Methods and compositions for treating biofilm-associated diseases

Abstract

A method for inhibiting the formation of a bacterial-based biofilm in a subject, the method comprising administering to the subject an effective amount of a pharmaceutical composition comprising one or more Lactobacillus species selected from the group consisting of L. rhamnosus ATCC2836, L. plantarum ATCC8014, L. plantarum ATCC14917, and L. salivarius ATCC11741, wherein the bacterial-based biofilm comprises Streptococcus mutans and / or Candida albicans.

Description

[Technical field]

[0001] This application claims priority to U.S. Provisional Patent Application No. 63 / 268,420, filed February 23, 2022, which is incorporated by reference herein.

[0002] This invention was made with Government support under No. K23DE027412 awarded by the NIDCR and No. NSF-CCF-1934962 awarded by the National Science Foundation. The Government has certain rights in this invention.

[0003] The present invention relates generally to the field of treatment of biofilm-associated diseases. [Background technology]

[0004] Dental caries, an ecological dysbiosis of the oral microflora, is initiated by a pathogenic biofilm formed on the tooth surface, where cariogenic bacteria and fungi metabolize dietary carbohydrates and produce acid, leading to an irreversible outcome, namely demineralization of the tooth enamel. Streptococcus mutans is a well-known cariogenic pathogen due to its acidogenicity, acid resistance, and ability to synthesize dental plaque extracellular matrix. Furthermore, studies have also revealed the cariogenic role of oral Candida, which is acidogenic, acid resistant, and can dissolve hydroxyapatite in rat models, leading to more severe dental caries when infected together with Streptococcus mutans. Children with oral Candida albicans showed a >5-fold higher likelihood of experiencing early childhood caries (ECC) than children without this yeast strain. The presence of C. albicans in the oral cavity of preschool children was associated with oral bacterial dysbiosis with a higher abundance of taxa that had higher virulence and were more conducive to ECC. Furthermore, the occurrence of S. mutans by 1 year of age was 3.5 times higher in infants with early colonization of oral Candida than in infants without oral Candida. Therefore, simultaneous regulation of S. mutans and C. albicans in the oral cavity sheds light on caries prevention. Summary of the Invention [Means for solving the problem]

[0005] One aspect of the present invention relates to a method of disestablishing or inhibiting the formation of a bacterial-based biofilm in a subject, comprising administering to the subject an effective amount of (1) one or more Lactobacillius species and / or (2) plantaricin, wherein the one or more Lactobacillius species are selected from the group consisting of L. rhamnosus ATCC2836, L. plantarum ATCC8014, L. plantarum ATCC14917, and L. salivarius ATCC11741.

[0006] Another aspect of the present invention relates to a method of preventing or treating a biofilm-associated disease in a subject, comprising administering to the subject an effective amount of (1) one or more Lactobacillus species and / or (2) plantaricin, wherein the one or more Lactobacillus species are selected from the group consisting of L. rhamnosus ATCC2836, L. plantarum ATCC8014, L. plantarum ATCC14917, and L. salivarius ATCC11741.

[0007] Another aspect of the present invention relates to a method for preventing the occurrence of early childhood caries (ECC) in a neonatal subject, comprising administering to the mother of the neonatal subject during pregnancy an effective amount of a pharmaceutical composition comprising one or more Lactobacillus species selected from the group consisting of L. rhamnosus ATCC2836, L. plantarum ATCC8014, L. plantarum ATCC14917, and L. salivarius ATCC11741.

[0008] Another aspect of the present invention relates to a pharmaceutical composition comprising one or more Lactobacillus species selected from the group consisting of L. rhamnosus ATCC2836, L. plantarum ATCC8014, L. plantarum ATCC14917, and L. salivarius ATCC11741, and a pharma- ceutically acceptable carrier, formulated as a mouthwash, dental gel, or dental coating. [Brief description of the drawings]

[0009] [Figure 1]Figure 1 shows plots of growth curves for Candida albicans, Streptococcus mutans, and Lactobacillus spp. in multi-species planktonic and biofilm conditions. The control group consisted of Candida albicans and Streptococcus mutans. Groups with added Lactobacillus spp. are marked "with Lactobacillus spp." (Panel A) Lactobacillus spp. significantly inhibited the growth of Candida albicans by 1 log after 6 hours incubation and by 1-2 logs after 20 hours incubation. (Panel B) Lactobacillus spp. significantly inhibited the growth of Streptococcus mutans at 6 hours and 20 hours. Streptococcus mutans was inhibited to undetectable levels (<20 CFU / ml) after 20 hours incubation with L. plantarum 8014 and L. salivarius 11741. (Panel C) Lactobacillus spp. maintained stable growth in all groups. (Panels D-L) Growth curves of Candida albicans, S. mutans, and Lactobacillus spp. were plotted in multi-species biofilm conditions. (Panels D-F) Lactobacillus spp. (L. plantarum and L. salivarius) inhibited the growth of Candida albicans under high sucrose conditions (1% w / v) with a 3 log reduction by 72 hours compared to the control. No difference in the growth of Candida albicans was detected with the addition of L. rhamnosus under all sugar conditions. (Panels G-I) Lactobacillus spp. (L. plantarum and L. salivarius) inhibited the growth of S. mutans under high sugar conditions (1% w / v sucrose and 1% w / v glucose). Significantly, L. plantarum 8014 and 14917 inhibited mutans streptococci in biofilms to undetectable levels (<20 CFU / ml) as early as 48 hours, and treated biofilms maintained undetectable mutans streptococci (<20 CFU / ml) at 72 hours. L. rhamnosus performed poorly in inhibiting the growth of mutans streptococci under all sugar conditions. (Panels J-L) Lactobacillus maintained stable growth in all groups. * indicates that CFU values ​​in multi-species biofilms were significantly lower than the control group at all follow-up time points (p<0.05).# indicates that the CFU values ​​of the multi-species biofilm were significantly lower than the control group at the particular indicated time point (p<0.05). [Diagram 2] Quantitative measurement of microcolonies in 72-h multi-species biofilms (1% w / v sucrose). Control (Candida albicans and S. mutans) and experimental (with L. plantarum 14917) biofilms under 1% w / v sucrose were visualized by two-photon laser confocal microscopy. The three-dimensional structure of the biofilms was depicted using Amira software. L. plantarum 14917 dramatically reduced biofilm formation compared to the control. Biofilm dry mass was significantly reduced by the addition of L. plantarum 14917 (Panel A). *p<0.05. Biomasses of the two biofilm components, bacteria and exopolysaccharides (EPS), were calculated using the image processing software COMSTAT (Heydorn et al., 2000). L. plantarum 14917 significantly reduced the biomass of bacteria and EPS (Panel B). Confocal images show cross-sectional and sagittal views of microcolonies formed in the control (Streptococcus mutans and Candida albicans) and with the addition of L. plantarum 14917. Well-formed mushroom-like microcolonies were found in the control group, and the largest size microcolonies were found in the biofilms of Streptococcus mutans and Candida albicans. Microcolonies formed with the addition of L. plantarum 14917 were much less structured. Bacterial components were less encapsulated with EPS. The amount of colocalization between bacteria and EPS was calculated using DUOSTAT (Panel E) and was consistent with the findings revealed by the images (*p<0.05). Surface-attached and free-floating microcolonies were assessed using COMSTAT and DUOSTAT software. Panel F illustrates that biofilms treated with L. plantarum 14917 had significantly reduced microcolony size (p>0.05, all pairwise comparisons using ANOVA, Tukey-Kramer HSD). [Diagram 3] Figure 1. Inhibitory effect of L. plantarum against clinical isolates of Candida albicans and S. mutans from children with early childhood caries in multispecies planktonic conditions. Growth of Candida albicans, S. mutans, and L. plantarum in multispecies planktonic conditions is plotted. Clinical strains of Candida albicans and S. mutans were isolated from 10 children with early childhood caries (ECC). Experiments were repeated in triplicate. Each planktonic multispecies condition contained C. albicans and S. mutans isolated from the same ECC children, supplemented with L. plantarum 14917. Control groups consisted of C. albicans and S. mutans only. (Panel A) L. plantarum inhibited the growth of Candida albicans by <1 log at 6 hours and approximately 2 log at 20 hours (p<0.05 at 20 hours). (Panel B) L. plantarum significantly inhibited the growth of S. mutans by 1 log at 6 hours and completely inhibited the growth of S. mutans after 20 hours (p<0.05 at 20 hours). (Panel C) L. plantarum maintained stable growth during 20 hours of interaction with S. mutans and C. albicans. (Panel D) The pH of the culture medium was rapidly decreased with the addition of L. plantarum in the planktonic state, with both groups reaching the same acidic level at 20 hours. [Figure 4]Interaction of L. plantarum 14917 with clinical isolates of C. albicans and S. mutans in multi-species biofilms. Multi-species biofilms were formed with L. plantarum 14917 and clinical isolates of C. albicans and S. mutans from three children with ECC. Treated groups were grown with or without L. plantarum 14917. Growth of C. albicans, S. mutans, and L. plantarum 14917 in multi-species biofilms was plotted (Panels A-C). In the treated group with L. plantarum 14917, C. albicans was reduced by 3 logs compared to the control group (Panel A). At 48 hours after two doses of L. plantarum 14917, S. mutans was completely inhibited in the biofilms of the treated group (Panel B). L. plantarum decreased during the first 24 h due to low sucrose (0.1%) conditions and kept stable growth in high sucrose (1%) conditions after 24 h (Panel C). (Panel D) The pH of the culture medium was significantly lower at 24, 48, and 72 h with the addition of L. plantarum 14917 compared to the control group (p<0.05). (Panel E) The composition of the respective microorganisms. L. plantarum 14917 became the dominant species after 48 h of incubation. (Panel F) Biofilm formation was significantly decreased with the addition of L. plantarum 14917, with a drop in dry mass of almost 40% at 72 h. [Diagram 5]Figure 1. Changes in the 3D structure of multi-species biofilms by L. plantarum 14917. Biofilms formed by Candida albicans and S. mutans only (control) were treated with L. plantarum 14917 under 1% sucrose conditions and visualized by two-photon laser confocal microscopy at 72 hours. L. plantarum 14917 dramatically reduced the biomass of bacteria (panel A) and EPS (panel B). Significantly fewer bacteria were colocalized with EPS in the treated group (panel C). Biofilm parameters are calculated using data from three biofilms formed by Candida albicans and S. mutans isolated from three children with ECC. [Figure 6] Inhibition of Candida albicans hypha formation by L. plantarum 14917. (Panel A) Streptococcus mutans and Candida albicans grown in 1% glucose for 48 hours. (Panel B) Streptococcus mutans and Candida albicans grown in 1% glucose with the addition of L. plantarum 14917 for 48 hours. The addition of L. plantarum 14917 reduced the growth of Candida albicans and inhibited the switch from yeast to hyphal and pseudohyphal forms. [Figure 7] Figure 1. Regulation of mutans streptococcus and Candida albicans virulence genes by L. plantarum 14917 in multi-species biofilms. Compared to the control group, expression of mutans streptococcus genes related to carcinogenesis (gtfB, gtfC, and atpD) was reduced by approximately 50% in 72-h biofilms treated with L. plantarum 14917. [Figure 8] FIG. 1 shows a study design to determine the effect of Lactobacillus species against clinical isolates of Candida albicans and Streptococcus mutans from children with early childhood caries. [Figure 9]Figure 1 shows the inhibitory effect of Lactobacillus species on clinical isolates of Candida albicans and Streptococcus mutans from children with early childhood caries in a multi-species planktonic condition. Growth of Candida albicans, Streptococcus mutans, and Lactobacillus species in a multi-species planktonic condition is plotted. Clinical strains of Candida albicans and Streptococcus mutans were isolated from two children with early childhood caries (ECC). Experiments were repeated in triplicate. Each planktonic multi-species condition contained Candida albicans and Streptococcus mutans isolated from the same ECC child, supplemented with L. plantarum 14917. The control group consisted of Candida albicans and Streptococcus mutans only. The treated group that contained Lactobacillus was marked "with Lactobacillus species." (Panel A) All three Lactobacilli inhibited the growth of Candida albicans by <1 log after 6 hours of incubation and by 1-2 logs after 20 hours of incubation. (Panel B) All three Lactobacilli inhibited the growth of mutans streptococci. Performance is ranked with L. plantarum 14917 and L. salivarius 11741, as well as L. plantarum 8014. All three Lactobacilli significantly inhibited the growth of mutans streptococci by 2 logs at 6 hours. With the exception of L. plantarum 8014, L. plantarum 14917 and L. salivarius 11741 completely inhibited the growth of mutans streptococci after 20 hours. (Panel C) Lactobacilli maintained stable growth during the 20 hours of interaction with mutans streptococci and Candida albicans. (Panel D) The pH of the culture medium decreased rapidly upon addition of Lactobacillus in the planktonic state, reaching the same acidic level at 20 h in both the control and treated groups. [Figure 10]Figure 1. Inhibition of C. albicans and S. mutans by Lactobacillus in multi-species biofilms in children with ECC. Multi-species biofilms were formed by L. plantarum 14917 and L. salivarius 11741 and clinical isolates of C. albicans and S. mutans from two children with ECC. The control group consisted of C. albicans and S. mutans from the same children with ECC. (Panel A) L. plantarum and L. salivarius inhibited the growth of C. albicans by 3 logs compared to the control group. (Panel B) At 48 hours after two doses of L. plantarum and L. salivarius, S. mutans was completely inhibited in the biofilms of the treatment group. (Panel C) Lactobacillus maintained stable growth during the 72-hour incubation period, with L. plantarum being significantly higher than L. salivarius. (Panel D) The pH of the culture medium was significantly lower at 24, 48, and 72 hours with the addition of Lactobacillus compared to the control group (p<0.05). (Panels E-F) The composition of each microbial species was plotted. Lactobacillus became the dominant species after 48 hours of incubation. [Figure 11-1] Figure 1. Differential gene expression of mutans streptococci grown in treatment vs. control groups (part 1). Significant genes (>(-)1Log2 fold change and FDR p-value<0.05) that fit into KEGG pathways are shown. Control group: mutans streptococci + Candida albicans. Treatment group: L. plantarum 14917 + mutans streptococci + Candida albicans. All FDR p-values ​​less than 1E-15 are shown as 1E-15. [Figure 11-2] Differential gene expression of mutans streptococci grown in treatment vs. control groups (part 2). Significant genes (>(-)1Log2 fold change and FDR p-value<0.05) that fit into KEGG pathways are shown. Control group: mutans streptococci + Candida albicans. Treatment group: L. plantarum 14917 + mutans streptococci + Candida albicans. All FDR p-values ​​less than 1E-15 are shown as 1E-15. [Figure 11-3] Differential gene expression of mutans streptococci grown in treatment versus control groups (part 3). Significant genes (>(-)1Log2 fold change and FDR p-value<0.05) that fit into KEGG pathways are shown. Control group: mutans streptococci + Candida albicans. Treatment group: L. plantarum 14917 + mutans streptococci + Candida albicans. All FDR p-values ​​less than 1E-15 are shown as 1E-15. [Figure 11-4] Figure 4. Differential gene expression of mutans streptococci grown in treatment vs. control groups. Significant genes (>(-)1Log2 fold change and FDR p-value<0.05) that fit into KEGG pathways are shown. Control group: mutans streptococci + Candida albicans. Treatment group: L. plantarum 14917 + mutans streptococci + Candida albicans. All FDR p-values ​​less than 1E-15 are shown as 1E-15. [Figure 12-1] Differential gene expression of Candida albicans grown in treatment versus control groups (part 1). Significant genes (>(-)1Log2 fold change and FDR p-value<0.05) that fit into KEGG pathways are shown. Control group: mutans streptococcus + Candida albicans. Treatment group: L. plantarum 14917 + mutans streptococcus + Candida albicans. All FDR p-values ​​less than 1E-15 are shown as 1E-15. [Figure 12-2] Differential gene expression of Candida albicans grown in treatment vs. control groups (part 2). Significant genes (>(-)1Log2 fold change and FDR p-value<0.05) that fit into KEGG pathways are shown. Control group: mutans streptococcus + Candida albicans. Treatment group: L. plantarum 14917 + mutans streptococcus + Candida albicans. All FDR p-values ​​less than 1E-15 are shown as 1E-15. [Figure 12-3]Differential gene expression of Candida albicans grown in treatment versus control groups (part 3). Significant genes (>(-)1Log2 fold change and FDR p-value<0.05) that fit into KEGG pathways are shown. Control group: mutans streptococcus + Candida albicans. Treatment group: L. plantarum 14917 + mutans streptococcus + Candida albicans. All FDR p-values ​​less than 1E-15 are shown as 1E-15. [Figure 12-4] Differential gene expression of Candida albicans grown in treatment vs. control groups (part 4). Significant genes (>(-)1Log2 fold change and FDR p-value<0.05) that fit into KEGG pathways are shown. Control group: mutans streptococcus + Candida albicans. Treatment group: L. plantarum 14917 + mutans streptococcus + Candida albicans. All FDR p-values ​​less than 1E-15 are shown as 1E-15. [Figure 12-5] Figure 5. Differential gene expression of Candida albicans grown in treatment vs. control groups. Significant genes (>(-)1Log2 fold change and FDR p-value<0.05) that fit into KEGG pathways are shown. Control group: mutans streptococcus + Candida albicans. Treatment group: L. plantarum 14917 + mutans streptococcus + Candida albicans. All FDR p-values ​​less than 1E-15 are shown as 1E-15. [Figure 12-6] Figure 6. Differential gene expression of Candida albicans grown in treatment vs. control groups. Significant genes (>(-)1Log2 fold change and FDR p-value<0.05) that fit into KEGG pathways are shown. Control group: mutans streptococcus + Candida albicans. Treatment group: L. plantarum 14917 + mutans streptococcus + Candida albicans. All FDR p-values ​​less than 1E-15 are shown as 1E-15. [Figure 12-7]Figure 7. Differential gene expression of Candida albicans grown in treated vs. control groups. Significant genes (>(-)1Log2 fold change and FDR p-value<0.05) that fit into KEGG pathways are shown. Control group: mutans streptococcus + Candida albicans. Treated group: L. plantarum 14917 + mutans streptococcus + Candida albicans. All FDR p-values ​​less than 1E-15 are shown as 1E-15. [Figure 13-1] Differential gene expression of L. p14917 grown in treatment groups versus monospecies biofilm (part 1). Significant genes (FDR p-value < 0.05) that fit into KEGG pathways are shown. Treatment groups: L. plantarum 14917 + Streptococcus mutans + Candida albicans. Monospecies biofilm: L. plantarum 14917. All FDR p-values ​​below 1E-15 are shown as 1E-15. [Figure 13-2] Differential gene expression of L. p14917 grown in treatment groups versus monospecies biofilm (part 2). Significant genes (FDR p-value < 0.05) that fit into KEGG pathways are shown. Treatment groups: L. plantarum 14917 + Streptococcus mutans + Candida albicans. Monospecies biofilm: L. plantarum 14917. All FDR p-values ​​less than 1E-15 are shown as 1E-15. [Figure 13-3] Differential gene expression of L. p14917 grown in treatment groups versus monospecies biofilm (part 3). Significant genes (FDR p-value < 0.05) that fit into KEGG pathways are shown. Treatment groups: L. plantarum 14917 + Streptococcus mutans + Candida albicans. Monospecies biofilm: L. plantarum 14917. All FDR p-values ​​less than 1E-15 are shown as 1E-15. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0010] Reference is made in detail to certain aspects and exemplary embodiments of the present invention, examples of which are illustrated in the accompanying structures and figures. Aspects of the present invention are described in conjunction with exemplary embodiments, including methods, materials, and examples, and such descriptions are non-limiting, and the scope of the present invention is intended to encompass all equivalents, alternatives, and modifications commonly known or incorporated herein. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by a person skilled in the art to which the present invention belongs. Those skilled in the art will recognize many techniques and materials similar or equivalent to those described herein that can be used in the implementation of aspects and embodiments of the present invention. The described aspects and embodiments of the present invention are not limited to the described methods and materials.

[0011] As used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the content clearly dictates otherwise.

[0012] Ranges may be expressed herein as from "about" one particular value and / or to "about" another particular value. When such a range is expressed, another embodiment includes from the one particular value and / or to the other particular value. Similarly, when values ​​are expressed as approximations, it will be understood that by using the antecedent "about," the particular value forms another embodiment. It will be further understood that each endpoint of a range is significant both in relation to the other endpoint and independently of the other endpoint. It will also be understood that several values ​​are disclosed herein, and that each value is also disclosed herein as a value "about" that particular value, in addition to the value itself. For example, when a value "10" is disclosed, "about 10" is also disclosed. It will also be understood that when a value that is "less than or equal to" a value is disclosed, "greater than or equal to" the value, as well as possible ranges between the values, as would be well understood by one of ordinary skill in the art. For example, when a value "10" is disclosed, "less than or equal to 10" is also disclosed.

[0013] I. Definition As used herein, the term "bacteria" refers to any member of a large group of unicellular microorganisms that have a cell wall but lack organelles and an organized nucleus.

[0014] As used herein, the term "gram-positive bacteria" refers to bacteria that are characterized by having peptidoglycan and polysaccharides and / or teichoic acid as part of their cell wall structure and by their blue-purple color reaction in the Gram staining procedure. Representative gram-positive bacteria include Actinomyces species, Bacillus species, Bifidobacterium species, Clostridium species, Corynebacterium species, Enterococcus species, Erysipelothrix species, Eubacterium species, Gardnerella species, Gemella species, and the like. Examples of species of the genera Gemella, Leuconostoc, Mycobacterium, Nocardia, Peptococcus, Peptostreptococcus, Proprionibacterium, Sarcina, Staphylococcus, and Streptococcus.

[0015] As used herein, the term "gram-negative bacteria" refers to bacteria characterized by the presence of a double membrane surrounding each bacterial cell. Representative gram-negative bacteria include Acinetobacter species, Actinobacillus species, Aggregatibacter species, Aeromonas species, Alcaligenes species, Bacteroides species, Bartonella species, Bordetella species, Borrelia species, Branhamella species, Brucella species, Campylobacter species, and the like. Campylobacter spp., Chlamydia spp., Chromobacterium spp., Citrobacter spp., Eikenella spp., Enterobacter spp., Escherichia spp., Flavobacterium spp., Fusobacterium spp., Haemophilus spp., Helicobacter spp., Klebsiella pneumoniaePneumoniae, Klebsiella spp., Legionella spp., Leptospira spp., Moraxella spp., Morganella spp., Mycoplasma spp., Neisseria spp., Pasteurella spp., Plesiomonas spp., Prevotella spp., Proteus spp. , Providencia spp., Pseudomonas spp., Rickettsia spp., Rochalimaea spp., Salmonella spp., Salmonella spp., Serratia spp., Shigella spp., Treponema spp., Veillonella spp., Vibrio spp., and Yersinia spp.

[0016] As used herein, the term "biofilm" refers to a sessile community of microorganisms attached to a substratum or interface, or to each other, embedded in a matrix of extracellular polymers (more specifically, cell-produced extracellular polymers), and characterized by cells that exhibit an altered phenotype with respect to growth rate and gene transcription (e.g., compared to their "non-biofilm," free-floating, or planktonic counterparts).

[0017] As used herein, the term "dental caries" refers to a biofilm-mediated, sugar-driven, multifactorial, dynamic disease that results in phasic demineralization and remineralization of dental hard tissues. Dental caries can occur throughout life in both the primary and permanent dentition, damaging the crowns and potentially resulting in exposure of the root surfaces later in life.

[0018] As used herein, the term "early childhood caries (ECC)" is formerly known as nursing bottle caries, baby bottle caries, nighttime bottle mouth, and nighttime bottle caries, and is a disease affecting the teeth of children between the ages of birth and 71 months. ECC is characterized by the presence of one or more carious (noncavitary or cavitary lesions), missing (caused by caries), or filled tooth surfaces in any primary tooth.

[0019] As used herein, the term "dental coating" refers to materials used in dentistry as a protective layer on tooth surfaces.

[0020] The phrase "pharmaceutically acceptable carrier or diluent" refers to any substance suitable for use in administration to an animal. In certain embodiments, the pharmaceutically acceptable carrier or diluent is sterile saline. In certain embodiments, such sterile saline is pharmaceutical grade saline.

[0021] As used herein, the term "subject" refers to mammals, such as humans, companion animals (e.g., dogs, cats, birds, etc.), livestock (e.g., cows, sheep, pigs, horses, poultry, etc.), and laboratory animals (e.g., rats, mice, guinea pigs, birds, etc.). A "subject in need thereof" refers to a subject who may have, has been diagnosed with, is suspected of having, or is in need of prevention of a biofilm-related disease or condition.

[0022] An "effective amount" or "therapeutically effective amount" is defined herein in relation to the treatment or prevention of a biofilm-associated disease or condition, and is an amount effective when administered to a cell, tissue, or subject, alone or in combination with another therapeutic agent, to reduce, reduce, inhibit, or otherwise inhibit the occurrence of a biofilm-associated disease or condition. An "effective amount" further refers to an amount of a compound sufficient to cause amelioration, e.g., treatment, cure, prevention, or amelioration of the symptoms of a biofilm-associated disease or condition, or to increase the rate of treatment, cure, prevention, or amelioration of a biofilm-associated disease or condition. When applied to an individual compound (active ingredient) administered alone, an "effective amount" refers to that ingredient alone. When applied to a combination, an "effective amount" refers to the combined amount of active ingredients that results in a therapeutic effect, whether administered in combination, sequentially, or simultaneously. An "effective amount" varies depending on the biofilm-associated disease or condition and the severity of the biofilm-associated disease or condition, as well as the age, weight, etc., of the subject to be treated. Furthermore, an "effective amount" may vary depending on the dosage form used and the route of administration utilized. A physician or veterinarian having ordinary skill in the art can readily determine and prescribe the effective amount of the active ingredient required (e.g., the ED50). For example, the physician or veterinarian can start dosages of the administered compound at levels lower than those required to achieve the desired therapeutic effect and gradually increase the dosage until the desired effect is achieved.

[0023] II. Methods of inhibiting the formation of microbial-based biofilms and treating biofilm-associated diseases One aspect of the present invention is a method for preventing or treating biofilm-associated disease in a subject. The method comprises administering to the subject an effective amount of a composition comprising (1) one or more Lactobacillus species and / or (2) plantaricin. The biofilm-associated disease can be caused by bacteria and / or yeast. In some embodiments, the biofilm-associated disease is selected from the group consisting of dental caries, oral yeast infection, denture stomatitis, periodontal disease, peri-implantitis, and burning mouth syndrome. In certain embodiments, the biofilm-associated disease is early childhood caries (ECC).

[0024] In some embodiments, the biofilm-associated disease is caused by a microbial biofilm. The microbial biofilm may be a biofilm formed from any bacteria or yeast. In some embodiments, the microbial biofilm comprises bacteria selected from the group consisting of Streptococcus, Candida, Veillonella, Rothia, Actinomyces, Prevotella, Tannerella, Treponema, and Campylobacter, Lautropia. In some embodiments, the microbial biofilm comprises Streptococcus mutans and / or Candida albicans.

[0025] In some embodiments, the composition comprises one or more Lactobacillus species selected from the group consisting of L. rhamnosus ATCC2836, L. plantarum ATCC8014, L. plantarum ATCC14917, and L. salivarius ATCC11741.

[0026] In some embodiments, the composition is administered orally. In some embodiments, the composition is administered orally in the form of an orally consumable composition, such as a gum, a candy, or a beverage. In some embodiments, the composition is a pharmaceutical composition of the invention, such as a mouthwash, a dental gel, or a dental coating.

[0027] Another aspect of the invention relates to a method of disestablishing or inhibiting the formation of a bacterial-based biofilm in a subject, the method comprising administering to the subject an effective amount of a composition of the invention.

[0028] Another aspect of the present invention is a method of preventing or treating dental caries in a subject, comprising administering to the subject an effective amount of a composition comprising (1) one or more Lactobacillus species selected from the group consisting of L. rhamnosus ATCC2836, L. plantarum ATCC8014, L. plantarum ATCC14917, and L. salivarius ATCC11741, and / or (2) plantaricin.

[0029] Another aspect of the present invention is a method for inhibiting the growth of mutans streptococci and / or Candida albicans in a subject. The method comprises administering to the subject an effective amount of a pharmaceutical composition comprising one or more Lactobacillus species selected from the group consisting of (1) L. rhamnosus ATCC2836, L. plantarum ATCC8014, L. plantarum ATCC14917, and L. salivarius ATCC11741, and / or (2) plantaricin. Another aspect of the present invention is a method for inhibiting the growth of mutans streptococci and / or Candida albicans in the oral cavity of a subject. The method comprises administering to the subject an effective amount of a composition comprising one or more Lactobacillus species selected from the group consisting of L. rhamnosus ATCC2836, L. plantarum ATCC8014, L. plantarum ATCC14917, and L. salivarius ATCC11741, and administering the composition orally.

[0030] In some embodiments, the composition used in the above-mentioned method comprises two, three, or four Lactobacillus species selected from the group consisting of L. rhamnosus ATCC2836, L. plantarum ATCC8014, L. plantarum ATCC14917, and L. salivarius ATCC11741. In some embodiments, the composition used in the above-mentioned method further comprises an antifungal agent. In some embodiments, the antifungal agent is nystatin. In some embodiments, the composition used in the above-mentioned method comprises L. plantarum 14917 and nystatin.

[0031] Another aspect of the present invention is a method for preventing or treating dental caries in a subject. The method comprises administering to the subject an effective amount of an agent that inhibits the activity or expression of a pharmaceutical composition comprising a bacterial gene. In some embodiments, the bacterial gene is selected from the group consisting of (1) gtfB, gtfC, and atpD genes of Streptococcus mutans, and (2) AAT22, ADE8, ALD5, AYR2, CAT1, CHA1, CHT2, ECM38, ERG4, FDH1, FOL1 GCV1, GCV2, HAL22, HPD1, IST1, LSC1, LSM6, MAL2, PCK1, PEX11, POX1-3, PXP2, SOD3, TEM1, THI20, THI6, URA3, HPW1, and ECE1 genes of Candida albicans. In certain embodiments, the dental caries is early childhood caries (ECC). In some embodiments, the agent is selected from the group consisting of Lactobacillus sp. and plantaricin. In some embodiments, the agent is selected from the group consisting of (1) Lactobacillus sp. selected from the group consisting of L. rhamnosus ATCC2836, L. plantarum ATCC8014, L. plantarum ATCC14917, and L. salivarius ATCC11741, and (2) plantaricin.

[0032] Another aspect of the present invention is a method of preventing or treating dental caries in a subject, comprising administering to the subject an effective amount of a pharmaceutical composition comprising one or more Lactobacillus species selected from the group consisting of L. rhamnosus ATCC2836, L. plantarum ATCC8014, L. plantarum ATCC14917, and L. salivarius ATCC11741.

[0033] Another aspect of the present invention relates to a method for preventing the occurrence of early childhood caries (ECC) in a neonatal subject. The method comprises administering to the mother of the neonatal subject an effective amount of a composition of the present invention during pregnancy. In some embodiments, the composition comprises one or more Lactobacillus species. In some embodiments, the one or more Lactobacillus species are selected from the group consisting of L. rhamnosus ATCC2836, L. plantarum ATCC8014, L. plantarum ATCC14917, and L. salivarius ATCC11741.

[0034] In some embodiments, the composition or pharmaceutical composition described above is orally administered in the form of a mouthwash, dental gel, or dental coating. In some embodiments, the mouthwash, dental gel, or dental coating contains one or more Lactobacillus species, 7 ~10 8 CFU / ml, 10 7 ~10 9 CFU / ml, 10 7 ~10 10 CFU / ml, 10 7 ~10 11 CFU / ml, 10 7 ~10 12 CFU / ml, 10 8 ~10 9 CFU / ml, 10 8 ~10 10 CFU / ml, 10 8 ~10 11 CFU / ml, 10 8 ~10 12 CFU / ml, 10 9 ~10 10 CFU / ml, 10 9 ~10 11CFU / ml, 10 9 ~10 12 CFU / ml, 10 10 ~10 11 CFU / ml, 10 10 ~10 12 CFU / ml, or 10 11 ~10 12 Included in individual or total doses of CFU / ml.

[0035] In some embodiments, the mouthwash, dental gel, or dental coating comprises one or more Lactobacillus species selected from the group consisting of L. rhamnosus ATCC2836, L. plantarum ATCC8014, L. plantarum ATCC14917, and L. salivarius ATCC11741, at least 10% by weight of Lactobacillus spp. 7 ~10 8 CFU / ml, 10 7 ~10 9 CFU / ml, 10 7 ~10 10 CFU / ml, 10 7 ~10 11 CFU / ml, 10 7 ~10 12 CFU / ml, 10 8 ~10 9 CFU / ml, 10 8 ~10 10 CFU / ml, 10 8 ~10 11 CFU / ml, 10 8 ~10 12 CFU / ml, 10 9 ~10 10 CFU / ml, 10 9 ~10 11 CFU / ml, 10 9 ~10 12 CFU / ml, 10 10 ~10 11 CFU / ml, 10 10 ~10 12 CFU / ml, or 10 11 ~10 12 Included in individual or total doses of CFU / ml.

[0036] In some embodiments, the mouthwash, dental gel, or dental coating comprises L. plantarum 14917, 10 7 ~10 8 CFU / ml, 10 7 ~10 9 CFU / ml, 10 7 ~10 10 CFU / ml, 10 7 ~10 11 CFU / ml, 10 7 ~10 12 CFU / ml, 10 8 ~10 9 CFU / ml, 10 8 ~10 10 CFU / ml, 10 8 ~10 11 CFU / ml, 10 8 ~10 12 CFU / ml, 10 9 ~10 10 CFU / ml, 10 9 ~10 11 CFU / ml, 10 9 ~10 12 CFU / ml, 10 10 ~10 11 CFU / ml, 10 10 ~10 12 CFU / ml, or 10 11 ~10 12 Included in the dose of CFU / ml.

[0037] In some embodiments, the mouthwash, dental gel, or dental coating comprises two Lactobacillus genera selected from the group consisting of L. rhamnosus ATCC2836, L. plantarum ATCC8014, L. plantarum ATCC14917, and L. salivarius ATCC11741, and the two Lactobacillus genera are present in the mouthwash, dental gel, or dental coating in a CFU ratio ranging from 1:10 to 10:1, or 1:5 to 5:1, or 1:3 to 3:1. In one embodiment, the mouthwash, dental gel, or dental coating comprises L. plantarum ATCC14917 and L. plantarum ATCC8014 in a 1:1 CFU ratio (e.g., 10 8L. plantarum ATCC14917 and 10 CFU / ml 8 CFU / ml of L. plantarum ATCC 8014).

[0038] In some embodiments, the mouthwash, dental gel, or dental coating contains plantaricin at a concentration of 20-40 ng / ml, 20-100 ng / ml, 20-200 ng / ml, 20-400 ng / ml, 20-1000 ng / ml, 20-2000 ng / ml, 40-100 ng / ml, 40-200 ng / ml, 40-400 ng / ml, 40-1000 ng / ml , 40-2000ng / ml, 100-200ng / ml, 100-400ng / ml, 100-1000ng / ml, 100-2000ng / ml, 200-400ng / ml, 200-1000ng / ml, 200-2000ng / ml, 400-1000ng / ml, 400-2000ng / ml, or 1000-2000ng / ml. In some embodiments, the mouthwash, dental gel, or dental coating comprises plantaricin at a concentration in the range of 200-400ng / ml.

[0039] In some embodiments, the mouthwash, dental gel, or dental coating is applied once a day, twice a day, or three times a day for a period of 1-60, 1-45, 1-30, 1-15, 1-10, 1-5, or 1-3 days. In some embodiments, the mouthwash, dental gel, or dental coating is applied once a day, twice a day, or three times a day for a period of at least 1, 2, 3, 4, 5, 6, 7, or 8 weeks.

[0040] In some embodiments, the composition is orally administered in the form of a mouthwash, dental gel, or dental coating. In some embodiments, the mouthwash, dental gel, or dental coating is applied once a day, twice a day, or three times a day for a period of 1-60, 1-45, 1-30, 1-15, 1-10, 1-5, or 1-3 days. In some embodiments, the mouthwash, dental gel, or dental coating is applied once a day, twice a day, or three times a day for a period of at least 1, 2, 3, 4, 5, 6, 7, or 8 weeks.

[0041] One of skill in the art will appreciate that the compositions used in the methods described herein may be delivered in forms including, but not limited to, oral formulations, capsule formulations, tablet formulations, infusions, etc. One of skill in the art will appreciate that the particular formulation or delivery method of the composition is not limiting to the methods described herein.

[0042] III. Compositions of the Invention Another aspect of the present invention relates to a composition that can be used to prevent or treat biofilm-associated diseases. The composition comprises (1) one or more Lactobacillus species and / or (2) plantaricin. In some embodiments, the one or more Lactobacillus species are selected from the group consisting of L. rhamnosus ATCC2836, L. plantarum ATCC8014, L. plantarum ATCC14917, and L. salivarius ATCC11741. In some embodiments, the composition comprises (1) one or more Lactobacillus species selected from the group consisting of L. rhamnosus ATCC2836, L. plantarum ATCC8014, L. plantarum ATCC14917, and L. salivarius ATCC11741, and (2) plantaricin. In some embodiments, the composition comprises two or more Lactobacillus species selected from the group consisting of L. rhamnosus ATCC 2836, L. plantarum ATCC 8014, L. plantarum ATCC 14917, and L. salivarius ATCC 11741. In some embodiments, the composition comprises (1) two or more Lactobacillus species selected from the group consisting of L. rhamnosus ATCC 2836, L. plantarum ATCC 8014, L. plantarum ATCC 14917, and L. salivarius ATCC 11741, and (2) plantaricin.

[0043] In some embodiments, the composition further comprises an antifungal agent. In some embodiments, the antifungal agent is nystatin.

[0044] In some embodiments, the composition of the present invention is in the form of an orally consumable product.The term "orally consumable product" refers to a composition that can be drunk, eaten, swallowed, orally ingested, or otherwise contacted with the mouth of a human or animal.Oral consumable products are safe for human or animal consumption when used within generally acceptable limits.Examples of oral consumable products include, but are not limited to, candy, gum, beverages, and dairy products such as yogurt.

[0045] In some embodiments, the compositions of the present invention are formulated as a pharmaceutical composition comprising a pharma- ceutically acceptable carrier.In some embodiments, the pharmaceutical composition is formulated for oral administration.In some embodiments, the pharmaceutical composition is formulated as a mouthwash, a dental gel, or a dental coating.

[0046] In some embodiments, the compositions of the present invention are formulated as liquid or hydrogel formulations. In some embodiments, the liquid or hydrogel formulations have a pH value in the range of 3-9, 3-8, 3-7, 3-6, 3-5, 3-4, 4-9, 4-8, 4-7, 4-6, 4-5, 5-9, 5-8, 5-7, 5-6, 6-9, 6-8, 6-7, 7-9, 7-8, or 8-9. In some embodiments, the liquid or hydrogel formulations have a pH value in the range of 4-7. In some embodiments, the liquid or hydrogel formulations are in the form of a mouthwash, a dental gel, or a dental coating.

[0047] In some embodiments, the compositions of the present invention are formulated as liquid or hydrogel formulations containing 0.1-0.3%, 0.1-0.6%, 0.1-1%, 0.1-3%, 0.1-6%, 0.1-10%, 0.3-0.6%, 0.3-1%, 0.3-3%, 0.3-6%, 0.3-10%, 0.6-1%, 0.6-3%, 0.6-6%, 0.6-10%, 1-3%, 1-6%, 1-10%, 3-6%, 3-10%, 6-10% (w / w or w / v) sugar. Examples of sugars include, but are not limited to, sucrose, glucose, galactose, fructose, and galactooligosaccharides. In some embodiments, the liquid or hydrogel formulation composition contains about 1% (w / w or w / v) sugar. In some embodiments, the liquid or hydrogel formulation composition comprises (1) L. plantarum and (2) 1% (w / w or w / v) sucrose, or 1% (w / w or w / v) glucose, or 1% (w / w or w / v) galactooligosaccharides.

[0048] In some embodiments, the compositions of the present invention are formulated as pharmaceutical compositions that contain one or more carriers suitable for delivering therapeutic agents (e.g., Lactobacillus, plantaricin, and / or antifungal agents) to target tissue / organ, such as teeth or gum tissue.Exemplary carriers for delivery include solutions, hydrogels, nanoparticles, lipids, liposomes, micelles, polymers, polymeric micelles, emulsions, polyelectrolyte complexes, microcapsules, and combinations thereof, and their PEGylated derivatives.

[0049] Exemplary nanoparticles include paramagnetic nanoparticles, superparamagnetic nanoparticles, metal nanoparticles, polymeric nanoparticles, nanoworms, nanoemulsions, nanogels, fullerene-like materials, inorganic nanotubes, dendrimers (such as those with covalently bound metal chelates), nanocapsules, nanospheres, nanofibers, nanohoms, nanoonions, nanorods, nanoropes, and quantum dots.

[0050] In other embodiments, the polymeric nanoparticles are made from synthetic biodegradable polymers, natural biodegradable polymers, or combinations thereof. Synthetic biodegradable polymers can include polyesters, such as poly(lactic-co-glycolic acid) (PLGA) and polycaprolactones, polyorthoesters, polyanhydrides, polydioxanones, poly-alkyl-cyano-acrylates (PACs), polyoxalates, polyiminocarbonates, polyurethanes, polyphosphazenes, or combinations thereof. Natural biodegradable polymers can include starch, hyaluronic acid, heparin, gelatin, albumin, chitosan, dextran, or combinations thereof.

[0051] In certain embodiments, the pharmaceutical compositions provided herein comprise one or more therapeutic agents (e.g., Lactobacillus sp., plantaricin, and / or antifungal agents) and one or more excipients.Exemplary excipients include water, salt solution, alcohol, polyethylene glycol, gelatin, lactose, amylase, magnesium stearate, talc, silicic acid, viscous paraffin, hydroxymethylcellulose, polyvinylpyrrolidone, and combinations thereof.

[0052] In some embodiments, the pharmaceutical composition comprises a buffering agent to maintain a desired pH range.In some embodiments, the buffering agent is a buffering agent for oral use.Examples of buffering agents for oral use include, but are not limited to, phosphate buffered saline (PBS), potassium chloride, sodium chloride, magnesium chloride, calcium chloride, potassium thiocyanate, and sodium bicarbonate.

[0053] The pharmaceutical composition of the present invention is formulated according to a specific route of administration. In some embodiments, the pharmaceutical composition of the present invention is formulated for oral administration. In some embodiments, the pharmaceutical composition of the present invention is formulated as a mouthwash, a dental gel, or a dental coating.

[0054] The present invention is further illustrated by the following examples, which should not be construed as limiting. The contents of all references, patents, and published patents cited throughout the present invention and in the figures and tables are hereby incorporated by reference. EXAMPLES

[0055] Example 1 material and method Bacterial strains and starting preparations The microorganisms used in the study were Streptococcus mutans UA159, Candida albicans SC5314, L. rhamnosus ATCC2836, L. plantarum ATCC8014, L. plantarum ATCC14917, and L. salivarius ATCC11741. Candida albicans, Streptococcus mutans, and Lactobacillus were recovered from frozen stocks using YPD agar (BD Difco™, 242720), blood agar (TSA with sheep blood, Thermo Scientific™ R01202), and MRS agar (BD Difco™, 288210), respectively. After 48 hours of incubation, 3-5 colonies of each species were inoculated into 10 ml of broth and incubated overnight (5% CO2, 37°C). Candida albicans was grown in YPD broth (BD Difco™, 242820), Streptococcus mutans was grown in TSBYE broth (3% tryptic soy, 0.5% yeast extract broth, BD Bacto™ 286220, and Gibco™ 212750) with 1% glucose, and Lactobacillus species was grown in MRS broth (BD Difco™, 288130). The following day, 0.5 ml of the overnight starts were added to individual glass tubes containing fresh broth and incubated for 3-4 hours to reach mid-log phase with the desired optical density. The morning starts were then ready for preparation of the planktonic and biofilm models described below.

[0056] Planktonic model First, the interactions between Candida albicans, Streptococcus mutans, and Lactobacillus species were evaluated in planktonic conditions. 3 CFU / ml) and inoculum size of mutans streptococci (10 5 CFU / ml were selected to simulate high caries conditions in a clinical setting. The inoculum doses (10 8 CFU / ml) were significantly higher than those of commercially available probiotic products (10 9 ~10 12This is the lowest dose of probiotics used in terms of CFU. Candida albicans, Streptococcus mutans, and one of the Lactobacillus species were grown in 10 ml of TSBYE broth containing 1% glucose for 20 h (5% CO2, 37°C). In addition, L. plantarum 14917 (10 4 ~10 7 The effect of dose titration of the culture medium (inoculation of CFU / ml) was evaluated. Growth and pH values ​​of each organism were measured at multiple time points.

[0057] Mixed-species biofilm model The study then used a mixed-species biofilm model to evaluate the effect of Lactobacillus on biofilm formation by Streptococcus mutans and Candida albicans. Biofilms were formed on saliva-coated hydroxyapatite disks (0.50 inch diameter x 0.05 inch thickness, Clarkson Chromatography Products, Inc., South Williamsport, PA). To mimic the caries-prone smooth tooth surfaces in the oral cavity, the disks were placed in a vertical position using a custom-made disk holder (Xiao, J. et al., (2012). PLoS Pathog 8(4), e1002623).

[0058] A mixture of Streptococcus mutans, Candida albicans, and Lactobacillus sp. was inoculated into 2.8 ml of TSBYE broth containing 0.1% (w / v) sucrose and incubated at 37°C and 5% CO2. During the first 24 h, the organisms were allowed to grow undisturbed to allow for initial biofilm formation. At 24 h, the biofilms were transferred to fresh culture medium containing 1% (w / v) sucrose or 1% (w / v) glucose to induce a cariogenic challenge, while an additional set of biofilms was grown in 0.1% (w / v) sucrose. Culture medium was changed every 24 h until the end of the experimental period (72 h). Lactobacillus sp. (10 8CFU / ml) were added daily to fresh culture medium. The pH of the culture medium was measured at selected time points. Biofilms underwent microbiological, dry mass, and confocal imaging assays at 24, 48, and 72 h, transcriptomic analysis via RNA-Seq at 48 h, and qRT-PCR validation at 48, 50, and 52 h. Methods are detailed in Xiao et al. (2012) supra. Duplicate discs were used in each run. Independent assays were repeated three times.

[0059] Inhibition of Candida albicans and Streptococcus mutans by L. plantarum supernatant Supernatants of L. plantarum 8014 and 14917 overnight cultures were harvested and sterilized using a vacuum filter system (0.22 μm PES, Corning™ Disposable Vacuum Filter System, USA). 1~8 , 10 for Candida albicans 1~6 Mutans Streptococcus and Candida albicans bearing L. plantarum were treated with the supernatant and grown in 96-well plates in TSBYE containing 1% (w / v) glucose or 1% (w / v) sucrose conditions for 24 h. Clear cultures indicated the absence of microbial growth.

[0060] Inhibition of Streptococcus mutans and Candida albicans by plantaricin The bacteriocin, an antimicrobial molecule produced by L. plantarum, is known as plantaricin. A plantaricin solution was prepared by dissolving the peptide plantaricin-149 (acetate) powers (Creative Peptides, Shirley, USA) in ddH2O. Streptococcus mutans (3.6 × 10 3 CFU / ml) and Candida albicans (3.1 × 10 1CFU / ml) were selected and treated with plantaricin in a range of concentrations (0-400 μg / ml). Mixtures of plantaricin with mutans streptococcus or Candida albicans were grown in 96-well plates in TSBYE containing 1% glucose for 24 hours. A clear culture after 24 hours of incubation indicated no growth of the microorganisms. Thus, the minimum inhibitory concentration (MIC) of plantaricin-149 was defined as the lowest concentration that inhibited the growth of mutans streptococcus and Candida albicans.

[0061] RNA-seq - Transcriptome analysis by RNA library preparation and sequencing Biofilm mass was harvested from four discs for each condition. Discs were soaked in RNALater (Applied Biosystems / Ambion, Austin, TX, USA) for 1 h, followed by removal of biomass with a spatula. RNA was extracted and purified with MasterPure complete DNA and RNA purification kit (epicenter, Lucigen, Wisconsin, USA). Raw RNA products were quantified using a NanoDrop One Microvolume UV-Vis spectrophotometer (Thermo Scientific™, Wilmington, DE, USA). rRNA depletion was performed using Ribozero rRNA removal kit (Illumina, San Diego, CA, USA). RNA sequencing libraries were prepared using the NEBNext Ultra RNA Library Prep Kit for Illumina by following the manufacturer's recommendations (NEB, Ipswich, MA, USA). Sequencing libraries were multiplexed and clustered onto one lane of a flow cell and loaded onto an Illumina HiSeq instrument according to the manufacturer's instructions.

[0062] RNA sequencing libraries were prepared using the NEBNext Ultra RNA Library Preparation Kit for Illumina by following the manufacturer's recommendations (NEB, Ipswich, MA, USA). Briefly, enriched RNA was fragmented at 94°C for 15 min. First and second strand cDNA were synthesized. cDNA fragments were end-repaired and adenylated at the 3' end, and universal adapters were ligated to the cDNA fragments, followed by indexing and library enrichment using limited cycle PCR. Sequencing libraries were validated using an Agilent Tapestation 4200 (Agilent Technologies, Palo Alto, CA, USA) and quantified by using a Qubit 2.0 fluorometer (Invitrogen, Carlsbad, CA, USA) and by quantitative PCR (Applied Biosystems, Carlsbad, CA, USA).

[0063] Sequencing libraries were multiplexed and clustered on one lane of a flow cell and loaded onto an Illumina HiSeq instrument according to the manufacturer's instructions. Samples were sequenced using 2 × 150 paired end (PE) configurations. Image analysis and base calling were performed using HiSeq Control software (HCS). Raw sequence data generated from the Illumina HiSeq were converted to FASTQ files and demultiplexed using Illumina's bcl2fastq 2.17 software. One mismatch was allowed for index sequence identification. After demultiplexing, sequence data were inspected for overall quality and yield. Sequence reads were trimmed to remove possible adapter sequences and poor quality nucleotides using Trimmomatic version 0.36. Trimmed reads were mapped against the reference genome using STAR aligner version 2.5.2b (Dobin, A. et al. (2013). Bioinformatics 29(1), pp. 15-21). Unique gene hit counts were calculated by using feature Counts from the Subread package version 1.5.2. Only unique reads within exonic regions were counted. Gene hit counts were extracted and the gene hit count table was used for downstream differential expression analysis.

[0064] Comparison of gene expression between sample groups was performed using DESeq2. Wald test was used to generate p-values ​​and fold Log2 changes. Streptococcus mutans and Candida albicans genes with adjusted p-values ​​(false discovery rate (FDR) p-value) <0.05 and fold absolute log2 changes >2, and L. plantarum 14917 genes with FDR p-value <0.05 and fold absolute log2 changes >1 were called differentially expressed genes (DEGs) for each comparison. Gene Ontology (GO) analysis was performed on statistically significant gene sets by running the software CeneSCF version 1.1 (Subhash, S. et al., (2016). Bmc Bioinformatics 17). The GO list was used to cluster gene sets based on their biological processes and determine their statistical significance. Principal component analysis (PCA) was performed using the "plot PCA" function in the DESeq2 R package. The plot shows the sample in a 2D plane spanned by its first two principal components. The top 500 genes selected by highest row variance were used to generate the plot. Volcano plots were generated by VolcaNoseR (Goedhart, J. et al., (2020). Scientific Reports 10(1)). Kyoto Encyclopedia of Genes and Genomes pathways were generated by KEGG mapper (genome.jp) and Cytoscape software version 3.8.2.

[0065] Real-time reverse transcription polymerase chain reaction (qRT-PCR) cDNA was then synthesized using 0.2 μg of purified RNA and a BioRad iScript cDNA synthesis kit (Bio-Rad Laboratories, Inc., Hercules, CA). The resulting cDNA and negative controls were amplified using Applied Biosystems™ PowerTrack™ SYBR Green Master Mix and a QuantStudio™ 3 Real-Time PCR System (Thermo Fisher Scientific, USA) under quantitative amplification conditions. Each 20 μL reaction mixture contained template cDNA, 10 μM of each primer, and 2×SYBR-Green mix (containing SYBR-Green and Taq DNA polymerase). Unique core genes of Streptococcus mutans, Candida albicans, and L. plantarum were used as internal references for comparative expression calculations: gyrA (Zeng, L. et al., (2013). J Bacteriol 195(4), 833-843) for Streptococcus mutans, ACT1 for Candida albicans, and ropB for L. plantarum.

[0066] Laser scanning confocal fluorescence microscopy (LCSFM) imaging of biofilm substrates In the study, two essential components of the biofilm matrix, namely bacteria and exopolysaccharides (EPS), were evaluated using LCSFM, as described above (Xiao et al., 2012). Briefly, for visualization of exopolysaccharides, 1 μM Alexa Fluor® 647-labeled dextran conjugate (Molecular Probes, Invitrogen Corp., Carlsbad, CA) was added to the culture medium at the initiation and during biofilm development. Bacterial and fungal species were labeled with SYTO® 9 green fluorescent nucleic acid stain (485 / 498 nm, Molecular Probes). Images were obtained using an Olympus FV 1000 two-photon laser scanning microscope (Olympus, Tokyo, Japan) equipped with a 10× (0.45 numerical aperture) water immersion objective. Each biofilm formed on the HA disk was randomly scanned at five positions (Xiao, J. et al., (2010). J Appl Microbiol 108(6), pp. 2103-2113). Three independent biofilm experiments were performed and 10 image stacks were collected for each experiment. Amira 5.0.2 (Mercury Xomputer Systems Inc., Chelmsford, Mississippi) was used to create 3D renderings of EPS and bacteria in the biofilm as previously detailed (Klein, MI et al., (2011). J Vis Exp (47)). COMSTAT and DUOSTAT (http: / / www.imageanalysis.dk) were used for quantitative analysis of the biofilms including biomass, number, and size (volume, diameter, and height) of microcolonies, as well as colocalization of EPS and bacteria across the biofilm, as described above (Xiao et al., 2012).

[0067] Microbiological analysis of mixed-species bacterial populations Biofilms were homogenized by sonication. The homogenized suspension was used to determine the number of viable cells by plating on blood agar using an automated EddyJet Spiral Plater (IUL, SA, Barcelona, ​​Spain). The three species were differentiated by colony morphology combined with microscopic examination of cells from selected colonies (Guggenheim, B. et al., (2001). (J Dent Res 80(1), 363-370).

[0068] statistical analysis To compare the abundance of Streptococcus mutans, Candida albicans, and Lactobacillus spp. in planktonic and biofilm conditions, CFU values ​​were first transformed to natural logarithm, with 0 values ​​remaining 0. Log values ​​were compared after assessing the normality of the data between each group treated with Lactobacillus spp. and the control group using the Mann-Whitney U test. Normality tests were first performed for the biomass (bacteria and EPS), number, and size of microcolonies at specific time points, as well as pH values ​​of specific biofilms and other measurements. For normally distributed data, comparisons between groups were tested using t-tests for two groups and one-way ANOVA for more than two groups, followed by post hoc tests. For data that were not normally distributed, Kruskal-Wallis was used to compare results of more than two groups, and Mann-Whitney U test was used to compare two groups. Statistical tests were two-sided, with a significance level of 5%. IBM SPSS was used for statistical analysis. Example 2

[0069] Macrophage inhibition of Candida albicans and Streptococcus mutans by Lactobacillus species in the planktonic state All four Lactobacillus species significantly inhibited the growth of C. albicans in the planktonic state by 1 log at 6 hours and 2 logs at 20 hours (Figure 1, Panel A). All Lactobacillus species tested significantly inhibited the growth of S. mutans at 6 hours and 20 hours (Figure 1, Panel B). In contrast to the inhibited growth of C. albicans and S. mutans, the growth of Lactobacillus in the multi-species state did not differ from its growth in the single Lactobacillus species state (Figure 1, Panel C). The pH of the culture medium decreased more quickly with the addition of Lactobacillus species, but reached the same acidity (~4) at 20 hours across all conditions (p>0.05). A dose-dependent effect was seen, with a minimal inoculum of L. plantarum 14917 at 100 mg / mL, which demonstrated inhibition against the growth of S. mutans and C. albicans. 8 CFU / ml. Example 3

[0070] Inhibition of Candida albicans and Streptococcus mutans by Lactobacillus species in multispecies biofilms The growth of Candida albicans and S. mutans streptococci was significantly inhibited by L. salivarius 11741, L. plantarum 8014, and L. plantarum 14917 in multi-species biofilms (Figure 1D-L). Interestingly, rich sucrose conditions (1% sucrose vs. 0.1% sucrose) enhanced the performance of Lactobacillus species. Interestingly, L. plantarum 8014 and 14917 inhibited S. mutans streptococci to undetectable levels (<20 CFU / ml) as early as 48 h, and the inhibitory effect remained up to 72 h. On the other hand, L. rhamnosus did not inhibit the growth of S. mutans streptococci except under the condition of 1% (w / v) glucose. The dynamic changes in microbial composition in each condition were plotted. In biofilms treated with L. salivarius 11741, L. plantarum 8014, and L. plantarum 14917 (1% (w / v) sucrose and 1% (w / v) glucose conditions), Lactobacillus became the dominant species after 48 h. The pH of the culture medium was significantly lower with the addition of Lactobacillus compared to the control at 24, 48, and 72 h (p<0.05). Example 4

[0071] Inhibition of cariogenic biofilm formation by L. plantarum L. plantarum 8014 and 14917 demonstrated better inhibition of C. albicans and S. mutans in planktonic and biofilm conditions, and therefore these two strains were advanced for biofilm structure analysis. L. plantarum 8014 and 14917 significantly reduced cariogenic biofilm formation measured by bacterial and EPS biomass and biofilm dry mass compared to the control group (C. albicans-S. mutans bispecies biofilm) (p<0.05). The 72-hour biofilm is shown in Figure 2, Panel A, and the dynamic changes of biofilm formation from 24 to 72 hours are shown in Figure 2, Panel B. The vertical distribution of bacteria and EPS further demonstrates the altered biofilm assembly (Figure 2, Panel C). The control group formed the thickest biofilm under 1% sucrose conditions, with the majority of the biofilm accumulating approximately 150–250 μm above the biofilm-HA disk interface. Conversely, the biofilm treated with L. plantarum 14917 was the thinnest and had the least horizontal coverage, with approximately 15% coverage and 19% EPS in the most abundant layer (20 μm above the biofilm-HA disk interface).

[0072] Microcolonies are considered to be the pathogenic and functional structures of biofilms. Surface-attached and free-floating microcolonies were identified in the biofilms. Well-formed mushroom-like microcolonies were formed in the control group (Figure 2, Panel A). Microcolonies formed with the addition of L. plantarum 14917 were less structured and had fewer bacterial components entangled with EPS (Figure 2, Panel E) (p<0.05). Furthermore, biofilms treated with L. plantarum 14917 had significantly fewer surface-attached and free-floating microcolonies and were reduced in size (Figure 2, Panel F). Example 5

[0073] Inhibition of the growth of Candida albicans and Streptococcus mutans by L. plantarum supernatant The supernatant of L. plantarum 14917 demonstrated antibacterial and antifungal activity against Candida albicans and Streptococcus mutans. Specifically, the supernatant of L. plantarum 14917 showed antibacterial and antifungal activity against Candida albicans and Streptococcus mutans under 1% sucrose conditions at 10 4 It inhibits the growth of mutans streptococci at starting concentrations of less than CFU / ml and inhibits the growth of 10 1 It inhibited the growth of Candida albicans at starting concentrations below 100 CFU / ml. The supernatant of L. plantarum 8014 had no inhibitory effect on Candida albicans. The inhibitory effect was identified as bacteriostatic and fungistatic. Example 6

[0074] Transcriptomic analysis Principal component analysis (PCA) and hierarchical clustering analysis showed unique transcriptomic profiles of biofilms treated with L. plantarum 14917. Overall, 441 genes of mutans streptococcus and 232 genes of C. albicans had differential expression between L. plantarum 14917-treated multi-species biofilms and the control group, while 391 genes of L. plantarum 14917 were differentially expressed between the multi-species group and the L. plantarum 14917 single-species biofilm (Figures 11-13). Notably, genes related to resistance to antifungal drugs (ERG4), fungal cell wall chitin remodeling (CHT2), and resistance to oxidative stress (CAT1) in C. albicans were significantly downregulated when treated with L. plantarum 14917.

[0075] KEGG pathway analysis was further performed with 441 mutans streptococcus DEGs, 232 Candida albicans DEGs, and 391 L. plantarum 14917 DEGs, resulting in 33 pathways in mutans streptococcus, 66 pathways in Candida albicans, and 31 pathways in L. plantarum 14917. Transcriptomics analysis revealed that the addition of L. plantarum disrupted the interkingdom interactions of mutans streptococcus and Candida albicans. Genes in mutans streptococcus and Candida albicans involved in metabolic pathways (e.g., EPS formation, carbohydrate metabolism, glycan biosynthesis and metabolism) were significantly downregulated. In contrast, L. plantarum 14917 genes in pathways of genetic information processing, environmental information processing, cellular processes, and metabolism (lipids, carbohydrates, glycans, energy) were significantly upregulated.

[0076] To determine the transcriptomic dynamic changes in genes of interest during specific stages of biofilm formation, especially with a significant decrease in pH value in the culture medium, qRT-PCR was performed on biofilms at 50 and 52 h (2 and 4 h after culture medium change). Mutans Streptococcus genes related to EPS formation (gtfB and gtfC) were significantly downregulated at 50 h. Candida albicans genes related to resistant fungal cell wall chitin remodeling (CHT2), and resistance to oxidative stress (CAT1) were also significantly downregulated after culture medium change. Lactobacillus genes plnD, plnG, and plnN, which contribute to the antimicrobial peptide plantaricin, were significantly upregulated.

[0077] The study results revealed the antimicrobial properties of overnight culture supernatants of L. plantarum. Furthermore, the study demonstrated dose-dependent inhibition of L. plantarum against the growth of Streptococcus mutans and Candida albicans, and a threshold concentration (10 8CFU / ml were required. Ecological shifts in the microbial community were observed in the model. High doses of L. plantarum (≥ 10 8 Despite the inhibition of Streptococcus mutans and Candida albicans by 10 CFU / ml, the low dose of L. plantarum (10 4~6 CFU / ml) promoted the growth of mutans streptococci and Candida albicans in planktonic conditions on 1% glucose, indicating a mechanistic interaction between L. plantarum and other species. Of the four Lactobacillus species tested, L. plantarum 14917 showed superior inhibitory properties, while L. rhamnosus, a commonly used probiotic in commercial products, failed to inhibit the growth of Candida albicans and mutans streptococci in cariogenic biofilms. In addition to its antifungal and antibiofilm activity observed in this study, L. plantarum has various potential pharmaceutical uses to prevent and treat respiratory diseases, irritable bowel syndrome, depression, etc. Without being bound by theory, the mechanism of action may be related to the production of plantaricin, altered fitness and virulence of Streptococcus mutans, altered virulence of Candida albicans, production of other antimicrobial products such as hydrogen peroxide and lactic acid, and sugar metabolism upon addition of L. plantarum 14917. Example 7

[0078] Effect of the probiotic L. plantarum on clinical isolates of Streptococcus mutans and Candida albicans from children with early childhood caries This study was designed in six steps to screen the best performing probiotic Lactobacillus species against Streptococcus mutans and Candida albicans clinical isolates. The study scheme is shown in Figure 8. In step 1, the inhibitory effects of Lactobacillus were evaluated against Candida albicans and Streptococcus mutans from two S-ECC children in planktonic state. The best performing Lactobacillus was carried forward to step 2 to verify its inhibitory effect against Candida albicans and Streptococcus mutans from eight more S-ECC children. In step 3, two of the three Lactobacillus species with higher inhibition against Streptococcus mutans and Candida albicans in planktonic state were further tested with Candida albicans and Streptococcus mutans isolated from two S-ECC children in multi-species biofilm state. In step 4, the better performing Lactobacilli were advanced to step 5 to evaluate their effect on cariogenic biofilm structure. Furthermore, molecular assays were used to evaluate the mechanistic interactions between Lactobacilli, Streptococcus mutans, and Candida albicans in the biofilms in step 5. Finally, the effect of plantaricin, an antimicrobial peptide produced by L. plantarum, on the growth of Streptococcus mutans and Candida albicans was examined in step 6. Example 8

[0079] Characteristics of children with S-ECC who isolated Streptococcus mutans and Candida albicans The demographics-socioeconomic-oral health status of S-ECC children from whom Candida albicans and S. mutans were isolated are shown in Table 1. The S-ECC children were 3.5 ± 1.0 years old and there were equal numbers of boys and girls. The majority of children brushed their teeth daily and did not attend day care. The plaque index was 1.8 ± 0.6. The mean number of carious teeth and carious surfaces was 11.7 ± 5.1 and 27.2 ± 17.4, respectively.

[0080] [Table 1] Example 9

[0081] L. plantarum 14917 inhibited the growth of clinical isolates of Streptococcus mutans and Candida albicans in the planktonic state The growth of C. albicans, S. mutans, and L. plantarum 14917 in the multispecies planktonic condition is plotted in Figure 3. Clinical strains of C. albicans and S. mutans were isolated from 10 children with early childhood caries (ECC). Experiments were repeated in triplicate. Each planktonic multispecies condition contained C. albicans and S. mutans isolated from the same ECC children, plus L. plantarum 14917. The control group consisted of C. albicans and S. mutans only. L. plantarum inhibited the growth of C. albicans by <1 log at 6 hours and approximately 2 log at 20 hours (p<0.05 at 20 hours). L. plantarum significantly inhibited the growth of mutans streptococci by 1 log at 6 hours and completely inhibited the growth of mutans streptococci after 20 hours (p<0.05 at 20 hours). L. plantarum maintained stable growth during 20 hours of interaction with mutans streptococci and Candida albicans. The pH of the culture medium was rapidly decreased with the addition of L. plantarum in the planktonic state, and both groups reached the same acidic level at 20 hours.

[0082] Of note, among the three Lactobacillus species tested in the screening step against Candida albicans and S. mutans isolated from two S-ECC children, the inhibitory effects of L. plantarum 14917 and L. salivarius were similar but superior to L. plantarum 8014 (Figure 9). Example 10

[0083] L. plantarum 14917 inhibited biofilm formation by clinical isolates of Streptococcus mutans and Candida albicans During the screening step, L. plantarum 14917 and L. salivarius 11741 were added to biofilms formed by mutans streptococci and Candida albicans isolated from two S-ECC children, respectively. Both L. plantarum 14917 and L. salivarius 11741 inhibited the growth of Candida albicans and S. mutans streptococci (Figure 10). Both L. plantarum 14917 and L. salivarius 11741 became the dominant species after 48 h of incubation, but at 24 h, L. plantarum 14917 had a higher composition (40%, Figure 10, panel E) compared to L. salivarius 11741 (20%, Figure 10, panel F). Furthermore, growth of L. plantarum 14917 within multispecies biofilms remained 2 logs higher at 72 h than L. salivarius 11741 (Figure 10, panel C). For the reasons discussed above, L. plantarum 14917 was advanced for evaluation of its effects on biofilm structure and for evaluation of mechanistic interactions.

[0084] The growth of Candida albicans and S. mutans was significantly inhibited by L. plantarum 14917 and is plotted in Figure 4. In the group treated with L. plantarum 14917, C. albicans was reduced by 3 logs compared to the control group. At 48 hours after two doses of L. plantarum 14917, S. mutans was completely inhibited in the biofilm of the treated group. L. plantarum was reduced during the first 24 hours due to the low sucrose (0.1%) condition and kept stable growth in the high sucrose (1%) condition after 24 hours. The pH of the culture medium was significantly lower at 24, 48, and 72 hours with the addition of L. plantarum 14917 compared to the control group (p<0.05). L. plantarum 14917 became the dominant species after 48 hours of incubation. Example 11

[0085] L. plantarum 14917 altered the 3D structure of biofilms formed by Streptococcus mutans and Candida albicans clinical isolates As L. plantarum 14917 demonstrated better inhibition of C. albicans and S. mutans isolates in planktonic and biofilm conditions, we proceeded to evaluate its effect on biofilm structure and evaluate mechanistic interactions. L. plantarum 14917 significantly reduced cariogenic biofilm formation, as measured by bacterial and EPS biomass, compared to the control group (C. albicans-S. mutans bispecific biofilm). In contrast to the complex and thick biofilms formed in the control group, biofilms treated with L. plantarum 14917 significantly reduced biofilm thickness and both bacterial and EPS biomass (Figure 5, Panels A and B, p<0.05). The horizontal coverage of the control group was also much more extensive than that of the treatment group, with nearly 60% bacterial coverage and 40% EPS coverage in the most abundant layer (approximately 30-40 um above the substrate), while the treated group only had 20% coverage in the most abundant layer (approximately 10 um above the substrate). Furthermore, EPS-localized bacteria were significantly less abundant in the treatment group (p<0.05).

[0086] Furthermore, the addition of L. plantarum 14917 also had a significant effect on microcolony formation in 72-h multi-species biofilms. In contrast to well-formed mushroom-like microcolonies in the control group, biofilms treated with L. plantarum 14917 had significantly impaired microcolony structure. Microcolonies are considered to be pathogenic and functional structures of biofilms. Surface-attached and free-floating microcolonies were quantified, and their numbers and sizes were compared between control and L. plantarum 14917-treated biofilms. Numerous and large microcolonies were detected in the control group, while L. plantarum 14917 intervention resulted in fewer and smaller sized microcolonies (Table 2).

[0087] [Table 2] Example 12

[0088] Plantaricin inhibited the growth of clinical isolates of Streptococcus mutans and Candida albicans Streptococcus mutans (3.6 × 10 3 CFU / ml) and Candida albicans (3.1 × 10 1 CFU / ml were treated with plantaricin ranging from 0 to 400ng / ml and grown for 24 hours in 1% glucose. The MICs of plantaricin were 400ng / ml for Streptococcus mutans and 200ng / ml for Candida albicans. Clear cultures were plated and further incubated for 48 hours. Results showed that the inhibitory effect on Streptococcus mutans and Candida albicans was bacteriostatic and fungistatic. Example 13

[0089] L. plantarum 14917 down-regulated virulence genes of Candida albicans and Streptococcus mutans in biofilms Expression of mutans streptococcus genes associated with oncogenicity (gtfB, gtfC, and atpD) was reduced by approximately 50% in 72-h biofilms treated with L. plantarum 14917 compared to the control group (see Figure 7). Example 14

[0090] Inhibition of hypha formation of Candida albicans by L. plantarum When treated with L. plantarum 14917, inhibition of the yeast to hyphal morphology switch of Candida albicans was observed in the planktonic state (Figure 6).

[0091] L. plantarum 14917 demonstrated equal efficacy in inhibiting clinical isolates of Candida albicans and S. mutans from children with S-ECC compared to wild-type reference strains, indicating that L. plantarum 14917 has strong potential to be incorporated into future clinical regimens for caries prevention and control from targeting cariogenic pathogens.

[0092] The findings of the study showed a significant inhibitory effect of L. plantarum 14917 against Streptococcus mutans and Candida albicans clinical isolates resulting in a reduced biofilm structure with significantly less microorganisms and extracellular matrix as well as a less pathogenic microcolony structure. Mechanistic evaluation showed that L. plantarum 14917 had a positive inhibitory impact on the expression of virulence genes of Streptococcus mutans and Candida albicans as well as on pathogenic structures such as hypha formation of Candida albicans. Future utilization of L. plantarum 14917 and / or its antimicrobial peptide plantaricin may bring about a new paradigm shift in the prevention of dental caries.

[0093] While various embodiments have been described above, it should be understood that such disclosure is presented by way of example only, and not of limitation. Thus, the breadth and scope of the subject compositions and methods should not be limited by any of the above-described exemplary embodiments, but should be defined only in accordance with the following claims and their equivalents.

[0094] The above description is intended to teach those skilled in the art how to carry out the invention, and is not intended to detail all obvious modifications and variations that will become apparent to a skilled artisan upon reading the description. However, it is intended that all such obvious modifications and variations be included within the scope of the present invention, as defined by the following claims. Unless the context specifically indicates otherwise, the claims are intended to cover the components and steps in any sequence that is effective to fulfill its intended purpose.

Claims

1. A pharmaceutical composition for preventing the establishment or inhibiting the formation of bacterial biofilms, comprising: (1) one or more species of Lactobacillus, and / or (2) Plantaricin Contains the one or more Lactobacillus species are selected from the group consisting of L. rhamnosus ATCC 2836, L. plantarum ATCC 8014, L. plantarum ATCC 14917, and L. salivarius ATCC 11741; Bacterial-based biofilms include bacteria and yeast; Pharmaceutical compositions.

2. The pharmaceutical composition of claim 1 , wherein a bacterial biofilm is formed on the surface of a tooth.

3. The pharmaceutical composition of claim 1 , which is for oral administration.

4. The pharmaceutical composition of claim 1, wherein the bacterial biofilm comprises Streptococcus mutans (S. mutans) and / or Candida albicans (C. albicans).

5. 2. The pharmaceutical composition of claim 1, wherein the one or more Lactobacillus species includes L. plantarum ATCC 14917.

6. The pharmaceutical composition of any one of claims 1 to 5, further comprising one or more additional agents that inhibit biofilm formation, Optionally, the one or more additional agents that inhibit biofilm formation include an antifungal agent and / or nystatin. Pharmaceutical compositions.

7. A pharmaceutical composition for preventing or treating a biofilm-associated disease, comprising: (1) one or more species of Lactobacillus, and / or (2) Plantaricin Contains the one or more Lactobacillus species are selected from the group consisting of L. rhamnosus ATCC 2836, L. plantarum ATCC 8014, L. plantarum ATCC 14917, and L. salivarius ATCC 11741; Bacterial-based biofilms include bacteria and yeast; Pharmaceutical compositions.

8. the biofilm-associated disease is selected from the group consisting of dental caries, oral yeast infection, periodontal disease, peri-implantitis, and burning mouth syndrome; Optionally, the dental caries is early childhood caries (ECC), The pharmaceutical composition of claim 7.

9. The pharmaceutical composition according to claim 7, wherein the biofilm-associated disease is caused by a biofilm based on bacteria including Streptococcus mutans and / or Candida albicans.

10. 8. The pharmaceutical composition of claim 7, wherein the one or more Lactobacillus species includes L. plantarum ATCC 14917.

11. Further comprising one or more additional agents that inhibit biofilm formation, 8. The pharmaceutical composition of claim 7, wherein optionally, the one or more additional agents that inhibit biofilm formation include an antifungal agent.

12. 1. A pharmaceutical composition for administration to the mother of a newborn subject during pregnancy to prevent the occurrence of early childhood caries (ECC) in the newborn subject, comprising: A pharmaceutical composition comprising one or more Lactobacillus species selected from the group consisting of L. rhamnosus ATCC 2836, L. plantarum ATCC 8014, L. plantarum ATCC 14917, and L. salivarius ATCC 11741.

13. one or more Lactobacillus species selected from the group consisting of Lactobacillus rhamnosus ATCC 2836, Lactobacillus plantarum ATCC 8014, Lactobacillus plantarum ATCC 14917, and Lactobacillus salivarius ATCC 11741; a pharmaceutically acceptable carrier; A pharmaceutical composition comprising: The pharmaceutical composition is formulated as a mouthwash, dental gel, or dental coating.

14. having a pH value in the range of 4 to 7; and / or 13. The pharmaceutical composition of claim 12, comprising 1% sugar (w / v).

15. 15. The pharmaceutical composition of claim 13 or 14, comprising L. plantarum ATCC 8014 and L. plantarum ATCC 14917 in a CFU ratio of L. plantarum ATCC 8014:L. plantarum ATCC 14917 ranging from 1:10 to 10:1.

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