Strains and culture conditions
Oral probiotics, along with their culture conditions listed in supplementary table S1, were isolated and preserved in our laboratory, potential pathogenic bacteria including Prevotella copri CNGBCC1802008 (P. copri), Porphyromonas gingivalis PG (P. gingivalis), Fusobacterium nucleatum ATCC25586 (F. nucleatum), Streptococcus mutans XS30M7 (S. mutans), Escherichia coli NCTC12900 (E. coli), Staphylococcus aureus (S. aureus), Candida albicans (C. albicans), Pseudomonas aeruginosa PAO1 (P. aeruginosa) used in this study were purchased and preserved in our laboratory.
Lactobacillus and Bifidobacterium were grown in MRS medium (HuanKai Microbial) at 37℃ without agitation overnight in an anaerobic environment (80%N2, 10%H2, 10%CO2) for 24hr. Streptococcus were grown aerobically in tryptic soy broth (TSB, HuanKai Microbial) at 37℃ for 24hr. F. nucleatum and P. gingivalis were grown anaerobically in brain heart infusion broth (BHI broth, OXOID) supplemented with 0.1% L-Cysteine hydrochloride monohydrate (Sangon Biotech), 10 µm/mL chlorohemin (Solarbio, IC1380) at 37℃ for 72 h. S. mutans were grown aerobically in BHI broth supplemented with 10 µm/mL chlorohemin at 37℃ for 24 h. E. coli, S. aureus, C. albicans and P. aeruginosa were grown aerobically in LB broth at 37℃ for 16 h.
All the strains were inoculated on growth medium agar under aerobic/anaerobic conditions at 37℃. The solitary colony was picked to growth broth and incubated at 37℃ for a suitable time respectively. After subculturing twice, the inoculum was adjusted to an OD600 of 1.0 (~ 109 CFU/mL) using their growth medium for bacterial suspension.
The 5% bacterial suspension was inoculated in MRS broth, modified MRS broth with cholic acid added to adjust the pH to 2.0, and MRS broth containing 0.3% choline salt, respectively. The viable cell counts were measured after two hours of treatment.
Isomalt, fructooligosaccharide, xylitol, mint powder, mango juice powder, resistant dextrin, inulin, isomitose oligosaccharides, onion extracts, xylooligosaccharide, galactooligosaccharide (Guangzhou Zhier Chuangyan Biotechnology Co., Guangzhou Hengyuan Biotechnology Co.) was dissolved in distilled water at a concentration of 0.05g/mL each and filtered through a 0.22um filter membrane to create the prebiotic solution. Bacteria suspension was diluted 20 times with growth medium adding 10% prebiotic solution, in which the same bacteria diluent without prebiotics was used as the control. After 24 hours of culture, the bacteria in each sample were detected for comparison.
The transwell co-culture system was used to evaluate the interaction of two bacterial strains. A 0.22-µm chamber was used to establish a co-culture system of strains A and B so that they would not migrate through the polycarbonate membrane. The A and B were inoculated in the upper and lower chamber respectively. The upper layer was inoculated with 200 µL suspension of A (inoculated 5%), the lower layer was inoculated with 700 µL suspension of B (inoculated 5%), and the lower layer was inoculated with 700 µL blank medium of B as the control [29].
Co-aggregation experiment
Equal volumes of suspension of the probiotics and oral pathogen were mixed and incubated at 37℃ with agitation at 110 rpm/min, while they were incubated respectively as the control group. After 30 min, the inoculums were statically cultured at room temperature for 3 minutes. Then, we took 200 µL of supernatant and measured its absorbance at 600 nm with a microplate reader (TECAN, SPARK, 10M). The co-aggregation ratio (%) was calculated as follows [30, 31]:
Condensation (%) = [(AProbiotics + Apathogenic bacteria) /2-AMIX] / [ (AProbiotics + Apathogenic bacteria) /2] ×100
Hydrogen peroxide (H2O2) estimation
The probiotics suspension was centrifuged at 5000 rpm for 10 min at 4℃ and take the supernatant. Subsequently, the supernatant was neutralized to pH 7.0 with NaOH and filtered through a 0.45-µm membrane, the filtrate was assayed based on colorimetry using a hydrogen peroxide assay kit (Abcam, Cambridge, MA, USA, ab102500). The optical density was read at 570 nm by a microplate reader (TECAN, SPARK 10M) using 100 µL of supernatant placed in a 96-well enzyme-linked immunosorbent assay (ELISA) microplate [32].
Antibacterial activity
The inhibition of pathogenic bacteria by probiotics was detected using the double-layer agar method. 1 mL of probiotics suspension was added into 10 mL of solid MRS medium cooled to 50℃ (with an agar content of 1.5%). The mixture was then shaken and poured into a 60 mm petri dish. A plate full of candidate bacteria was obtained after incubating anaerobically at 37℃ overnight, afterward drilled holes with a 1 mL pipette tip for the probiotics cake. We poured blank solid medium (1.5% agar) into the plates as the lower layer. After the lower layer of the medium has solidified, semi-solid medium (0.75% agar) containing 1% pathogen suspension was poured into the plate as the upper layer, and a 1 mL pipette tip was used to punch holes in the double-layer agar plate. The probiotic cake was then placed into each hole created by removing pathogenic bacteria from the double-layered plate, which was cultured overnight at 37℃ to assess its inhibitory effect on pathogens [30, 33–35].
In vitro assay for antibiofilm formation
The probiotics and pathogenic bacteria suspensions were diluted 20 times with their corresponding growth medium, 200 µL of probiotics diluent was added to the upper layer of the 24-well transwell plate, and 1 mL of pathogenic diluent was added to the lower layer, an equal volume of MRS broth was added to the upper layer as the control. After incubation at 37℃ for 48h, the lower plate wells were washed with sterilized distilled water and air-dried for 10 min, and then stained with 1 mL 0.1% crystal violet aqueous solution (BBI, E607309-0100) for 15 min. After staining and washing two times with sterile water, the biofilm formed on the side of each well was dissolved in 1 mL DMSO (dimethyl sulfoxide) and measured at 570 nm using a microplate reader (TECAN, SPARK 10M)[31, 34–36].
In vitro assays for H2S, NH3 and indole inhibition
Probiotics inoculum was filtered through a 0.22-µm membrane and added to oral pathogens suspension at a proportion of 20%, an equal volume of MRS broth was used in place of the probiotic inoculum as a control. After anaerobic incubation at 37℃ for 48 h, the gas in each anaerobic tube was sucked with a syringe. The content of hydrogen sulfide and ammonia in the gas was measured with hydrogen sulfide and ammonia detector (BOSEAN ELECTRONIC, BH-90) [31]. The culture was centrifuged at 8000 rpm for 5 min at 4℃ and 2 mL of the supernate was evenly mixed with 1.2 mL of Ehrlich Reagent (Solarbio, G1290). Then, the mixture was heated in a water bath at 40℃ for 10 min and then cooled quickly with running water. Next, 200 µL of the reaction solution was measured at 564 nm with microplate readers (TECAN, SPARK 10M).
Rats and experimental procedures
A total of twenty-five SPF 6-week-old male SD healthy rats, weighing 180 ± 20 g, were individually maintained in ventilated cages under controlled environmental conditions [room temperature (21 ± 2℃) with 40 ± 5% humidity and controlled 12-h light/dark cycle and were fed total pathogen-free diet and water ad libitum].
After 3 days of adaptive feeding, 20 rats were randomly selected and anesthetized by intraperitoneal injection of 10% chloral hydrate at a dose of 300 mg/kg to establish the periodontitis model [37, 38], while the remaining 5 rats were used as the normal control group. The bilateral maxillary second molars of the rats were ligated with absorbable medical silk thread (4 − 0), which was placed into the gingival sulci as far as possible. If the thread fell off, it was necessary to ligate again. After ligation, the whole mouth was coated with P. gingivalis (OD600 = 0.8-1) wrapped in 2% sodium carboxymethyl cellulose solution every other day, and fed with the high sugar diet (Keyes diet 2000).
On the fourteenth day of modeling, the model of periodontitis in rats was determined by PPD index, B. animalis ZK-77, L. salivarius ZK-88 and S. salivarius ZK-102 were cultured anaerobically at 37℃ overnight for 100 mL, then centrifuged and resuspended in 100 mL saline to prepare the probiotics suspension for the experimental group. Then, 20 rats with periodontitis were randomly assigned to four groups (n = 5/group). Group M + B (B. animalis ZK-77), M + L (L. salivarius ZK-88) and M + S (S. salivarius ZK102) received a single probiotic intervention each, group M was given normal saline as a model control, and group N was the normal control group without any treatment. The rats were fed with probiotics suspension every two days while being given a normal diet.
Euthanasia and collection of plasma
After two weeks of intervention, the animals were euthanized with a lethal dose (150 mg/kg body weight) of sodium thiopental at the end of the experiment. 500 µL of tail venous blood was collected into a 1.5 mL centrifuge tube pretreated with heparin, kept at room temperature for 30min, and then centrifuged at 5000 rpm for 2min, the upper plasma was taken and stored at -80℃ until testing.
Cytokine measurements
The concentration of cytokines IL-6, IL-10, MCP, IFN-γ, tumor necrosis factor (TNF) and IL-12p70 in the plasma samples were measured using the BD™ Cytometric Bead Array (CBA) Mouse Inflammation Kit (cat. no.552364, BD Biosciences). Protocols were performed according to the manufacturer’s instructions. Briefly, 50 µL of the sample was mixed with 50 µL of the mixed capture beads and 50 µL of the mouse detection reagent. After incubation at RT for 2 h in the dark, the samples were washed and suspended in 300 µL of wash buffer and acquired on CytoFLEX LX Flow Cytometer (Beckman Coulter, USA). At least 10,000 events were collected for each sample. The data were analyzed using the CBA analysis software (FCAP Array; BD Biosciences). The concentration of each cytokine was calculated using the corresponding standard curve.
Periodontitis measurement and microcomputed tomography (Micro-CT) analysis
During the intervention period, the periodontal probing depth (PPD), bleeding on probing (BOP) and weight of each rat were observed in the 1st, 3rd, 5th, 7th and 14th days
Alveolar bone specimens were fixed in 4% paraformaldehyde (PFA) for 24–48 h and sent for section-based scanning. The alveolar bone of rats was scanned by CT imaging, the scanning parameters included a 360° rotation with a spatial resolution of 39um, and 3D images were obtained through reconstruction.
16S rRNA sequencing data analysis
After probiotics intervention, 1 mL of saline was injected into the mouth of rats, and then the fluid was sucked out and frozen at -80°C immediately. Genomic DNA from gingival crevicular fluid of different groups was extracted by CTAB or SDS and PCR amplified with primers for 16S V4 region (515F and 806R), library construction using the TruSeq® DNA PCR-Free Sample Preparation Kit library building kit and sequencing using Novaseq PE250. After obtaining the raw data, the reads were spliced using FLASH to obtain the raw Tags data (Raw Tags); the assembled Raw Tags needs to be strictly filtered to obtain high-quality Tags data (Clean Tags). Referring to the Tags quality control process of Qiime, we performed the following operations: a) Tags intercept: the first low quality base site of Raw Tags from continuous low-quality value (default quality threshold is < = 19) bases to a set length (default length value is 3); b) Tags length filtering: Tags data set obtained after Tags interception, further filter out Tags with continuous high quality base length less than 75% of the Tags length. After the above treatment, the Tags need to remove the chimera sequence. The Tags sequence is compared with the Gold database by UCHIME Algorithm, and the chimera sequences are finally removed to obtain the final valid data (Effective Tags). All Effective Tags were clustered by Uparse software. By default, the sequences were clustered as OTUs (Operational Taxonomic Units) with 97% consistency. Meanwhile, the representative sequences of OTUs were selected. According to the algorithm principle, the sequence with the highest frequency in OTUs was selected as the representative sequence of OTUs. Species annotation of OTUs, species annotation analysis by Mothur method with SSUrRNA database (set threshold of 0.8 ~ 1), taxonomic information was obtained and the community composition of each sample was counted at each taxonomic level: kingdom, phylum, class, order, family, genus and species. Phylogenetic relationships with all OTUs were obtained using PyNAST software for rapid phylogenetic alignment with the "Core Set" data information in the GreenGene database. Finally, the data of each sample were homogenized, using the standard of the least amount of data in the sample.
Cats and experimental procedures
Equal amounts of B. animalis ZK-77 and L. salivarius ZK-88 (total 5×109 CFU/mL) were combined with 10% fructooligosaccharides. The cats drank probiotics combination water at regular intervals every day. The mouth of cats was photographed on 0 d, 7 d, 14d and 21 d and scored.
Scoring halitosis and teeth color in cats
After the intervention, the cat’s oral odour was checked at intervals of more than 30 minutes. The examiner will score the cat's oral odour from a distance of 10 cm using organoleptic scoring [39], 0, absence of odour, the odour cannot be detected, 1, questionable odour, where odour is detectable, although the examiner could not recognize it as malodour, 2, slight malodour, where odour is deemed to exceed the threshold of malodour recognition, 3, moderate malodour, malodour is definitely detected, 4, strong malodour, where strong malodour is detected, but can be tolerated by examiner, 5, severe malodour, overwhelming malodour is detected and cannot be tolerated by the examiner (examiner instinctively averts the nose). Tooth color cards (Vita) were used to assess the tooth color of cats before and after probiotic combination intervention.
Whole genome sequence analysis of B. animalis ZK-77 and L salivarius ZK-88
After obtaining the whole genome second-generation sequencing data of the two strains, we first quality controlled the raw sequencing reads using fastqc software and filtered the data according to the report content of the web page. Later, the raw sequencing data were filtered using fastp software to remove the low-quality reads and adapter to obtain cleandata. The cleandata obtained by fastp filtering was used for sequence splicing directly. Using megahit to perform sequence splicing to obtain the contigs. After obtaining contigs, we used abricate and to predict the upper virulence factors and antibiotic resistance genes of the genome according to the VFDB and CARD databases, and used prokka to rapidly annotate the prokaryotic genome with prokka. To annotate the function of protein coding genes, we used the blastKOALA server to annotation CDS by align with the Kyoto Encyclopedia of Genes and Genomes (KEGG) database. In addition, we also used the eggNOG-mapper to annotate CDS by the alignment with the Clusters of Orthologous Groups (COGs) database.
Statistical Analysis
In multi-target screening, each index was meticulously evaluated and scored using a standardized system of 5 equal points, ensuring that each aspect was given the same level of importance. The scores were then tallied up to determine the total score for each strain.
The values of measurements were expressed as means ± SEM. Comparisons between the two groups were performed using an unpaired two-tailed Student’s t-test. The two-way ANOVA was used to evaluate differences between more than two groups. Results were considered statistically significant at * P < 0.05, ** P < 0.01, *** P < 0.001, and **** P < 0.0001.