Impact of Diode Laser, Ozonated Water, and Chlorhexidine on
Oral Microbiota: Insights from Proteomic Analysis.
Ziba Najmi1, Emanuele Ruga2, Andrea Melle3, Rachele Pertusati3, Anna Maria Agnone4, Andrea Cochis1, Alessandro Calogero Scalia1*
1. Department of Health Sciences, Center for Translational Research on Autoimmune and Allergic Diseases CAAD, Università del Piemonte Orientale UPO, Italy.
2. DDS; MSc Oral Surgery, Aesthetic Medicine; Specialist in Oral Surgery, SCDU ODONTOSTOMATO-LO- GIA Osp. S. Andrea, Vercelli. ASLVC., Università del Piemonte Orientale UPO, Italy https://orcid.org/0000-0002-6401-9689
3. RDH; MSc; SCDU ODONTOSTOMATOLOGIA Osp. S. Andrea, Vercelli. ASLVC., Università del Piemonte Orientale UPO, Italy.
4. DDS; Specialist in Oral Surgery; Contract Professor University of Eastern Piedmont. Division of Dentistry, Sant'Andrea Hospital Vercelli, Corso Mario Abbiate, 21, Vercelli 13100, Italy.
*Correspondence to: Alessandro Calogero Scalia, Department of Health Sciences, Center for Translational
Research on Autoimmune and Allergic Diseases CAAD, Università del Piemonte Orientale UPO, Italy.
Copyright.
© 2025 Alessandro Calogero Scalia, This is an open access article distributed under the Creative Commons
Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided
the original work is properly cited.
Received: 25 September 2025
Published: 01 November 2025
ABSTRACT
Objectives: The human oral microbiota is composed of fungi, viruses, protozoa, and more than 700 bacterial species living in eubiosis. Poor oral hygiene, medications, and un-healthy lifestyle may lead to dysbiosis with local (e.g., periodontitis) and systemic diseases(e.g., rheumatoid arthritis, and Alzheimer's). This study evaluates the effectiveness of three professional treatments, diode laser, ozonated water, and 0.20% chlorhexidine, on human cariogenic bacteria present in the oral microbiota extracted from plaque samples, focusing on their ability to target pathogens selectively.
Materials and Methods: Oral plaque samples were collected from three healthy volun-teers, pooled and incubated anaerobically for 24 hours. The dentin discs, used as substrate, were inoculated with oral plaque and incubated for 24 hours. Samples were then treated with 20 seconds of diode laser, 30 seconds of ozonated water, or 30 seconds of 0.20% chlorhexidine. Peptides from bacterial proteins were analysed by proteomics, and populations were assessed using the Human Oral Microbiome Database.
Results: The bacterial populations isolated from dentin samples after treatments were first compared to those from untreated oral plaque, and then compared to each other to assess shifts in microbial composition due to the specific treatment. In the first comparison, all three treatments led to a marked reduction in several bacterial species, including Fusobac- terium nucleatum and Prevotella oris, commonly associated with oral infections. In the com- parison between treatments, laser and ozonated water appeared to limit the overgrowth of Granulicatella adiacens, which overgrowth may contribute to systemic issues.
Conclusions: These findings suggest that while all treatments exert an antimicrobial effect, they differ in their selective pressure on specific bacterial taxa, ultimately influencing the overall microbial composition.
Keywords: Oral microbiota, diode laser, ozonized water, chlorhexidine, bovine dentin.
Introduction
The human oral microbiota is a complex ecological community composed of commensal, symbiotic, and pathogenic microorganisms inhabiting the oral cavity. Among these, more than 700 distinct bacterial species have been identified, coexisting in a typically balanced ecosystem called eubiosis. In addition to bacteria, the oral microbiota includes archaea, fungi, mycoplasmas, protozoa, and a transient viral population. The composition of this microbial community is highly dynamic and evolves in response to changes in the biological environment of the oral cavity [1–5]. The oral microbiota comprises a diverse array of Gram-positive and Gram-negative bacterial species, including both facultative and obligate anaerobes. These bacteria are classified into 13 phyla, which include Firmicutes, Bacteroidetes, Actinobacteria, Proteobacteria, Spirochaetes, Fusobacteria, Synergistes, SR1, TM7, Chloroflexi, Deinococcus, Acidobacteria, and Cyanobacteria [4–6].
The oral cavity comprises multiple distinct niches that support the growth of diverse microbial communities. These anatomical habitats include the teeth, the gingival sulcus, the tongue, cheeks, saliva, hard and soft palate, and tonsils [7]. Microbial colonization within these niches is modulated by several physicochemical factors, including oxygen availability, pH, temperature, redox potential, ionic strength, and osmotic pressure [8-10].
Moreover, exogenous factors such as the administration of antibiotics (e.g., tetracyclines) and the use of systemic or topical antiseptics can significantly disrupt the composition and balance of the resident oral microbiota [11]. The oral microbiota predominantly exists in the form of biofilms, which are essential for maintaining oral homeostasis, providing protection against external insults, and limiting the development of pathogenic conditions. Biofilms are structured microbial communities embedded within a self-produced extracellular polysaccharide matrix, which facilitates firm adhesion to both biotic and abiotic surfaces in the oral cavity. Within this complex architecture, microorganisms interact through chemical signaling, metabolic cooperation, physical associations, and molecular communication, promoting the stability and resilience of the community. Mature oral biofilms typically consist of approximately one-third microbial cells and two-thirds extracellular components, including bacterial metabolic byproducts, host-derived exudates, entrapped food debris, and water [12–17].
Under conditions of proper oral hygiene and a balanced diet, the oral biofilm remains in a state of symbiotic equilibrium, contributing to the maintenance of oral health. However, poor oral hygiene practices and frequent intake of fermentable carbohydrates can disrupt this balance, leading to a shift in the microbial composition, a phenomenon known as dysbiosis [18]. This ecological shift favors the proliferation of acidogenic and aciduric pathogenic species at the expense of commensal microorganisms, promoting the development of oral diseases such as dental caries and periodontal disease [19]. The persistence of a dysbiotic biofilm fosters a pro-inflammatory environment and enhances tissue degradation, ultimately compromising both hard and soft oral tissues [20]. Dental caries is one of the most prevalent oral diseases worldwide and represents a leading cause of tooth pain and tooth loss in humans [15]. Beyond the progressive destruction of dental hard tissues, caries can result in pulpal and periapical infections, potentially leading to systemic complications. The condition exhibits a high incidence and affects individuals across all age groups, from early childhood through advanced age [16,21]. Streptococcus 84 mutans is widely recognized as a key etiological agent in the initiation of dental caries. As 85 the lesion advances, the microbial composition of the biofilm shifts, with a progressive 86 transition from early colonizers such as S. mutans and Actinomyces spp. to aciduric and ac- 87 idogenic genera like Lactobacillus and Bifidobacterium [3,16,22-24].
Periodontitis is a chronic, multifactorial inflammatory disease that affects the supporting structures of the teeth and represents the primary cause of tooth loss globally. In contrast to gingivitis, which is confined to reversible inflammation of the gingival tissues, periodontitis is characterized by the progressive destruction of the gingiva, periodontal ligament, and alveolar bone. The disease is associated with a dysbiotic shift in the subgingival microbiota, favoring the proliferation of pathogenic anaerobic species. Key bacterial taxa implicated in periodontitis include Porphyromonas gingivalis, Treponema denticola, Tannerella forsythia, Porphyromonas endodontalis, Prevotella denticola, and Dialister spp. Among the modifiable risk factors, tobacco smoking plays a major role in disease onset and progression, exerting both direct immunosuppressive effects and altering the subgingival microbial composition in favor of pathogenic species [3,25].
The oral microbiota has been increasingly linked to the pathogenesis of several systemic diseases, including cardiovascular disease, pneumonia, rheumatoid arthritis, oral squamous cell carcinoma (OSCC), pancreatic cancer, colorectal cancer, esophageal cancer, stroke, and adverse pregnancy outcomes [14,26]. Given its widespread influence on host physiology and its compositional shifts in disease states, the oral microbiota has been proposed as a potential biomarker for various human pathologies. Oral squamous cell carcinoma (OSCC) is among the most prevalent malignant neoplasms of the oral cavity. Emerging evidence suggests that the biofilm associated with OSCC lesions harbors an increased load of both aerobic and anaerobic bacteria. Notably, higher abundances of Veillonella, Fusobacterium, Prevotella, Porphyromonas, Actinomyces, Clostridium, Haemophilus, Enterobacteriaceae, and various Streptococcus species have been observed in OSCC related biofilms. Furthermore, salivary profiles of patients with oral can- 111 cer often show elevated levels of Prevotella melaninogenica and Streptococcus mitis, suggesting their potential as microbial indicators of malignancy [3,27].
Chlorhexidine is considered the gold standard disinfectant for antimicrobial rinses due to its broad-spectrum activity and prolonged substantivity [27]. It is highly effective in controlling bacterial proliferation and preventing dental caries. However, its effect on already established carious lesions is limited. Ozonated water and diode lasers present several advantages, and studies have demonstrated their efficacy not only as preventive agents but also as therapeutic tools [28-30]. In particular, they are recommended as supplementary antibacterial surface pretreatment methods to enhance the removal of cariogenic bacteria.
Diode lasers, in particular, offer multiple benefits in both periodontal and surgical applications. They are increasingly employed in periodontal therapy as adjuncts to conventional treatments such as scaling and root planing (SRP) [30]. Diode lasers have shown antibacterial effects against a variety of oral pathogens and are commonly used before and after cavity preparation to reduce postoperative sensitivity and microbial load.
The 915 nm diode laser wavelength is especially noted for its beneficial properties, including enhanced bacterial reduction, decreased inflammation, and accelerated healing. These effects are partly attributed to its biostimulatory impact on the viability 130 and regenerative capacity of human gingival fibroblast cells [31].
This research project aimed to evaluate the effectiveness of three professional treatments diode laser, ozonated water, and 0.20% chlorhexidine each with distinct mechanisms of action, on cultures of human cariogenic bacteria on dentin slices by assessing their impact on bacterial populations collected from the oral plaque of healthy donors. Furthermore, using a high-throughput analytical approach, the study identified which pathogenic and commensal bacterial species were most affected by each treatment, thereby highlighting their potential targeted effects.
Results
Population analysis
The results of the comparison between the original bacterial populations identified in the oral plaque exploited for the infection of the dentin slices and those present on the surface after infection’s eradication with the diode laser, ozonated water (O? water), or chlorhexidine (CHX) are presented in the Figure 1. Only bacterial populations with a relative abundance greater than 1% were considered. A total of 19 bacterial populations were included in the comparison between the original plaque and the treated samples, with the results shown in Figure 1. Similarly, 8 populations were selected for the comparison among the three treatments, as reported in Figure 2. The complete list of identified bacterial species is provided in Supplementary Table 1 (ST1).
In general, according to the results shown in Figure 1, all three treatments substantially reduced the abundance of several bacterial strains, including Fusobacterium nucleatum and Prevotella oris, both of which are commonly associated with endodontic and periodontal infections [32]. Conversely, an increase was observed in other taxa such as Granulicatellav adiacens and Veillonella atypica.
Then, the proteomic analysis was focused on the impact of each treatment in terms of targeted bacteria. Figure 2a shows the relative abundance, in terms of percentage of total population, of bacterial phyla identified in plaque samples treated with Laser (left panel), O? water (middle panel), and CHX (right panel). Firmicutes