In the context of an increasingly escalating antibiotics crisis, phototherapy has emerged as a promising therapeutic approach due to its inherent advantages, including high selectivity, noninvasiveness, and low drug resistance. Photothermal therapy (PTT) and photodynamic therapy (PDT) are two complementary and promising phototherapies albeit with inherent limitations, noted as the challenges in achieving precise heat confinement and the associated risk of off-target damage for PTT, while the constraints due to the hypoxic microenvironment are prevalent in biofilms faced by PDT. Herein, we have designed a supramolecular nanoformulation that leverages the complexation-induced quenching of guanidinium-modified calix[5]arene grafted with fluorocarbon chains (GC5AF5), the efficient recognition of adenosine triphosphate (ATP), and the oxygen-carrying capacity of the fluorocarbon chain. This intelligent nanoformulation enables the adaptive enhancement of both photothermal therapy (PTT) and photodynamic therapy (PDT), allowing for on-demand switching between the two modalities. Our nanoformulation utilizes ATP released by dead bacteria to accelerate the elimination of biofilms, rendering bacteria unable to resist while minimizing harm to healthy tissues. This research highlights the particular recognition and assembly capabilities of macrocycles, offering a promising strategy for creating potent, combined antibiofilm therapies.

Dental caries is a common disease caused by plaque biofilms, which are important pathogenic factors in many diseases. When hosts are overexposed to dietary sugars, pathogens such as Streptococcus mutans (S. mutans) and other cariogenic bacteria, metabolically assemble an extracellular matrix rich in exopolysaccharides to form a disease‐causing biofilm, in which the microenvironment is characterized by regional hypoxia, low pH, and nutritional deprivation. Current antimicrobials with inadequate penetration and a lack of pathogens targeting the biofilm do not degrade the protective matrix within the biofilm. In this study, a guanidine and galactose decorated nanophotosensitizer with oxygen self‐sufficient capability, p(GF/GEF)‐I, is developed to enhance the permeability of biofilms by positively charging the particle surface and easily binding to the bacteria within the membrane through electrostatic interactions. 90% of the biofilm on enamel surface is eliminated after treatment with p(GF/GEF)‐I under laser irradiation. Notably, the nanophotosensitizer inhibits the recolonization of dental biofilms by S. mutans , preventing secondary infections. Furthermore, dental caries in a rodent model are reduced with exposure to nanophotosensitizer. p(GF/GEF)‐I is a significantly higher efficacy without damaging the surrounding soft tissue. With further development and optimization, p(GF/GEF)‐I shows significant potential as a phototherapeutic agent for the treatment of biofilm‐induced diseases.

The efficacious delivery of antimicrobial drugs to intractable oral biofilms remains a challenge due to inadequate biofilm penetration and lack of pathogen targeting. Herein, we have developed a microenvironment-activated poly(ethylene glycol) (PEG)-sheddable nanoplatform to mediate targeted delivery of drugs into oral biofilms for the efficient prevention of dental caries. The PEGylated nanoplatform with enhanced biofilm penetration is capable of deshielding the PEG layer under slightly acidic conditions in a PEG chain length-dependent manner to re-expose the bacteria-targeting ligands, thereby facilitating targeted codelivery of ciprofloxacin (CIP) and IR780 to the bacteria after accumulation within biofilms. The nanoplatform tends to induce bacterial agglomeration and suffers from degradation in the acidic oral biofilm microenvironment, triggering rapid drug release on demand around bacterial cells. The self-modulating nanoplatform under near-infrared (NIR) irradiation accordingly displays greatly augmented potency in oral biofilm penetration and disruption compared with drugs alone. Topical oral treatment with nanoplatforms involving synergetic pharmacological and photothermal/photodynamic trinary therapy results in robust biofilm dispersion and efficacious suppression of severe tooth decay in rats. This versatile nanoplatform can promote local accumulation and specific drug transport into biofilms and represents a new paradigm for targeted drug delivery for the management of oral biofilm-associated infections.

Objective: The aim of this study was to observe the effect of V. parvula on the physiological activity of S. mutans and elucidate the role of V. parvula on dental caries. Design: We constructed dual-species biofilms formed by V. parvula and S. mutans, and measured the pH dynamics, biofilm growth, Extracellular Polysaccharide (EPS) synthesis, and expression of S. mutans EPS synthesis-associated genes affected by V. parvula. Results: pH dynamics were not altered when V. parvula and S. mutans were co-cultured during a 120 -h test period. However, S. mutans cell number and EPS synthesis in dual-species biofilms were found to be significantly higher than in single-species biofilms. Moreover, expression levels of genes encoding glucosyltransferases (gtfs), gtfB and gtfC specifically, were up-regulated when S. mutans was co-cultured with V. parvula. Conclusions: Our findings indicate that V. parvula is not, as previously thought, protective and associated with caries health. On the contrary, V. parvula might participate in caries development through interactions with S. mutans. This study suggests that V. parvula may have an impact on the pathogenesis of dental caries.

The COX-2/PGE2 axis can play roles in mediating the progression of tumor. COX-2 induction was observed in oral cancer. In our previous study, we found Staphylococcus aureus, a pathogen prevalent in oral cancer, can activate the COX-2/PGE2 pathway in human oral keratinocyte (HOK) cells. Here, we investigated the proliferation of HOK cells affected by COX-2 induction and the role of COX-2 induction in the malignant transformation of HOK cells. We found S. aureus was able to facilitate HOK cell proliferation through upregulating COX-2 expression. With the induction of COX-2, expression of oral cancer-associated genes cyclin D1 was upregulated and p16 was downregulated. Transcriptome analysis showed that the “NF−kappa B signaling pathway” and “TNF signaling pathway” had the highest enrichment of differentially expressed genes (DEGs) with COX-2 over-expression. Seven upregulated genes (jun, tlr4, cxcl1, lif, cxcl3, tnfrsf1β, and il1β) in these two pathways were critical for the increased proliferation of HOK cells and might be associated with COX-2. Malignant transformation of cells was evaluated by soft agar colony formation assay and S. aureus infection promoted HOK cell colony formation. These results suggest the potential of S. aureus to induce the infection-associated malignant transformation of oral epitheliums through COX-2 activation.

Objective This study used finite element analysis (FEA) to assess the von Mises stresses of a mandibular first premolar after removing a separated instrument with an ultrasonic technique. Methods FEA models of the original and treated mandibular first premolar were reconstructed, and three models (the original canal, size 30/taper 0.04 canal, and separated instrument removal canal) were created. Two-direction (vertical and lateral) loading patterns were simulated with a 175-N force. The maximum von Mises stresses of the models within the roots from the apex to the cervical region were collected and summarized. Results Under vertical and lateral loads, all maximal values in the three models were localized in the straight-line access region. Compared with the original model (model 1), the treated models (models 2 and 3) had greater maximum stress values from the apex to the cervical region. Greater differences in the maximum von Mises stresses between models 2 and 3 were present in the straight-line access region. Conclusions Separated instrument removal caused changes in stress distribution and increases in stress concentration in the straight-line access region of roots.

The luxS gene is present in a wide range of bacteria and is involved in many cellular processes. LuxS mutation can cause autoinducer(AI)-2 deficiency and methyl metabolism disorder. The objective of this study was to demonstrate that, in addition to AI-2-mediated quorum sensing (QS), methyl metabolism plays an important role in LuxS regulation in Streptococcus mutans. The sahH gene from Pseudomonas aeruginosa was amplified and introduced into the S. mutans luxS-null strain to complement the methyl metabolism disruption in a defective QS phenotype. The intracellular activated methyl cycle (AMC) metabolites [S-adenosylmethionine (SAM), S-adenosylhomocysteine (SAH), homocysteine (HCY), and methionine] were quantified in wild-type S. mutans and its three derivatives to determine the metabolic effects of disrupting the AMC. Biofilm mass and structure, acid tolerance, acid production, exopolysaccharide synthesis of multispecies biofilms and the transcriptional level of related genes were determined. The results indicated that SAH and SAM were relatively higher in S. mutans luxS-null strain and S. mutans luxS null strain with plasmid pIB169 when cultured overnight, and HCY was significantly higher in S. mutans UA159. Consistent with the transcriptional profile, luxS deletion-mediated impairment of biofilm formation and acid tolerance was restored to wild-type levels using transgenic SahH. These results also suggest that methionine methyl metabolism contributes to LuxS regulation in S. mutans to a significant degree.

Caries is one of the most prevalent and costly infectious diseases affecting humans of all ages. It is initiated by cariogenic supragingival dental plaques forming on saliva-coated tooth surfaces, yet the etiology remains elusive. To determine which microbial populations may predispose a patient to caries, we report here an in-depth and comprehensive view of the microbial community associated with supragingival dental plaque collected from the healthy teeth of caries patients and healthy adults. We found that microbial communities from caries patients had a higher evenness and inter-individual variations but simpler ecological networks compared to healthy controls despite the overall taxonomic structure being similar. Genera including Selenomonas, Treponema, Atopobium, and Bergeriella were distributed differently between the caries and healthy groups with disturbed co-occurrence patterns. In addition, caries and healthy subjects carried different Treponema, Atopobium, and Prevotella species. Moreover, distinct populations of 13 function genes involved in organic acid synthesis, glycan biosynthesis, complex carbohydrate degradation, amino acid synthesis and metabolism, purine and pyrimidine metabolism, isoprenoid biosynthesis, lipid metabolism, and co-factor biosynthesis were present in each of the healthy and caries groups. Our results suggested that the fundamental differences in dental plaque ecology partially explained the patients' susceptibility to caries, and could be used for caries risk prediction in the future.

Staphylococcus aureus is a major pathogen of varieties of oral mucous infection. Prostaglandin E2 (PGE(2)) is a pro-inflammatory factor and Cyclooxygenase 2 (COX-2) is a critical enzyme of PGE(2) biosynthesis. The purpose of this study is to investigate whether Staphylococcus aureus can increase PGE(2) production of oral epithelial cells and how PGE(2) functions in the growth and adherence of Staphylococcus aureus. mRNA levels of COX-2, fnbpA and fnbpB were estimated by quantitative PCR. PGE(2) production was measured by Enzyme Linked Immunosorbent Assay (ELISA). The binding biomass of Staphylococcus aureus to human fibronectin was investigated by crystal violet staining and confocal laser scanning microscopy and the adherent force was measured by atomic force microscope (AFM). The COX-2 mRNA level and PGE(2) production were increased by Staphylococcus aureus. PGE(2) promoted the growth and biofilm formation of Staphylococcus aureus, enhanced the attachment of Staphylococcus aureus to the human fibronectin as well as to the HOK cells. The transcription of fnbpB was up-regulated by PGE(2) in both early and middle exponential phase but not fnbpA. These results suggest that the activation of COX-2/PGE(2) pathway in oral epithelial cell by Staphylococcus aureus can in turn facilitate the growth and the ability to adhere of the pathogen. These findings uncover a new function of PGE(2) and may lead to the potential of COX-2/PGE(2) targeting in the therapy of inflammation and cancer in both which the COX-2/PGE(2) pathway were observed activated.

The genus Veillonella is a member of the normal oral flora and is highly abundant in the human oral cavity.Veillonella utilizes lactate,which ameliorates the caries process.However,results of in vitro studies and rat model systems are ambiguous.With the development of human oral microbiome research,several studies reported that the frequency of Veillonella in caries-activated individuals is higher than that in caries-free ones,and the distribution of Veillonella is highly related to some cariogenic Streptococcus species.Veillonella facilitates the action of some cariogenic Streptococcus species,such as Streptococcus mutans in caries development.These findings create new focus on the genus Veillonella as a factor relating to caries.This paper provides a review on the distribution of Veillonella in human oral cavity,the relationship between Veillonella and caries,and the relationship between Veillonella and Streptococcus species related to caries.

OBJECTIVETo evaluate the effect of quorum sensing luxS gene on biofilm formation through construction of a luxS overexpression strain by Streptococcus mutans (Sm).METHODSIn order to construct pIB-luxS plasmid, the luxS gene fragment amplified by PCR was inserted into the shuttle plasmid pIB169 by corresponding double digests. The pIB-luxS plasmid was linearized electro-transformed into Sm cell and the overexpression strain was selected on chloramphenicol plate and testified by electrophoresis and western blot. The growth rate of both Sm wild type strain and its luxS overexpression strain were observed. Methyl thiazolyl tetrazolium (MTT) assay method was used to compare the biofilm formation quantification by both strains at different time points and containing different sucrose. The structures of the biofilms were observed by using confocal laser scanning microscopy, and biofilm-related gene expressions were investigated by real-time PCR. All experiments were performed in triplicate.RESULTSThe luxS overexpression strain was successfully constructed and confirmed by electrophoresis and Western blotting. The planktonic growth mode of the wild-type and luxS overexpression strain showed no difference, but biofilm formed by Sm overexpression strain was 0.400 ± 0.009 and 0.609 ± 0.041 at 14 and 24 h, higher than the wild type strain biofilm at the same time point (0.352 ± 0.028 and 0.533 ± 0.014, respectively, P < 0.05). After adding 0.125% sucrose, biofilm formed by Sm overexpression strain raised to 1.041 ± 0.038, higher than that by the wild type strain (0.831 ± 0.020, P < 0.05). The biofilm formed by both strains were also increased with the sucrose concentration increase, but there was no difference between them. The overexpression strain aggregated into distinct clusters on structure, genes expression including gtfB, ftf, gbpB, relA, brpA, smu630, comDE, vicR were increased (6.10 ± 0.12, 3.34 ± 0.07, 8.75 ± 0.13, 2.96 ± 0.04, 5.20 ± 0.19, 2.20 ± 0.06, 2.32 ± 0.07 and 10.67 ± 0.57 fold) compared to the wild-type strain (P < 0.05).CONCLUSIONSQuorum sensing luxS gene can promote the biofilm formation of Sm.

PUPPOSE To construct quorum sensing luxS knockout mutants of Enterococcus faecalis through homologous recombination. METHODS The upstream and downstream flank DNA fragments of E. faecalis luxS gene (up, dn) and erythromycin resistance gene (erm) were amplified by PCR. In order to construct recombination plasmid Puemrd, these DNA fragments were inserted into the plasmid pUC18 by corresponding double digests. After allelic exchange, the luxS knockout mutants strains were selected on 30 μg/mL erythromycin plates. RESULTS With endonuclease reaction and DNA sequencing, it was proved that the objective plasmid, Puemrd, was constructed correctly. The luxS knockout mutants strains were confirmed by PCR. CONCLUSIONS Enterococcus faecalis luxS gene has been successfully disrupted with homologous recombination. This mutant strain sets a good foundation for further functional study.

PURPOSE:To complement the activated methyl cycle (AMC) pathway at an AI-2 defect background in Streptococcus mutans (S. mutans) luxS null strain.METHODS:A sahH gene was amplified from Pseudomonas aeruginosa and introduced into the S. mutans luxS null strain to complement the methyl-metabolic disruption at an AI-2 defect background. Western blot, reverse-transcription PCR and AI-2 bioassay were performed to confirm the heterogenous expression of SahH in S. mutans luxS null strain. The data was statistically analyzed by SAS8.0 software package.RESULTS:LuxS and SahH were detected to express in Escherichia coli BL21 as well as their mRNA were confirmed to be successfully transcribed in S. mutans luxS null strain. AI-2 production was found in wide type S. mutans and its luxS-introduced luxS null strain but not found in the luxS null strain and its sahH and empty plasmid-introduced strains.CONCLUSIONS:A new S. mutans derivative with the AMC pathway complements while the AI-2 defect is constructed.

OBJECTIVETo investigate the predominant contribution of methyl-metabolism pathway to the regulation of LuxS of Strecptococcus mutans.METHODSThe differences in biofilm formation and aciduricity of Strecptococcus mutans among the methyl-metabolism-complementation strain (KO-S), the parental wide-type strain (WT) and the luxS null strain (KO) were observed by real-time PCR for monitoring the transcriptional level of genes related to biofilm formation (smu.238, gtfD) and aciduricity (smu.44, smu.46) of the studied strains, methyl thiazolyl tetrazolium (MTT) for quantifying the biofilm of the exhibited strains and confocal laser scanning microscopy for estimating the structure of the biofilm.RESULTSThe transcriptional level of smu.44, smu.46, smu.238, gtfD in WT were 1.289 ± 0.051, 1.694 ± 0.140, 1.565 ± 0.107, 1.667 ± 0.196 respectively; in KO were 1.001 ± 0.045, 1.007 ± 0.151, 1.000 ± 0.021, 1.012 ± 0.196 respectively, downregulated compared with WT (P < 0.05); in KO-S were 4.662 ± 0.091, 5.019 ± 0.258, 3.462±0.029, 3.071 ± 0.136 respectively, upregulated compared both with KO and with WT (P < 0.05). The quantity of biofilms formed by the studied strains were WT (1.592 ± 0.213), KO (0.939 ± 0.029), KO- S (2.177 ± 0.226), KO- P (1.020 ± 0.093), respectively, representing a less quantity by KO and KO-P than WT (P < 0.05) and a more quantity by KO-S than other three stains (P < 0.05). According to the observation of biofilms texture by confocal laser scanning microscopy, the WT biofilm was condensed and even. In contrast, fissures and gaps were found scattered in biofilms of KO, KO-P while lessened in that of KO-S, in which high-density bacterial aggregates were observed. The acid assay indicated a smaller biofilm decrease by WT and KO-S than that by KO and KO- P(P < 0.05).CONCLUSIONSThe methyl- metabolism pathway contributes to LuxS regulation on biofilm formation and auiduricity of Strecptococcus mutans.

Objective:To research the effect of quorum sensing inhibitor furanone C-30 on initial biofilm formation of Streptococcus mutans.Methods:The in vitro synthetic quorum sensing inhibitor furanone C-30 was prepared with BHI medium in which Streptococcus mutans was cultivated into different final concentration of 10μmol/L,100μmol/L.Biofilms were formed after being incubaed at 37℃ for 24 h,followed which the kinetic quantification of biofilm formation was detected by BioFilm Ring Test.Results:The furanone C-30 group with a concentration of 100μmol/L was shown to inhibit the biofilm formation by Streptococcus mutans significantly(P0.05).The time of magnetic beads immobilized and the value of BFI were both different between the 100μmol/L furanone C-30 group and the control groups.Conclusions:Quorum sensing inhibitor furanone C-30 in the concentration of 100μmol/L can inhibit the biofilm formation of Streptococcus mutans.It may direct a new way for the prevention and therapy of caries.