11-Inhibitory and preventive effects of Lactobacillus plantarum FB-T9 on dental caries in rats - Farmacologia I (2024)

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Full Terms & Conditions of access and use can be found athttps://www.tandfonline.com/action/journalInformation?journalCode=zjom20Journal of Oral MicrobiologyISSN: (Print) (Online) Journal homepage: www.tandfonline.com/journals/zjom20Inhibitory and preventive effects of Lactobacillusplantarum FB-T9 on dental caries in ratsQiuxiang Zhang, Sujia Qin, Yin Huang, Xianyin Xu, Jianxin Zhao, Hao Zhang &Wei ChenTo cite this article: Qiuxiang Zhang, Sujia Qin, Yin Huang, Xianyin Xu, Jianxin Zhao, HaoZhang & Wei Chen (2020) Inhibitory and preventive effects of Lactobacillus plantarumFB-T9 on dental caries in rats, Journal of Oral Microbiology, 12:1, 1703883, DOI:10.1080/20002297.2019.1703883To link to this article: https://doi.org/10.1080/20002297.2019.1703883© 2019 The Author(s). Published by InformaUK Limited, trading as Taylor & FrancisGroup.Published online: 25 Dec 2019.Submit your article to this journal Article views: 2629View related articles View Crossmark dataCiting articles: 9 View citing articles https://www.tandfonline.com/action/journalInformation?journalCode=zjom20https://www.tandfonline.com/journals/zjom20?src=pdfhttps://www.tandfonline.com/action/showCitFormats?doi=10.1080/20002297.2019.1703883https://doi.org/10.1080/20002297.2019.1703883https://www.tandfonline.com/action/authorSubmission?journalCode=zjom20&show=instructions&src=pdfhttps://www.tandfonline.com/action/authorSubmission?journalCode=zjom20&show=instructions&src=pdfhttps://www.tandfonline.com/doi/mlt/10.1080/20002297.2019.1703883?src=pdfhttps://www.tandfonline.com/doi/mlt/10.1080/20002297.2019.1703883?src=pdfhttp://crossmark.crossref.org/dialog/?doi=10.1080/20002297.2019.1703883&domain=pdf&date_stamp=25 Dec 2019http://crossmark.crossref.org/dialog/?doi=10.1080/20002297.2019.1703883&domain=pdf&date_stamp=25 Dec 2019https://www.tandfonline.com/doi/citedby/10.1080/20002297.2019.1703883?src=pdfhttps://www.tandfonline.com/doi/citedby/10.1080/20002297.2019.1703883?src=pdfInhibitory and preventive effects of Lactobacillus plantarum FB-T9 on dentalcaries in ratsQiuxiang Zhanga,b,c, Sujia Qina,b, Yin Huanga,b, Xianyin Xud, Jianxin Zhaoa,b, Hao Zhanga,b,e,f and Wei Chena,b,e,f,gaState Key Laboratory of Food Science and Technology, Jiangnan University, Wuxi, Jiangsu, P. R China; bSchool of Food Science andTechnology, Jiangnan University, Wuxi, Jiangsu, P. R. China; cInternational Joint Research Laboratory for Probiotics, Jiangnan University,Wuxi, Jiangsu, P.R. China; dDepartment of Stomatology, Wuxi Children’s Hospital, Wuxi, Jiangsu, P.R. China; eNational EngineeringResearch Center for Functional Food, Jiangnan University, Wuxi, Jiangsu, P.R. China; fBeijing Innovation Centre of Food Nutrition andHuman Health, Beijing Technology and Business University (BTBU), Beijing, P.R. China; gWuxi Translational Medicine Research Centerand Jiangsu Translational Medicine Research Institute Wuxi Branch, Wuxi, P.R. ChinaABSTRACTStreptococcus mutans is recognized as the main cause of dental caries, and the formation ofa plaque biofilm is required for caries development. This study aimed to determine theinhibitory effect of Lactobacillus plantarum FB-T9 on S. mutans biofilm formation in vitroand on the prevention and treatment of dental caries in rats. During in vitro experiments, FB-T9 exhibited good bacteriostatic ability in a plate competition assay. This strain also signifi-cantly reduced the biomass and viability of S. mutans biofilms and induced structural damageduring the early (6 h), middle (12 h) and late (24 h) stages of biofilm formation. In a 70-dayin vivo experiment, FB-T9 significantly reduced the levels of S. mutans on the dental surfacesof rats by more than 2 orders of magnitude of the levels in the dental caries model group (p <0.05). Moreover, FB-T9 significantly reduced the caries scores (modified Keyes scoringmethod) in both the prevention and treatment groups (p < 0.05) and had great colonizationpotential in the oral cavity. These results indicate the potential usefulness of L. plantarum FB-T9 as a probiotic for the prevention and treatment of caries.ARTICLE HISTORYReceived 10 July 2019Revised 4 September 2019Accepted 7 November 2019KEYWORDSCaries; Streptococcus mutans;dental plaque; biofilm;probioticsIntroductionCaries is a common type of oral bacterial infectiousdisease. According to the World Health Organization(WHO), caries is the third most common non-communicable disease after cancer and cardiovasculardisease. According to data from a previous Lancet pub-lication [1], dental caries of the permanent teeth was themost prevalent disease afflicting humans. In 2016, cariesin permanent teeth and caries in deciduous teethranked second and fifth, respectively, among the 10diseases with the highest global incidence.The formation of a plaque biofilm of bacteria isa prerequisite for the occurrence of a caries.A mature dental plaque biofilm is a three-dimensional micro-ecological environment compris-ing various bacteria embedded in a matrix mainlycomposed of water-insoluble polysaccharides witha certain thickness. In a normal dental plaque bio-film, oral microflora exist in a dynamic equilibrium.When host, dietary or microbial growth factors alterthe ecological balance, acidifying bacteria in theplaque decrease the pH of the biofilm environment.Subsequently, the demineralization–remineralizationbalance in the tooth shifts toward mineral loss,leading to tooth decay [2]. Streptococcus mutans,which has strong acid-producing and acid-resistantcapacities, has been recognized as the main type ofcariogenic bacteria [3]. A large number of extracel-lular polysaccharides synthesized by S. mutans areimportant to the complex tri-dimensional structureof a dental plaque. Indeed, many studies havereported the relationship between the presence ofS. mutans, biofilm formation and the related riskof caries [4–6].Currently, dental plaque is mainly controlled andprevented using mechanical removal techniques andantimicrobial agents. However, tooth brushing, themost common mechanical plaque removal method,cannot fundamentally reduce the number of cariogenicbacteria such as S. mutans. Indeed, the applications ofsome mechanical treatments in pediatric and elderlypopulations are limited by personal preferences, andthe effects are relatively superficial. The use of antibio-tics is also limited by the close structure of dental plaquebiofilm that makes the bacteria embedded in this com-plex community more resistant to antibiotics comparedwith the planktonic state. Accordingly, the concentra-tion of antibiotics required to kill bacteria in a biofilm ishundreds of times higher than that required to killplanktonic bacteria [7]. In addition, the long-term useCONTACT Jianxin Zhao jxzhao@jiangnan.edu.cn State Key Laboratory of Food Science and Technology, Jiangnan University, Wuxi, Jiangsu214122, P. R ChinaJOURNAL OF ORAL MICROBIOLOGY2019, VOL. 12, 1703883https://doi.org/10.1080/20002297.2019.1703883© 2019 The Author(s). Published by Informa UK Limited, trading as Taylor & Francis Group.This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permitsunrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.http://www.tandfonline.comhttps://crossmark.crossref.org/dialog/?doi=10.1080/20002297.2019.1703883&domain=pdf&date_stamp=2019-12-25of antibiotics may destroy the balance of the oral eco-system and increase bacterial resistance. Therefore,research into caries prevention and treatment funda-mentally relies on a safe and effective caries preventionmethod.Probiotics have long been thought to contribute tohuman gastrointestinal health. To date, studies haveconfirmed that probiotics can help to prevent andtreat diarrhea due to rotavirus infection, antibioticuse, ulcerativecolitis and pouch enteritis [8,9].Probiotics can also prevent respiratory tract infec-tions and osteoporosis in middle-aged women, regu-late the balance of oral flora and prevent dentalcaries, periodontitis and other oral diseases [10–12].Previous studies have demonstrated thatLactobacillus could inhibit the growth of S. mutansbiofilm and caries-related multispecies biofilmsin vitro [13–15]. Clinical data have revealed the abil-ity of some probiotics to colonize the oral cavity [16]and significantly reduce the oral population ofS. mutans. Lee and Kim found that Lactobacillusrhamnosus LGG suppressed S. mutans biofilm forma-tion by reducing glucan production and antimicrobialactivity [17]. Skim soy milk fermented withL. paracasei subsp. paracasei NTU101 could consid-erably reduce the population of S. mutans in rats andinhibit the development and deterioration of dentalcaries [18]. Hence, the screening of candidate oralprobiotics based on their abilities to inhibitS. mutans growth and biofilm formation may repre-sent an alternative means of preventing tooth decay.In our previous study, L. plantarum FB-T9 isolatedfrom healthy human feces exhibited an excellent anti-bacterial ability against S. mutans. In this study, weevaluated the antibacterial ability of L. plantarum FB-T9 on a half-strength plate and investigated the effect ofthis strain on S. mutans biofilm formation over differenttime periods. We also examined oral S. mutans coloni-zation and caries formation in rats using bacterialcounts and caries scores to evaluate the effect ofL. plantarum FB-T9 on the prevention or treatment ofcaries in vivo. These results will support and inform thedevelopment of probiotics to prevent caries.Materials and methodsBacterial strainsL. plantarum FB-T9 was isolated from healthy humanfeces and inoculated in De Man, Rogosa and Sharpe(MRS) broth (DifcoTM, Detroit, MI, USA) underanaerobic conditions at 37°C. S. mutans ATCC25,175 was purchased from the China CommonMicrobial Species Preservation and ManagementCenter (CGMCC, Beijing China) and cultured inTryptic Soy Broth (TSB, DifcoTM, Detroit, MI, USA)under anaerobic conditions at 37°C. Another strain,L. plantarum 5D-3 (5D-3), was selected froma healthy oral cavity and used as a control strain inanimal tests. All strains were frozen in 30% (v/v)glycerol broth at −80°C and routinely streaked oncorresponding ager plates. The plates were culturedin an anaerobic incubator (Electrotek AW500SG,England) at 37°C for 48 h. A single colony wasinoculated into 5 mL corresponding broth tubesanaerobically at 37°C for 16 h and sub-culturedtwice using 2% (v/v) inoculum in 5 mL brothbefore use.Competition assay on plateThe experimental method broadly followed Tonget al. [19]. After 16 h culture in broth tubes, thebacterial cultures were centrifuged at 3000 × g for10 min and washed twice with sterile saline solution.The bacteria were then re-suspended in saline solu-tion and diluted to a suspension with 1 × 105 livingcells by colony counting prior to use. FB-T9 andS. mutans (10 µL of each) were inoculated on trypticsoy ager plates (TSA) or half-strength TSA (1/2TSA)simultaneously or sequentially. Under the formercondition, 10 µL of both strains suspension wasinoculated simultaneously and co-cultured at 37°Cfor 32 h. Under the latter condition, 10 µL of thefirst colonizer (FB-T9/S. mutans) was inoculated andgrown for 16 h, after which 10 µL of the secondcolonizer (S. mutans/FB-T9) was inoculated near tothe first organism and incubated for another 16 h. Allthe plates were cultured anaerobically at 37°C.Biofilm formation assayA biofilm formation assay was conducted as proposedby Loo, with slight modification [20]. After 16 hculture, FB-T9 and S. mutans were centrifuged andre-suspended in corresponding medium supplemen-ted with 0.2% sucrose, then diluted to a concentrationof 1 × 105 CFU/mL by colony counting as describedabove. Next, a 100-μL aliquot of S. mutans suspen-sion was added to each well of a 96-well microplate(Costar, USA) and incubated at 37°C for 24 h. To testthe intervening effects of FB-T9 on the formation ofthe S. mutans early-stage biofilm, 40 μL of FB-T9suspension were added to each well at 0, 6 or 12 hand cultured to a total time interval of 24 h. To assessthe effects of FB-T9 on S. mutans middle- andmature-stage biofilms, S. mutans was initially cul-tured for 24 or 48 h. After removing the supernatant,200 μL of FB-T9 suspension was added, and thecultures were incubated for another 24 h.Subsequently, planktonic bacteria were gently aspiredfrom the microplates, which were rinsed with sterilephysiological saline solution. Each well was thenstained with 200 μL of crystal violet for 30 min at2 Q. ZHANG ET AL.room temperature. After two rinses with saline solu-tion, 100 μL of 95% alcohol was added to release thedye. Biofilm formation was then quantified by mea-suring optical density at 600 nm (OD600) in each wellusing a microplate reader (BioTek, Winooski, VT,USA). The negative control group was treated withthe same volume of saline instead of FB-T9suspension.Biofilm viability assayAn in vitro biofilm model was established usinga cover slide as carrier [21]. A sterile cover glasswas placed in a glass Petri dish with a 6-cm diameter,to which 4 mL of a S. mutans suspension was added.At 0, 6 or 12 h, 200 μL of FB-T9 suspension wasadded to the dish, and the culture was incubated upto a total of 24 h. Otherwise, a S. mutans biofilm wasallowed to form for 24 or 48 h, after which 4 mL ofFB-T9 suspension was added. The culture was thenincubated for another 24 h before the biofilm waswashed twice with phosphate-buffered saline. Finally,the glass slides were removed and subjected to fluor-escence staining as described by Khan [22].Carboxyfluorescein diacetate, succinimidyl ester(CFDA-SE, 65-0850-84, InvitrogenTM) was used tolabel all viable bacteria with green fluorescence,while propidium iodide (PI, P3566, InvitrogenTM)was used to label in red all bacteria with damagedmembranes (i.e., non-viable bacteria). The biofilmstructure was observed using a confocal laser scan-ning microscope (CLSM; LSM 710, Zeiss, Germany),and the Z-stack analysis was performed using Zen2010 software (Carl Zeiss, Germany) [23]. The areasof viable and non-viable bacteria and the biofilmthickness were recorded. The biofilm activity wascalculated as the percentage of viable bacteria. Allcontrol groups were administered an equivalentvolume of saline instead of FB-T9 suspension.Caries reduction in vivoAnimals and general proceduresFemale SPF Wistar rats (21 days old) were purchasedfrom Charles River Laboratories (Beijing, China) andreared with approval according to the guidelines ofthe Animal Care and Use Committee at the JiangsuInstitute of Parasitic Diseases. And the procedureswere carried out in accordance with EuropeanCommunity guidelines (Directive 2010/63/EU) forthe care and use of experimental animals. All of therats were allowed to adapt to the diet and environ-ment for 3 days and were then divided randomly intoeight groups, comprising three treatment groups,three prevention groups and one group each for thenaïve controls and caries models. A flow chart ofgroup allocation is shown in Figure 1. The rats inthe dental caries control group (C) were subjected to5 consecutive days of dental caries modeling. Briefly,after 16 h culture, S. mutans cultures were centri-fuged and diluted to 108 CFU/mL in saline solution.A sterile cotton swab saturated with this bacterialsuspension then applied to the rat oral cavity for 15s per quadrant, as described by Beiraghi et al. [24].On day 6, oral swabs were spread on mitis salivariusagar plates supplemented with 200 μg/mL streptomy-cin sulfate (MMS, DifcoTM, Detroit, MI, USA) toconfirm S. mutans colonization in the oral cavity.The intervention groups were treated with chlorhex-idine (0.02%) (T1),FB-T9 (T2) or 5D-3 (T3) foranother 5 consecutive days after S. mutans coloniza-tion and thrice weekly thereafter. The preventiongroups (P1 0.02% chlorhexidine treated; P2 FB-T9treated; and P3 5D-3 treated) were first colonizedwith 108 CFU/mL of lactobacilli for 5 consecutiveFigure 1. Experimental design for evaluating the induction of dental caries in SPF rats.JOURNAL OF ORAL MICROBIOLOGY 3days and then infected with S. mutans for another 5consecutive days. The caries-free control group (CF)consumed a regular diet and water throughout the10-week experimental period. All of the other groupswere fed a cariogenic diet 2000 (obtained fromNantong Trophy Feed Technology Co., Ltd.) supple-mented with a 5% (w/v) sucrose solution.Microbial analysis and caries scoringThe rats were weighed daily to monitor growth in eachgroup. During the experiment, the rats were sampled ondays 6, 13 and 70. The collected samples were plated ontwo types of agar [Tanzer et al., 2010]: MMS agar platewas used to enumerate S. mutans against streptomycin,while MRS supplemented with 12 μg/mL vancomycinwas used to enumerate lactobacilli [Montella et al., 2013].At the end of the 10-week experimental period, the ratswere anesthetized and sacrificed. The heart, liver, spleen,lungs and kidneys were harvested and weighed. Afterdecapitation, the skull was removed and placed in anautoclave at 121°C for 15 min. The attached soft tissuewas peeled off with a scalpel, and the jawwas cleaned anddried at room temperature. All of the specimens wereimmersed in a 0.4% ammonium purpurate staining solu-tion for 12 h, rinsed and semi-sectioned along the occlu-sal surfaces of maxillary and mandibular molars usinga diamond cutter (thickness: 0.1 mm). Caries on the ratmolars was observed and evaluated under a stereomicro-scope according to the caries diagnosis and scoringmethod reported by Keyes [25].Statistical analysisSPSS Statistics 22.0 (SPSS, Inc., Chicago, IL, USA)was used for the analysis. A single factor variancewas used to analyze the experimental results (one-way analysis of variance). A p-value <0.05 was con-sidered to indicate a statistically significant difference.Origin Pro 8.5 was used to map and analyze the data.ResultsAntagonism between L. plantarum andS. mutans on a growth plateFB-T9A plate competition experiment was used to evaluatethe competitive effects of FB-T9 and S. mutans in anin vitro environment with limited space and nutri-ents. Both FB-T9 and S. mutans grew well on TSAand 1/2TSA plates (Figure 2(a–d)). When FB-T9 andS. mutans were spotted on TSA plates simultaneously,the growth of S. mutans was suppressed near the FB-T9 colony (Figure 2(e)), but this suppression was notobvious on 1/2TSA (Figure 2(f)). Interestingly, thegrowth of S. mutans was inhibited severely when FB-T9 was inoculated first on both types of media(Figure 2(g–h)). However, no obvious competitiveinhibition was observed when S. mutans was inocu-lated first (Figure 2(i–j)), indicating that although FB-T9 could inhibit S. mutans under either nutrient-richor nutrient-deficient conditions, S. mutans had noinhibitory effect on FB-T9.S. Mutans biofilm formation mediated by FB-T9in vitroAccording to a report by Nobbs and colleagues [26],the formation of an early biofilm of S. mutans can beobserved at 24 h. The formation period includes fivekey time points: 0 h, initial bacterial adherence; 6 h:Figure 2. The inhibition effect of L. plantarum FB-T9 on S. mutans. (A and B) L. plantarum FB-T9 on TSA and 1/2 TSA plates; (Cand D) S. mutans on TSA and 1/2TSA plates. Competition between FB-T9 and S. mutans: (E, G, I) on TSA plates; (F, H, J) on 1/2TSA plates; (E and F) S. mutans and L. plantarum FB-T9 were inoculated on the plates at the same time; (G and H) L. plantarumFB-T9 was first inoculated on the plates; (I and J) S. mutans was first inoculated on the plates.4 Q. ZHANG ET AL.initial bacterial colonization; 12 h: initial early biofilmformation; 24 h: maturation of early-stage biofilmand 48 h: maturation of the later-stage biofilm. Inthis study, we added the FB-T9 fermentation super-natant at these five time points to mediate the for-mation of the S. mutans biofilm. The S. mutansbiofilm biomass, total number of bacteria in the bio-film and viability of biofilm were recorded underdifferent experimental conditions.The biomass of the S. mutans biofilm increasedgradually from 0 to 48 h (Figure 3). The addition ofFB-T9 at 0, 6 and 12 h significantly influenced theformation of the biofilm (p < 0.05). The most obviousdecreases were observed at 0 and 6 h. In particular,the S. mutans biomass at 6 h was only a third of thecontrol value. Moreover, the addition of FB-T9 at 24and 48 h significantly inhibited the S. mutans biofilmbiomass (p < 0.05).CLSM was used to visualize viable and non-viablebacteria. When FB-T9 was added to the S. mutanssuspension at 0 h, no attached bacteria were observedafter a 24-h culture (Figure 4(a–b)). Meanwhile, theareas of bacteria in the biofilms of cultures treated at 6and 12 h increased gradually, indicating that the bio-films accumulated gradually before the intervention(Figure 4(c–f)). Additionally, the biofilms formed inthe negative control group cultures (24 and 48 h) weredense and concentrated, with large areas containingviable bacteria (Figure 4(g,i,k,m)). In the cultures trea-ted at 24 and 48 h, larger proportions of non-viablebacteria were observed, and biofilm structure was rela-tively loose and dispersed (Figure 4(h,j,l,n)). Visually,no obvious differences were observed between thecontrol and mediation groups at 24 and 48 h. Hence,the total bacterial area and biofilm viability werecalculated.The bacterial areas of the early-treated (0, 6 and 12 h)biofilms ranged from 0.33 to 5.34 × 104 μm2 (Figure 5),while that of the 12-h negative control biofilmwas 16.08× 104 μm2. The bacterial areas of the treated biofilmswere significantly smaller than that of the control bio-film (p < 0.05), representing a reduction of more than65%. Furthermore, the medium-term (24 h) bacterialarea decreased by more than 50% after treatment. In thelater-stage of biofilm maturation, the total bacterialareas in the 48-h mediation groups decreased onlyslightly when compared with the 48-h negative control,and this difference was not significant (p = 0.063).Interestingly, the viability of the biofilm decreased gra-dually with the time interval after the intervention,whereas the value of the negative control increasedgradually. These results indicate that earlier FB-T9mediation led to a better inhibitory effect onS. mutans biofilm formation (p < 0.05).Evaluation of a caries-induced rat modelBacterial colonization of the rat oral cavityNo significant differences in weight gain and visceralcoefficients were observed between the groups of rats(p > 0.05, data not shown), and all animals appeared tobe in good physical condition throughout the experi-ment. The concentrations of S. mutans on the molarsof the dental caries model group and the interventiongroup ranged from 1 to 3 × 105 CFU/mL (Table 1),indicating that S. mutans had successfully colonized theoral cavities after 5 days of infection. When chlorhexi-dine was applied for another 5 days (second sampling),the S. mutans concentration decreased to 0.1 × 105 CFU/mL and stabilized at 0.2 × 105 CFU/mL at the end of thetreatment (third sampling). However, population ofS. mutans after FB-T9 treatment decreased two ordersof magnitude from 3.2 × 105 to 0.03 × 105 CFU/mL(third sampling) which showed a greater extent thanchlorhexidine (p < 0.05). Interestingly, treatment with5D-3 also reduced the population of S. mutans, althoughit could not inhibit biofilm formation in vitro (dataunpublished). Briefly, no significant differences in theeffects of the interventions were observed between thethree groups at the second and third samplings.To evaluate the preventiveeffects of lactobacilli, ratswere initially inoculated with lactobacilli for 5 daysand then continuously infected with S. mutans for 5days. Compared with the initial S. mutans infection(Table 1), the S. mutans population in preventiongroups decreased by 1–2 orders of magnitude (Figure6(a), second sampling). In particular, the population ofS. mutans in the FB-T9 prevention group (P2) wassignificantly lower than that in the other two groups(p < 0.05) at the second sampling and remained at thesame level after 2 months (third sampling). In contrast,the number of S. mutans in the other two preventionFigure 3. Biomass of S. mutansmediated by L. plantarum FB-T9at different stages. C12, biofilm of negative control at 12 h. C24,biofilm of negative control at 24 h. C48, biofilm of negativecontrol at 48 h. *P < 0.05 when compared with the controltreatment and compared between groups.JOURNAL OF ORAL MICROBIOLOGY 5groups increased significantly (p < 0.05) during theexperiment (P1, P3).Initially, FB-T9 colonized the oral cavity toa concentration of approximately 105 CFU/mL(Figure 6(b)), which was significantly higher thanthat induced by 5D-3 (104 CFU/mL). Meanwhile,the number of FB-T9 initially decreased inthe second sampling but increased in the third sam-pling. It was speculated that FB-T9 may successfullycolonize the oral environment via a competitive pro-cess. However, 5D-3 did not undergo a similar pro-cess, indicating that FB-T9 was generally retainedbetter than 5D-3 in the rat oral environment.Caries lesion evaluation by Keyes scoringThe cheek, tongue and adjacent surfaces and sulcalof a rat molar can be divided into several dentalcaries units according to the extent of cariesdamage, as shown in Table 2. Whereas Table 2demonstrates how the dental caries units weredetermined and scored, the score results for thecorresponding caries are shown in Table 3. Dentincaries (Dm) occurred in the model and treatmentgroups but not in the prevention groups, whereasdeep dentin caries (Dx) did not occur in anyexperimental groups. The FB-T9 prevention group(P2, 14.7) and intervention group (T2, 23.9)received the two lowest total dental caries scores(E; 14.7 and 23.9, respectively). Both of these scoreswere less than half of the score in the model group,indicating that FB-T9 significantly prevented andtreated dental caries in vivo. Overall, caries reduc-tion was more efficient in the prevention groupsthan in the intervention groups. However, theeffect in the 5D-3 prevention group (P3) did notsurpass that in the chlorhexidine prevention group(P4), and 5D-3 (T3) had no significant treatmenteffect (p > 0.05). Interestingly, the Dm score in theFB-T9 group was significantly lower than that inthe model group (p < 0.05), whereas no significantdifference was observed between the chlorhexidineand model groups (p > 0.05). FB-T9 appeared toFigure 4. CLSM of S. mutans biofilm on glass coverslips. Addition of FB-T9 bacterial suspension at 0 h (A and B), 6 h (C and D),12 h (E and F), 24 h (H and L) and 48 h (J and N) respectively. (G and K) S. mutans biofilm after incubation for 24 h with mediumalone. (I and M) S. mutans biofilm after incubation for 48 h with medium alone. And A, C, E, G, H, I, J are corresponding 3Dgraphs.6 Q. ZHANG ET AL.have a better therapeutic effect with respect to deepcaries, whereas chlorhexidine had a more super-ficial therapeutic effect.Discussion/conclusionDental caries is associated closely with cariogenicbacterial species, such as S. mutans. Although it isimpossible to eradicate such microorganisms comple-tely, interest in the potential use of probiotics toprevent oral caries has been increasing in recentyears. The inhibitory effects of probiotics againstS. mutans may be due to antimicrobial substancesproduced by probiotics, including organic acids,hydrogen peroxide, bacteriolytic enzymes, bacterio-cins and biosurfactants [27,28]. Organic acids andhydrogen peroxide can enable probiotics to toleratecertain acidic conditions and to survive in an oralenvironment containing a certain amount oflysozyme [3]. According to Figure 2, when FB-T9was spotted first on a culture plate, it had a strongerinhibitory effect on S. mutans than when S. mutanswas spotted first, implying that substances producedby FB-T9 may contribute to its effects againstS. mutans.Oral cavity is a nutrient fluctuation environmentwhich creates scarce resources such as nutrients andspace for bacteria, so they are more likely to metabolizesome inhibitory substances to suppress the surroundingmicroorganisms to ensure their nutritional needs andsurvival, which involves a mechanism called quorumsensing [29,30]. Nutritional limitations and environ-mental fluctuations stimulate the synthesis of signalmolecules in bacterial cells. When the concentrationsof these signal molecules reach a threshold, appropriatetarget genes are transcribed and used to produce activesubstances, thus inhibiting competitive bacteria [31,32].Kreth et al. [33] observed that the nutritional richnesscan interfere with the inhibitory capacity ofStreptococcus sanguinis against S. mutans. In general,under nutrient-rich conditions, the magnitudes of inhi-bition were higher than under nutrient-limited condi-tions, except for a few species in specific environmentalconditions. One can consider that under nutrient-limited conditions, the energy expenditure of the bac-teria was employed for bacterial survival and not for theproduction of H2O2 [34]. In our study, we use 1/2 TSAto simulate the condition of nutrient deficiencies andobserved the interaction between S. mutans andLactobacillus. The result showed that FB-T9 not onlyexerted a strong inhibitory effect on S. mutans, the maincariogenic bacteria, but also exhibited a strong compe-titive advantage under nutrient-deficient conditions.Therefore, we may infer that FB-T9 is a strong compe-titor against S. mutans for temporal and spatial niches.Dental caries is well established as a representativebiofilm-dependent oral disease. Although S. mutansis not always the most abundant bacteria in the oralcavity, it is a key producer of matrix and coordinatorof cariogenic biofilm formation [35]. Many studieshave proven the effects of Lactobacillus on S. mutansbiofilms at 24 h [36–38]. To date, however, themechanism by which Lactobacillus interferes withthe formation of S. mutans biofilms at different stagesand the correlation between the biofilm biomass andactivity at different stages remains unknown. In thisFigure 5. Total bacterial area of biofilm and biofilm viabilityafter intervention by L. plantarum FB-T9 at different stages.The left axis corresponded to the histogram, showing thebacterial area of biofilm (×104 μm2). Biofilm mediated by FB-T9 at 0 h, 6 h, 12 h was compared to the 12 h negativecontrol, while biofilm mediated by FB-T9 at 24 h and 48h were compared to the 24 h and 48 h negative control,respectively. The dashed line indicated the biofilm viability ofthree controls, and the solid line indicated the biofilm viabi-lity after treatment at different stages. C12, biofilm of nega-tive control at 12 h. C24, biofilm of negative control at 24h. C48, biofilm of negative control at 48 h. The assay wasperformed three times and data are expressed as mean ±standard error of the mean. *P < 0.05 when compared withthe control treatment and compared between groups.Table 1. S. mutans counts from rat dental samples at different periods.Caries-free group (CFU/mL) Caries group(×105CFU/mL) Intervention group(×105CFU/mL)Sampling times C3 C T1 T2 T31 <30Aa 2.56 ± 0.7Ba 3.37 ± 0.8Bb 3.20 ± 2.2Bb 1.23 ± 0.36Bb2 <30Aa 1.89 ± 0.8Ba 0.10 ± 0.0Ca 0.04 ± 0.0Ca 0.06 ± 0.0Ca3 <30Aa 1.40 ± 0.7Ba 0.20 ± 0.0Ca 0.03 ± 0.0Ca 0.15 ± 0.0CaA, B and C indicated the difference in the number of S. mutans in the oral cavity of rats in each row.a, b and c showed the difference of the number of S. mutansin the oral cavity of rats sampled three times in each group.Data are presented as the mean with the standard error of the mean in parentheses following the statistical analyses of all pairs using the Tukey–Kramermultiple comparison test (n = 8).JOURNAL OF ORAL MICROBIOLOGY 7study, S. mutans biofilm formation was mediated byFB-T9 at five key time points (0, 6, 12, 24 and 48 h),as described above. The following key indicators wereused: (i) crystal violet staining to characterize thebiofilm biomass, (ii) total bacterial area and propor-tions of viable and non-viable bacteria and (iii) bio-film structure. The results showed that earliermediation yielded better effects against the biofilm.In addition, almost no biofilm formation wasobserved at 0 h mediation (Figure3-5(a)), possiblybecause the initial adhesion of S. mutans was inhib-ited by Lactobacillus. FB-T9 remained effective evenagainst an initially maturing or already matureS. mutans biofilm, as demonstrated by the inactiva-tion of all bacteria in the mediated biofilm. Lin et al.[15] also used fluorescence staining to determine theviability of S. mutans biofilm after the intervention offive Lactobacillus strains, which indicated the inhibi-tory effect of five Lactobacillus strains on S. mutansbiofilm. Moreover, the structure of the mediated bio-film in our study was looser than that of the control,suggesting that Lactobacillus had a significant effect at48 h (Figure 4). We speculated that althoughS. mutans had formed a mature biofilm at 48 h,Lactobacillus inhibited the proliferation of S. mutansin the dense biofilm and destroy the dense, cross-linked biofilm structure. Many biofilm structurescontain channels through which environmental fluidscan move. These channels often act as transport ves-sels to deliver nutrients, remove waste products andserve as conduits for messenger molecules [39].Destroying the biofilm structure will affect the phy-siological activity of the biofilm. The analysis ofCLSM suggested that the effect of biofilm inhibitionis not necessarily to reduce the bacterial area ofbiofilm, but also to destroy the structure of biofilmand reduce its viability.The inhibitory effects of Lactobacillus on cario-genic bacteria and biofilm formation observedin vitro do not necessarily mean that Lactobacillushas the same effects in vivo. Therefore, this studyfurther examined the anti-caries activity of FB-T9in a rat model of dental caries. L. plantarum 5D-3that exhibited poor inhibitory activity against bio-films in previous experiments (data unpublished),was selected as control. During a 70-day experi-mental period, S. mutans counts and caries scoresused to evaluate the decay process demonstratedthat the number of S. mutans decreased from 105to 103 CFU/mL after intervention (Table 1). Ineach intervention group, the number of S. mutansdiffered significantly in the second and third sam-pling related to the first sampling (p < 0.05), indi-cating significant reductions in the population ofS. mutans. In particular, no S. mutans was detectedin the second and third samplings after FB-T9Figure 6. S. mutans & lactobacilli count from rat dental samples at different periods in prevention groups. (A) S. mutans counts;(B) lactobacilli counts; Data are expressed as mean ± standard error of the mean (n = 8).Table 2. Number of linear units assigned to each molar.Mandible MaxillaryCaries site 1st 2nd 3rd* 1st 2nd 3rd*Buccal 6 4 4 6 4 3Lingual 6 4 4 6 4 3Sulcal 7 5 2 5 3 2Proximal 1 2** 1 1 2** 1*1st, 2nd and 3rd denote the first, second and third molars, respectively;**The second molar contains a near-middle and a far-middleneighbourhood.Table 3. Effect of different groups on caries development(incidence and severity) in rats.Group NumberCaries levelE Ds DmCaries group C3 47.6 ± 0.7 28.6 ± 1.0 15.3 ± 1.3Intervention group T1 34.8 ± 1.4* 24.7 ± 1.3* 14.7 ± 0.9T2 23.9 ± 1.0* 14.4 ± 0.9* 8.6 ± 0.8*T3 40.7 ± 1.3 27.6 ± 1.1 13.5 ± 0.7Prevention group P1 28.0 ± 0.8* 10.6 ± 0.7* –P2 14.7 ± 0.9* 8.0 ± 0.6* –P3 30.7 ± 1.5* 14.5 ± 0.8* –Data are expressed as mean ± standard error of the mean (n = 8). E,enamel caries; Ds, dentin exposed; Dm, three-fourths of the dentinaffected. * P < 0.05.8 Q. ZHANG ET AL.mediation. In Figure 6(a), the number of S. mutansafter FB-T9 colonization was significantly lowerthan the number in the other two groups, suggest-ing that FB-T9 had more effectively prevented thecolonization of S. mutans than had chlorhexidineand 5D-3. Meanwhile, the FB-T9 prevention groupreceived the lowest caries score of all of the groups(Table 3). As shown in Figure 6(b), FB-T9appeared to better prevent caries because of itsstrong ability to colonize the rat oral cavity.Previous studies showed that L. rhamnosus SD11was detected in large quantities in the oral cavityeven 4 weeks after the cessation of consumptionand had a significant inhibitory effect on the popu-lation of S. mutans in the oral cavity [40,41], whichis similar to our findings. The ability of a probioticto colonize the oral cavity is key to its function,and this ability is strain-specific. For example, 5D-3may effectively prevent biofilm formation, perhapsbecause it had the advantage of pre-colonization ofthe oral cavity (Figure 6(a,b) and Table 3). Hence,the ability of Lactobacillus spp. to colonize oralcavity should be considered an indicator of oralprobiotics. FB-T9 exhibited excellent efficacy inboth the prevention and treatment groups anddemonstrated that a potential probiotic could out-perform chlorhexidine in the treatment of dentalcaries (Table 3). Current studies have shown thatprobiotic products can reduce the population ofS. mutans in the human oral cavity [42,43].In conclusion, this study enabled us to identifyL. plantarum FB-T9 as a highly suitable probiotic.This bacterium antagonized S. mutans in vitro,inhibited biofilm formation and reduced viabilityin the biofilm at different stages of formation.Moreover, the in vivo animal experiments revealedthat FB-T9 could significantly reduce the popula-tion of S. mutans in the rat oral cavity, as well asthe caries score. It appears that L. plantarum FB-T9is a potential new oral probiotic, which can befurther developed into a probiotic preparationwith practical application value to prevent dentalcaries.Disclosure statementNo potential conflict of interest was reported by theauthors.FundingThis work was supported by the National Key R&DProgram of China [2017YFD0400600], the NationalNatural Science Foundation of China [No. 31820103010,31530056], National First-class Discipline Program of FoodScience and Technology [JUFSTR20180102], andCollaborative innovation center of food safety and qualitycontrol in Jiangsu Province.References[1] Vos T, Abajobir AA, Abate KH, et al. Global, regional,and national incidence, prevalence, and years livedwith disability for 328 diseases and injuries for 195countries, 1990–2016: a systematic analysis for theGlobal Burden of Disease Study 2016. Lancet.2017;390(10100):1211–1259.[2] Featherstone JD. The continuum of dental caries–evi-dence for a dynamic disease process. 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ZHANG ET AL.AbstractIntroductionMaterials and methodsBacterial strainsCompetition assay on plateBiofilm formation assayBiofilm viability assayCaries reduction invivoAnimals and general proceduresMicrobial analysis and caries scoringStatistical analysisResultsAntagonism between L.plantarum and S.mutans on agrowth plateFB-T9S.Mutans biofilm formation mediated by FB-T9 invitroEvaluation of a caries-induced rat modelBacterial colonization of the rat oral cavityCaries lesion evaluation by Keyes scoringDiscussion/conclusionDisclosure statementFundingReferences