal doses of UVA light irradiation alone. The results are reported as mean ± SEM. * P < 0.05, ** P < 0.01: significant differences according to the control

Discussion

Preventing the formation of biofilms by P. aeruginosa is a critical aspect for combating persistent infections, particularly in healthcare settings. The ability of this opportunistic pathogen to form biofilms plays a significant role in its virulence, facilitating surface attachment and conferring resistance to treatments aimed at controlling its growth (Moradali et al. 2017; Shokri et al. 2018). Biofilms provide protective mechanisms to bacterial colonies, making them highly resistant to antibiotics and immune responses, resulting in persistent infections that are challenging to eliminate (Zafer et al. 2024). Consequently, current advancements in antimicrobial therapies are focused on disrupting biofilm stability and inhibiting their formation. These strategies are crucial for reducing the transmission and persistence of infections (Abdelhamid and Yousef 2023; Mishra et al. 2023). When comparing the MBIC of CDs used in different studies, it can be challenging to make a direct comparison due to variations in CD synthesis, functionalization, and experimental conditions. These factors can significantly influence the concentrations of CDs required to inhibit biofilm growth. For example, a study reported the synthesis of CDs from Citrus medica fruit juice, which demonstrated that CDs reduced P. aeruginosa biofilm production by 56% and 40% at concentrations of 0.1% and 0.07% (v/v), respectively (Selvaraju et al. 2022). This indicates that the source and method of CD preparation can have a substantial impact on their efficacy in inhibiting biofilm growth. PDT is currently approved and widely used for specific infectious skin diseases, showing favorable results (Luo et al. 2024). Moreover, several studies have demonstrated the efficacy of PDT in combating bacterial and biofilm infections (Hu et al. 2018; Songca and Adjei 2022; Akhtar et al. 2024). To our knowledge, no studies have investigated the effect of aPDT using antibiotic-derived CDs on P. aeruginosa biofilms. In this study, aPDT using CDs derived from gentamicin and imipenem as photosensitizers was investigated against P. aeruginosa biofilms. In the research conducted by Prochnow et al., it was observed that PDT using formulations containing MB and ethanol resulted in a significant reduction in the survival of P. aeruginosa biofilms, with an average microbial reduction of approximately 2.58 log₁₀ for MB/10% ethanol and 2.66 log10 for MB/20% ethanol compared to the control group. However, the reduction in biofilm survival following PDT with the toluidine blue (TB) and ethanol formulation did not reach statistical significance (Prochnow et al. 2016). In a study conducted by Perez-Laguna et al. in 2020, the effect of aPDT using methylene blue (MB) in combination with gentamicin and an LED lamp emitting light at a wavelength of 625 nm was investigated on P. aeruginosa ATCC 27853 biofilms. The results demonstrated that when aPDT was used with 64 µg/mL MB and 5 µg/mL gentamicin, there was a significant reduction of 3 Log₁₀ CFU/mL within the P. aeruginosa biofilm. Furthermore, the application of light with 2048 µg/mL MB and 20 µg/mL gentamicin resulted in a significant 6 Log₁₀ CFU/mL reduction within the P. aeruginosa biofilm. Interestingly, the combination of gentamicin and MB-aPDT did not significantly alter the photoinactivating efficacy of MB against Staphylococcus aureus biofilms (Pérez-Laguna et al. 2020). It has also been reported that the combination of MB-aPDT with ciprofloxacin and linezolid enhances the antibiofilm activity against S. aureus biofilms through a synergistic mechanism (Ronqui et al. 2016; Kashef et al. 2017). The research conducted by Orlandi et al. demonstrated that the use of dicationic diaryl porphyrin in photodynamic therapy (PDT) significantly reduced the viability of P. aeruginosa biofilms and prevented their formation. This study highlighted the effectiveness of PDT in combating bacterial growth (Orlandi et al. 2021). Hendiani et al. showed that sAPDI, using a concentration of 0.012 mM methylene blue and a light exposure of 23 J/cm², suppressed the biofilm-forming ability of P. aeruginosa ATCC 27853. The effectiveness of sAPDI was determined using both the triphenyl tetrazolium chloride (TTC) assay and scanning electron microscopy (Hendiani et al. 2019). Donnelly et al. demonstrated that MB-assisted PDT was highly effective in eliminating S. aureus biofilms, achieving a reduction of 88.19% (Donnelly et al. 2009). Pourhajibagher et al. showed a significant reduction in both bacterial count and Porphyromonas gingivalis biofilm formation when using sAPDI with 15.6 µg/mL indocyanine green and a fluence of 15.6 J/cm², compared to untreated bacteria (Pourhajibagher et al. 2017). In a 2022 study, researchers investigated the effectiveness of nano-quercetin (N-QCT)-mediated aPDT against Streptococcus mutans biofilms. The MBIC of N-QCT was found to be 128 µg/mL against S. mutans. Application of aPDT using half of the MBIC of N-QCT, in combination with a blue laser resulted in a significant reduction of 4 log₁₀ CFU/mL (equivalent to a 99.99% reduction) in S. mutans biofilm compared to the control group (P < 0.05) (Pourhajibagher et al. 2022). These findings align with the reduction observed in both biofilm mass and bacterial CFU/mL in the study. However, it is important to note that differences in results across studies could arise from various factors, such as the use of different photosensitizers, variations in bacterial strains, or differences in experimental conditions, including light sources and exposure times. In previous studies, the use of methylene blue-mediated APDI and toluidine blue-mediated APDI, either alone or in combination with linezolid, did not lead to a significant reduction (≥ 3 log₁₀) in the viability of S. aureus within the biofilm (Vilela et al. 2012; Kashef et al. 2017). FESEM images in the study showed that sAPDI inhibited biofilm formation compared to the control group. These results align with findings from other studies and suggest that sAPDI disrupts cell signaling and suppresses quorum sensing in bacterial cells (Ronqui et al. 2016; Hendiani et al. 2019). These observations further support the notion that aPDT can be a valuable alternative therapy with notable anti-biofilm activity (Hendiani et al. 2019; Mahmoudi et al. 2019). The pslA and pelA are involved in the biochemical pathways responsible for the production of Psl and Pel polysaccharides, respectively, which contribute to biofilm formation (Franklin et al. 2011). In the study, sAPDI treatment using sub-MBIC concentrations of CDsGEN-NH₂, CDsIMP-NH₂, CDsIMP-GEN, and CDsGEN-IMP, along with sub-lethal UVA light, resulted in decreased expression of the pslA and pelA genes in P. aeruginosa ATCC 27853. While treatment with sub-MBIC CDs and sub-lethal UVA light alone also led to reduced expression of biofilm-associated genes, the reduction observed was not statistically significant. These findings are consistent with a previous study by Hendiani et al., where reduced expression of pslA and pelA genes was observed following sAPDI treatment using methylene blue at a concentration of 0.012 mM and a light dose of 23 J/cm² in P. aeruginosa ATCC 27853 (Hendiani et al. 2019). In another study, toluidine blue-mediated aPDT demonstrated a significant decrease in the expression of genes associated with biofilm formation in S. aureus (Mahmoudi et al. 2019). However, it is important to note that the present study has limitations, such as the lack of in vivo evidence to confirm the in vitro results. Nonetheless, these findings can provide a valuable foundation for future clinical investigations regarding the effects of aPDT using different antimicrobial agents. The slight variations observed, such as differences in MBIC or sub-lethal doses of light, could be attributed to specific experimental setups, including the choice of photosensitizer and light source. Additionally, the genetic diversity of bacterial strains used across different studies may influence the outcomes of photodynamic therapy. Overall, the existing body of research shows a promising trend towards the use of photodynamic therapy in combating P. aeruginosa biofilms. However, further studies, particularly those utilizing in vivo models, will be crucial for translating these findings into clinical practice. Future research efforts should focus on optimizing photodynamic therapy parameters and exploring the combination of photodynamic therapy with other antimicrobial strategies to enhance efficacy.

The current study demonstrated the effectiveness of sAPDI using sub-MBIC CDsGEN-NH₂, CDsIMP-NH₂, CDsGEN-IMP, and CDsIMP-GEN, in combination with sub-lethal UVA light exposure, in reducing biofilm formation of P. aeruginosa. Furthermore, this treatment approach significantly decreased the expression of genes associated with biofilm formation. However, further studies involving animal models are necessary to validate these findings and assess the potential of this approach in a more complex biological system.