Periodontitis remains one of the most prevalent chronic infectious diseases, causing significant damage to periodontal supporting tissues [1]. Extensive research indicates a strong association between periodontitis and various diseases, including diabetes, rheumatoid arthritis, bacterial pneumonia, cardiovascular disease, and adverse pregnancy outcomes [[2], [3], [4], [5], [6]]. While its etiology is multifactorial, subgingival microorganisms have been identified as pivotal factors in the development and progression of periodontitis [7,8]. Studies have suggested that the early stages of plaque development involve microorganisms from periodontal pockets interacting continuously with host cells, thereby fostering a symbiotic relationship. However, as these microorganisms accumulate and the plaque matures, the microbiota undergoes an ecological shift to a dysregulated state. This shift disrupts the host's internal environmental homeostasis by triggering inflammation and evading immune responses, ultimately leading to periodontitis [9,10].

A significant member of this plaque is Fusobacterium nucleatum, an anaerobic gram-negative bacterium that acts as a bridge between early- and late-colonizing dental plaque [[11], [12], [13]]. Abundant in both healthy and diseased individuals, F. nucleatum has drawn attention due to its association with various diseases, such as head and neck infections, adverse pregnancy outcomes, and colorectal cancer [14,15]. This bacterium reduces oxygen levels, promoting the proliferation of less oxygen-tolerant and more pathogenic microorganisms, such as Porphyromonas gingivalis. Additionally, F. nucleatum provides physical protection to other vulnerable bacteria under adverse environmental conditions [14]. F. nucleatum interacts with pathogenic microorganisms and is believed to play a pivotal role in the maturation of dental plaque. This interaction disrupts the equilibrium within the plaque, contributing to the onset of periodontitis [[15], [16], [17]]. Consequently, controlling F. nucleatum in dental plaque may prove crucial for preventing and managing periodontitis. However, as a commonly used method, antibiotics can lead to the acquisition of resistance mutations and may suppress the oral microbiota, potentially contributing to elevated blood pressure [[18], [19], [20]].

Photodynamic therapy (PDT) has emerged as a highly promising and less invasive method for bactericidal treatment, with a lower likelihood of causing antibiotic resistance in bacteria [[21], [22], [23], [24], [25]]. PDT relies on specific wavelengths of light to excite photosensitizer molecules. The excited photosensitizer can generate reactive oxygen species that have multiple and complex targets for bactericidal action [26]. A previous study reported the effective inactivation of F. nucleatum after exposure to light-emitting diode (LED) irradiation of 50 J/cm2 at 400–410 nm wavelengths [27]. However, administering such a high irradiation dose in clinical settings proves time-consuming for patients, and intense light may cause harm to the surrounding tissues and non-target bacteria. Researchers commonly use exogenous photosensitizers to improve the bactericidal effect of PDT on F. nucleatum, such as methylene blue, Rose Bengal, and Curcuma longa gel [[28], [29], [30]]. However, exogenous photosensitizers have certain limitations. They often have low specificity for recognizing target bacteria and may enter non-targeted, healthy human cells during use, leading to off-target effects on healthy tissues. Additionally, the potential long-term effects of residual photosensitizers on tissues need to be considered.

To address these challenges, researchers have turned their attention to 5-aminolevulinic acid (5-ALA), a naturally occurring metabolite synthesized by various organisms, including plants, animals, bacteria, and fungi. It also acts as a precursor in the synthesis of vitamin B12 and hemoglobin [31]. Normally, intracellular 5-ALA synthesis is tightly regulated, leading to the limited production of porphyrins as byproducts in the 5-ALA metabolic pathway. However, the addition of exogenous 5-ALA bypasses normal metabolic regulation, leading to the accumulation of uroporphyrin (UP), coproporphyrin (CP), or protoporphyrin IX (PPIX) within bacterial cells [32,33]. In tumor cells, a reduction in ferrochelatase enzyme activity, which normally converts PPIX to heme, results in significant intracellular accumulation of PPIX [53]. These porphyrins play photosensitizer in various metabolic and photochemical processes [33,34]. These porphyrins play key roles as photosensitizers in various metabolic and photochemical processes [34,37]. PDT utilizing 5-ALA (ALA-PDT) exploits the intracellular accumulation of porphyrins. In human non-cancerous cells, 5-ALA is rapidly converted to protoporphyrin IX (PPIX) and subsequently to heme. Excess porphyrins, which are not converted, are efficiently expelled from the cell via a PPIX-specific efflux transporter, minimizing the risk of intracellular porphyrin accumulation [38,53].

In recent years, increasing attention has been paid to the bactericidal effects and potential impact of ALA-PDT on bacteria. Studies have shown that ALA-PDT exhibits a potent bactericidal effect on various bacteria, such as Staphylococcus aureus, Pseudomonas aeruginosa, and Candida albicans [[39], [40], [41]]. However, research on the bactericidal effects of ALA-PDT against oral bacteria, including F. nucleatum, remains limited.

Thus, this study aimed to explore whether the addition of 5-ALA could enhance the bactericidal efficiency of PDT against F. nucleatum. We investigated various conditions, such as 5-ALA concentration, culture time, and light intensity, to determine the optimal conditions for the bactericidal effect of ALA-PDT against F. nucleatum. Given the intricate interactions within the oral microbiota, we also examined the microbiota after ALA-PDT treatment using high-throughput sequencing technology to verify the impact of the treatment. Furthermore, we delved into the bactericidal mechanism underlying ALA-PDT by analyzing F. nucleatum after cultivation with 5-ALA through fluorescence spectroscopy and high-performance liquid chromatography (HPLC).