ORIGINAL ARTICLE Acidogenicity of dual-species biofilms of bifidobacteria and Streptococcus mutans Bruno Mello de Matos1,2 & Fernanda Lourenção Brighenti3 & Thuy Do4,5 & David Beighton6 & Cristiane Yumi Koga-Ito1,7 Received: 13 June 2016 /Accepted: 5 September 2016 /Published online: 23 September 2016 # Springer-Verlag Berlin Heidelberg 2016 Abstract Objective The aim of this study was to evaluate the acidogenicity of dual-species biofilms of bifidobacteria and Streptococcus mutans. Materials and methods The following strains were tested: Bifidobacterium dentium DSM20436, Parascardovia denticolens DSM10105, and Scardovia inopinata DSM10107. Streptococcus mutans UA159 and Lactobacillus acidophilus ATCC4356 were used as control. Bifidobacteria were studied planktonically as they were not able to form monospecies bio- film, they were grown in biofilms associated with S. mutans. Endogenous polysaccharide reserves of cultures at log phase were depleted. Standardized suspensions of the microorganisms were incubated in growth media supplemented with 10 mM glucose, lactose, raffinose, glucose, or xylitol. S. mutans biofilms were grown on glass cover slips for 24 h to which bifidobacteria were added. After 24 h, the dual-species biofilms were exposed to the same carbon sources, and after 3 h, the pH of spent culture media and concentrations of organic acids were measured. Statistical analyses were carried out using ANOVA and Tukey’s test (α = 0.05). Results A higher pH drop was observed when S. mutans was associated with P. denticolens or S. inopinata, in either plank- tonic or biofilm cultures, than with S. mutans alone. Bifidobacteria showed a higher pH drop in the presence of raffinose than S. mutans or L. acidophilus. Conclusions Dual-species biofilms of bifidobacteria and S. mutans produced more acid and greater pH drops than biofilms of S. mutans alone. Clinical relevance New insights on the complex process of caries pathogenicity contribute to the establishment of preven- tive and therapeutic measures, in particular in specific cases, such as in early childhood caries. Keywords Acidogenicity . Bifidobacteria . Biofilms . Dental caries Introduction The etiology of caries is undoubtedly complex. It is generally recognized that microbial, environmental, and host factors interact to contribute to dental caries development [1]. Although dental caries is a biofilm-mediated disease, it is un- likely that all members of the oral biofilm participate equally in the caries process. The ecological plaque hypothesis Electronic supplementary material The online version of this article (doi:10.1007/s00784-016-1958-1) contains supplementary material, which is available to authorized users. * Cristiane Yumi Koga-Ito cristiane@fosjc.unesp.br 1 Environmental Engineering Department, São José dos Campos Institute of Science and Technology, UNESP, Univ. Estadual Paulista, Avenida Engenheiro Francisco Jose Longo 777, São José dos Campos 12245-000, São Paulo, Brazil 2 UNIVASF, Univ. Federal do Vale do São Francisco, Rua da Alvorada, General Dutra, Paulo Afonso, BA 48607-190, Brazil 3 Araraquara School of Dentistry, UNESP, Univ. Estadual Paulista, Rua Humaitá, 1680, Centro, Araraquara, SP 14801-385, Brazil 4 School of Dentistry, Faculty of Medicine and Health, University of Leeds, Leeds, UK 5 Division of Oral Biology,Wellcome Trust Brenner Building, Level 7, St James’s University Hospital Campus, Leeds LS9 7TF, UK 6 Dental Institute, King’s College London, Bessemer Rd., Denmark Hill, London SE5 9RW, UK 7 Oral BiopathologyGraduate Program, São José dos Campos Institute of Science and Technology, Avenida Engenheiro Francisco Jose Longo 777, São José dos Campos 12245-000, Brazil Clin Oral Invest (2017) 21:1769–1776 DOI 10.1007/s00784-016-1958-1 http://dx.doi.org/10.1007/s00784-016-1958-1 http://crossmark.crossref.org/dialog/?doi=10.1007/s00784-016-1958-1&domain=pdf suggests that the cariogenic oral environment will select in- creased proportions of acidogenic and aciduric microbiota [2]. These microorganisms include lactobacilli, streptococci, Actinomyces spp., yeasts, and bifidobacteria [3]. The resultant pH dropmay induce dental enamel demineralization under the critical pH of 5.5 [4]. Aas et al. [5] using molecular techniques demonstrated that 10 % of subjects with rampant caries in secondary dentition did not have detectable oral levels of Streptococcus mutans in intact enamel and white-spot lesions. The authors suggested that at least half of the bacteria associated with dental caries have not yet been cultivated. Thus, there is a considerable body of evi- dence for the emergence of other taxa, in addition to S. mutans in a cariogenic oral environment or within carious lesions [3]. Many studies have reported the presence of bifidobacteria in the oral cavity of healthy and diseased children and adults. These bacteria were found in saliva, plaque, and dental caries [3, 6, 7]. Beighton et al. [8] demonstrated that the bifidobacteria levels in adults’ saliva were not significantly different from the levels of mutans streptococci. Similar ob- servations were described in caries-active children [9]. Bifidobacterium dentium is themost prevalent bifidobacterial species in the oral cavity [3] with Parascardovia denticolens and Scardovia inopinata also frequently isolated [6, 10]. B i f idobac t e r i aceae cons i s t s o f seven gene ra (Aeriscardovia, Alloscardovia, Bifidobacterium, Falcivibrio, Gardnerella, Parascardovia, Scardovia) and about 36 spe- cies, the majority of which have been described and isolated from the intestinal and caecal microbiota. The range of taxa reported to be oral commensal seems primarily restricted to Bifidobacterium dentium, Parascardovia denticolens, and Scardovia inopinata [3]. Considering that little is known about the influence of en- vironmental factors—dietary components in particular—on acid production by oral bifidobacteria and that acid production from carbon sources is an important cariogenic feature, the aim of this study was to evaluate the acidogenicity of bifidobacteria after exposure to different carbon sources and determine if bifidobacteria are able to increase the acidogenicity in single- and dual-species planktonic cultures or biofilms in association with S. mutans. Materials and methods Strains and incubation conditions The following type strains of bifidobacteria were used: Bifidobacterium dentium DSM 20436, Parascardovia denticolens DSM 10105, and Scardovia inopinata DSM 10107. Also, Streptococcus mutans UA 159 and Lactobacillus acidophilus ATCC 4356 were included. S. mutans is considered an important species related to dental caries initiation whilst lactobacilli are related to dental caries progression. Cultures were obtained for each species from two indepen- dent frozen stocks. S. mutans and B. dentium were grown in semi-defined medium broth supplemented with yeast extract (SDMY) and 0.2 % sucrose [11]. L. acidophilus , P. denticolens, and S. inopinata were grown in lactobacilli MRS broth (Difco, USA) developed by De Man, Rogosa, and Sharpe [12]. All strains were grown to log phase at 37 °C in anaerobic jars (10 % CO2, 10 % H2, 80 % N2). Strains were studied planktonically and in biofilms and all experiments were performed at least in duplicate on two dif- ferent occasions. Preparation of planktonic cultures Bacterial cultures were washed twice in cysteine peptone wa- ter (CPW; 5 g/L yeast extract, 1 g/L peptone, 8.5 g/L NaCl, 0.5 g/L L-cysteine-HCl). Depletion of endogenous carbohy- drate reserves in stationary phase cultures was performed by incubating the washed cells for 30 min in water bath at 37 °C. Standardized inocula of the microorganisms at OD620 = 0.7 were prepared in an artificial saliva medium modified by McBain et al. [13], pH 7.0, for pH drop evaluation. Pilot tests showed that, even though McBain medium is an artificial saliva medium, it causes undesirable interferences in organic acids analyses by capillary electrophoresis. During those tests, it was verified that the chemical composition of McBain medium produced peaks (observed on Millenium Chromatography Manager Software) that overlap the acid peaks making them difficult to identify. For this reason, SDM broth (modified version of SDMY broth prepared with- out yeast extract), supplemented with aqueous solutions of glucose, lactose, raffinose, sucrose, or xylitol in final concen- trations of 10 mM was used to organic acids analyses. Sterile distilled water was used as negative control. Dual-species suspensions were prepared using S. mutans inocula plus bifidobacteria at a 1:1 ratio. Preparation of biofilms Pilot studies showed that none of the bifidobacteria species and L. acidophilus was able to form single-species biofilms in the model used in the present study. It was therefore not pos- sible to assess the production of acid or utilization of carbo- hydrates by single-species biofilms composed of bifidobacteria or lactobacilli alone. Instead, we assessed the acidogenicity of dual-species biofilms formed inoculating ei- ther bifidobacteria or lactobacilli onto pre-formed S. mutans biofilms. To produce the pre-formed S. mutans biofilms, S. mutans cultures were standardized in SDMY (OD620 = 0.7) and dilut- ed 1:50 in SDMYplus 0.2 % sucrose. S. mutans biofilms were 1770 Clin Oral Invest (2017) 21:1769–1776 grown on glass coverslips (ø12 mm) using an active attach- ment model [14]. Twenty-four-well plates were filled with 1.5 mL of the diluted inocula per well and incubated for 24 h. The glass coverslips containing S. mutans biofilms were placed in 24-well plates that were filled with 1.5 mL of either bifidobacteria or L. acidophilus diluted inocula. The bifidobacteria (B. dentium, P. denticolens, or S. inopinata) or L. acidophilus cultures to be added to the S. mutans biofilms were prepared (OD620 = 0.7) and diluted 1:50. Inocula of B. dentium were prepared in SDMY plus 0.2 % sucrose and P. denticolens, S. inopinata, and L. acidophilus were prepared in lactobacilli MRS broth (Difco, USA). After 24 h of incubation, dual-species biofilms were washed twice in CPW. Depletion of endogenous polysaccha- ride reserves was performed by incubating the biofilms in CPW for 30 min in water bath at 37 °C. The depleted biofilms were placed in 1.5 mL McBain me- dium or SDMY broth supplemented with 10 mM glucose, lactose, raffinose, sucrose, or xylitol and incubated at 37 °C for 3 h. Assessment of biofilm viability Presence of S. mutans, lactobacilli, and bifidobacteria species in mixed biofilms was evaluated by culture method. Glass coverslips with biofilms were carefully detached from the clamps and placed in 2 mL CPW. Biofilms were dispersed by sonication on ice 120 times for 1 s at amplitude of 40 W (Vibra Cell™, Sonics and Materials Inc., Newtown, USA) [15]. Serially diluted samples were plated onto SB20 (sucrose bacitracin), Rogosa (Difco, USA), and MMTPY agar plates (modified version of mupirocin trypticase peptone yeast ex- tract) for isolation of S. mutans, lactobacilli, and oral bifidobacteria, respectively [8, 16]. The plates were incubated anaerobically (as previously described) at 37 °C for 48 h. Colonies were counted and expressed as colony forming units (CFU). Experiments were performed in six replicates on two different occasions. Assessment of suspensions and biofilms acidogenicity The pH of the culture medium was measured to estimate bio- film acidogenicity at 0 and 3 h and pH variations calculated. The measurements were performed with the aid of an elec- trode with a micro-bulb (Hanna, Woonsocket, Rhode Island, USA). The amount of organic acids was analyzed by capillary electrophoresis (Waters Capillary Ion Analyzer; Milford, MA, USA) in plates with SDM broth. Samples were run in duplicate, and Millenium Chromatography Manager Software, version 3.05 was used for data analysis. Peak iden- tification and peak area integration were manually corrected if necessary. Sodium salts of formic, acetic, propionic, butyric, succinic, and lactic acid were used to prepare single and mixed standard solutions in deionized water, ranging from 0.05 to 2 mM. Calibration curves were made for each acid separately. As an internal standard, 0.1 mM oxalic acid was included in all samples. Lactic, propionic, acetic, formic, butyric, and succinic acid concentrations were determined [17]. Experiments were performed in four replicates on two differ- ent occasions. Data analysis Initially, all data were compared to the appropriate water, no added carbohydrate control. Then for those cultures in which significant changes to pH or to acid levels occurred, the results obtained for bifidobacteria species were compared to control microorganisms (S. mutans or L. acidophilus). Statistical anal- ysis were carried out using Graphpad Prism 3 (ANOVA and Tukey’s test, α = 0.05). Results None of the bacteria in the planktonic phase or in biofilms produced significant changes to the pH of the media or to the concentrations of lactic or acetic acids when incubated with xylitol (data not shown). Table 1 shows the pH drop (ΔpH) after the addition of different carbon sources to single-species suspensions. For L. acidophilus, the pH drop was higher than S. mutans when glucose was used. Statistically significant ΔpH were observed, which indicate that the presence of raffinose seems to be better metabolized by the three bifidobacteria species than by S. mutans or L. acidophilus. Higher pH drop was observed when S. mutans was associ- ated with P. denticolens or S. inopinata, in either planktonic or biofilm cultures (Tables 1 and 2). The association between S. mutans and B. dentium in suspension promoted higher pH drop when lactose, raffinose, or sucrose was used in compar- ison to S. mutans or S. mutans and L. acidophilus (Table 1). However, the co-culture of S. mutans and B. dentium in biofilms promoted lower pH drop than S. mutans single- species biofilms or S. mutans and L. acidophilus biofilms. Final pH for enamel demineralisation was below critical (5.5) for all microorganisms and associations when culture media were supplemented with glucose or sucrose. Co- culture of S. mutans/P. denticolens and S. mutans/ S. inopinata led to the highest pH drop in presence of glucose (minimum final pH 4.3), sucrose (minimum final pH 4.2), and raffinose (minimum final pH 4.6). The pH (5.5) was not reached in the presence of lactose, xylitol, or control. Raffinose promoted a pH drop below critical pH in either single-species suspensions or associated to other species, both Clin Oral Invest (2017) 21:1769–1776 1771 in planktonic culture and biofilms. For this carbohydrate, final pH from S. mutans or S. mutans and L. acidophilus cultures remained above critical levels (pH 6.2 and 6.3, respectively) (supplementary material). Table 3 displays the organic acid production of single- species and dual-species suspensions. Butyric, formic, propionic, and succinic acids were below detection limit (0.01 mM, according to Kara et al. [17]). For B. dentium in single-species cultures, more lactate is produced in the pres- ence of raffinose or sucrose. When associated to S. mutans, lactate production was higher in the presence of raffinose. P. denticolens produces more acetate for all carbohydrates. The same pattern of lactate production is observed for ei- ther single-species or dual-species suspensions of S. inopinata. On the other hand, while acetate production in S. inopinata single-species is higher than S. mutans for glu- cose, raffinose, and sucrose, in dual-species cultures, signifi- cantly higher concentrations of acetate was found for all car- bon sources. In dual-species biofilms, the combination of S. mutans and B. dentium did not produce more acid than S. mutans or S. mutans and L. acidophilus biofilms, except for lactate pro- duction in the presence of raffinose. S. mutans and P. denticolens formed more lactate than S. mutans or S. mutans and L. acidophilus biofilms in the presence of glu- cose and sucrose. S. mutans and S. inopinata biofilms yielded more acetate and lactate in the presence of all carbon sources. When raffinose was added to the culture medium, S. mutans and S. inopinata biofilms produced 14 times more lactate and 48 times more acetate than S. mutans biofilms alone, even though this species participated with 1.46 % of the mixed biofilm (Tables 4 and 5). Discussion The present study adds important information to the existing evidence in the literature. This is the first time that B. dentium, P. denticolens, and S. inopinata are studied alone or in association with S. mutans, in either suspension or biofilms. Bacteria organized in biofilms are offered a higher antimicrobial resistance not only due its spatial or- ganization—that impairs the penetration of antimicrobial substances—but also due to the low growth rate and phe- notypical modifications and also because biofilms are ex- tremely organized communities, in which interaction be- tween cells confers an important resistance mechanism, as previously shown by Kara et al. [17]. Table 1 pH drop (ΔpH) (average ± sd) for single-species and dual-species suspensions (n = 4) Glucose Lactose Raffinose Sucrose Control S. mutans 2.06 ± 0.01 0.65 ± 0.01 1.06 ± 0.02 2.08 ± 0.01 0.53 ± 0.01 L. acidophilus 2.33 ± 0.03a 0.64 ± 0.01 0.94 ± 0.01ª 1.88 ± 0.03ª 0.45 ± 0.01 B. dentium 2.18 ± 0.02ª,b 1.12 ± 0.11ª,b 2.19 ± 0.02ª,b 2.21 ± 0.01ª,b 0.54 ± 0.02 P. denticolens 2.07 ± 0.01b 0.87 ± 0.01ª,b 1.55 ± 0.04ª,b 1.56 ± 0.01ª,b 0.68 ± 0.04 S. inopinata 0.55 ± 0.01a,b 0.57 ± 0.02 2.01 ± 0.06ª,b 2.12 ± 0.06b 0.49 ± 0.08 S. mutans + L. acidophilus 2.21 ± 0.01ª 0.57 ± 0.01 0.75 ± 0.02ª 2.28 ± 0.04ª 0.34 ± 0.04 S. mutans + B. dentium 2.11 ± 0.11 0.81 ± 0.04ª,c 2.04 ± 0.06ª,c 2.15 ± 0.10b 0.48 ± 0.02 S. mutans + P. denticolens 2.42 ± 0.05ª,c 1.02 ± 0.02ª,c 2.08 ± 0.05ª,c 2.44 ± 0.04ª,c 0.93 ± 0.05 S. mutans + S. inopinata 2.40 ± 0.01ª,c 0.82 ± 0.01ª,c 2.11 ± 0.02ª,c 2.45 ± 0.01ª,c 0.74 ± 0.01 Letters: significant differences within the same carbohydrate in relation to S. mutans (a), L. acidophilus (b), S. mutans + L. acidophilus (c); data in bold: no significant differences in the same line in relation to control; ANOVA/Tukey’s test, p < 0.05 Table 2 pH drop (ΔpH) (average ± sd) for dual-species biofilms (n = 4) Glucose Lactose Raffinose Sucrose Control S. mutans 1.66 ± 0.08 0.86 ± 0.05 1.28 ± 0.10 1.55 ± 0.05 0.76 ± 0.02 S. mutans + L. acidophilus 1.97 ± 0.03ª 0.93 ± 0.02 1.72 ± 0.06ª 2.02 ± 0.01ª 0.80 ± 0.01 S. mutans + B. dentium 1.56 ± 0.03b 0.92 ± 0.01 1.30 ± 0.01b 1.58 ± 0.08b 0.79 ± 0.02 S. mutans + P. denticolens 2.50 ± 0.04ª,b 1.17 ± 0.05ª,b 1.92 ± 0.08ª,b 2.37 ± 0.07ª,b 1.05 ± 0.02 S. mutans + S. inopinata 2.47 ± 0.06ª,b 1.11 ± 0.03ª,b 1.95 ± 0.10ª,b 2.35 ± 0.08ª,b 0.99 ± 0.04 Letters: significant differences within the same carbohydrate in relation to S. mutans (a), S. mutans + L. acidophilus (b); data in bold: no significant differences in the same line in relation to control; ANOVA/Tukey’s test, p < 0.05 1772 Clin Oral Invest (2017) 21:1769–1776 T ab le 3 L ac ta te an d ac et at e co nc en tr at io n (m M ;a ve ra ge ± sd ) in su sp en si on s (n = 8) G lu co se L ac to se R af fi no se Su cr os e C on tr ol L ac ta te A ce ta te L ac ta te A ce ta te L ac ta te A ce ta te L ac ta te A ce ta te L ac ta te A ce ta te S. m ut an s 3. 56 ± 0. 22 0. 36 ± 0. 03 0. 00 ± 0. 00 0. 02 ± 0. 04 0. 39 ± 0. 04 0. 32 ± 0. 07 3. 73 ± 0. 30 0. 32 ± 0. 08 0. 00 ± 0. 00 0. 01 ± 0. 02 L. ac id op hi lu s 6. 34 ± 0. 63 ª 0. 12 ± 0. 03 ª 0. 00 ± 0. 00 0. 02 ± 0. 04 0. 64 ± 0. 12 ª 0. 09 ± 0. 04 ª 0. 19 ± 0. 04 ª 0. 02 ± 0. 03 ª 0. 00 ± 0. 00 0. 00 ± 0. 00 B .d en tiu m 0. 91 ± 0. 10 ª,b 2. 74 ± 0. 13 ª,b 0. 01 ± 0. 02 0. 45 ± 0. 04 ª,b 1. 07 ± 0. 06 ª,b 2. 75 ± 0. 18 ª,b 1. 15 ± 0. 05 ª,b 3. 09 ± 0. 16 ª,b 0. 01 ± 0. 03 0. 00 ± 0. 00 P. de nt ic ol en s 0. 49 ± 0. 05 ª,b 1. 74 ± 0. 12 ª,b 0. 06 ± 0. 04 ª,b 0. 52 ± 0. 05 ª,b 0. 38 ± 0. 07 b 1. 11 ± 0. 11 ª,b 0. 43 ± 0. 06 ª 1. 16 ± 0. 08 ª,b 0. 19 ± 0. 03 0. 57 ± 0. 04 S. in op in at a 0. 00 ± 0. 00 a, b 0. 07 ± 0. 04 ª 0. 00 ± 0. 00 0. 00 ± 0. 00 1. 00 ± 0. 10 ª,b 2. 29 ± 0. 17 ª,b 0. 95 ± 0. 08 ª,b 2. 44 ± 0. 12 ª,b 0. 00 ± 0. 00 0. 00 ± 0. 00 S. m ut an s + L. ac id op hi lu s 4. 71 ± 0. 36 ª 0. 29 ± 0. 04 0. 00 ± 0. 00 0. 00 ± 0. 00 0. 69 ± 0. 08 ª 0. 28 ± 0. 05 5. 25 ± 0. 17 ª 0. 29 ± 0. 05 0. 00 ± 0. 00 0. 00 ± 0. 00 S. m ut an s + B .d en tiu m 3. 18 ± 0. 49 c 1. 82 ± 0. 10 ª,c 0. 00 ± 0. 00 0. 03 ± 0. 01 1. 32 ± 0. 08 ª,c 2. 47 ± 0. 22 ª,c 4. 21 ± 0. 70 c 2. 94 ± 0. 09 ª,c 0. 00 ± 0. 00 0. 00 ± 0. 00 S. m ut an s + P. de nt ic ol en s 5. 66 ± 0. 29 ª,c 1. 97 ± 0. 11 ª,c 0. 01 ± 0. 02 0. 47 ± 0. 03 ª,c 1. 01 ± 0. 09 ª,c 1. 46 ± 0. 11 ª,c 4. 45 ± 0. 22 ª,c 1. 51 ± 0. 13 ª,c 0. 01 ± 0. 01 0. 44 ± 0. 05 S. m ut an s + S. in op in at a 4. 50 ± 0. 55 ª 0. 62 ± 0. 04 ª,c 0. 00 ± 0. 00 0. 10 ± 0. 03 ª,c 1. 56 ± 0. 12 ª,c 1. 99 ± 0. 14 ª,c 4. 69 ± 0. 65 ª,c 1. 85 ± 0. 13 ª,c 0. 00 ± 0. 00 0. 21 ± 0. 02 L et te rs :s ig ni fi ca nt di ff er en ce s w ith in th e sa m e ca rb oh yd ra te in re la tio n to S. m ut an s (a ), L. ac id op hi lu s (b ), S. m ut an s + L. ac id op hi lu s (c ); da ta in bo ld :n o si gn if ic an td if fe re nc es in th e sa m e lin e in re la tio n to co nt ro l; A N O V A /T uk ey ’s te st ,p < 0. 05 T ab le 4 L ac ta te an d ac et at e co nc en tr at io n (m M ;a ve ra ge ± sd ) in bi of ilm s (n = 8) G lu co se L ac to se R af fi no se S uc ro se C on tr ol L ac ta te A ce ta te L ac ta te A ce ta te L ac ta te A ce ta te L ac ta te A ce ta te L ac ta te A ce ta te S. m ut an s 2. 96 ± 0. 28 0. 00 ± 0. 00 0. 00 ± 0. 00 0. 00 ± 0. 00 0. 15 ± 0. 13 0. 03 ± 0. 04 2. 25 ± 0. 44 0. 00 ± 0. 00 0. 00 ± 0. 00 0. 00 ± 0. 00 S. m ut an s + L. ac id op hi lu s 2. 15 ± 0. 49 ª 0. 38 ± 0. 12 ª 0. 05 ± 0. 05 0. 22 ± 0. 08 ª 0. 51 ± 0. 19 ª 0. 53 ± 0. 22 ª 3. 23 ± 1. 18 0. 22 ± 0. 09 ª 0. 05 ± 0. 06 0. 21 ± 0. 20 S. m ut an s + B .d en tiu m 2. 51 ± 0. 19 0. 09 ± 0. 06 b 0. 03 ± 0. 07 0. 06 ± 0. 08 0. 40 ± 0. 09 ª 0. 23 ± 0. 05 b 2. 28 ± 0. 59 0. 07 ± 0. 06 b 0. 00 ± 0. 00 0. 03 ± 0. 06 S. m ut an s + P. de nt ic ol en s 9. 50 ± 0. 90 ª,b 0. 34 ± 0. 05 ª,b 0. 07 ± 0. 04 0. 26 ± 0. 05 ª,b 1. 72 ± 0. 21 ª,b 1. 24 ± 0. 14 ª,b 12 .6 1 ± 1. 01 ª,b 0. 34 ± 0. 07 ª,b 0. 11 ± 0. 06 0. 28 ± 0. 02 S. m ut an s + S. in op in at a 9. 23 ± 0. 39 ª,b 0. 55 ± 0. 02 ª,b 0. 25 ± 0. 09 ª,b 0. 45 ± 0. 15 ª,b 2. 16 ± 0. 18 ª,b 1. 46 ± 0. 14 ª,b 11 .7 9 ± 0. 72 ª,b 0. 38 ± 0. 14 ª,b 0. 09 ± 0. 16 0. 31 ± 0. 17 L et te rs : si gn if ic an t di ff er en ce s w ith in th e sa m e ca rb oh yd ra te in re la tio n to S. m ut an s (a ), S. m ut an s + L. ac id op hi lu s (b ); da ta in bo ld : no si gn if ic an t di ff er en ce s in th e sa m e lin e in re la tio n to co nt ro l; A N O V A /T uk ey ’s te st ,p < 0. 05 Clin Oral Invest (2017) 21:1769–1776 1773 Pilot studies showed that bifidobacteria are not able to form single-species biofilms in the model used in the present study (data not shown). This is the reason why bifidobacteria single- species biofilms were not evaluated in the present study. This is also a notable finding because it shows the importance of the interaction of bifidobacteria species with other oral microorgan- isms. Amore detailed study of bifidobacteria biofilms, including other analyses (i.e., confocal analyses), may generate important data on this interaction and should be conducted in the future. The ability of bifidobacteria in suspension form to produce acids was already demonstrated in previous studies. However, to the best of our knowledge, this is first report on biofilms of bifidobacteria co-cultured with S. mutans. Haukioja et al. [18] showed that four different bifidobacteria strains were also able to promote a pH drop below critical pH for enamel demineralisation (5.5) when different carbon sources are used. Moynihan et al. [19] showed that B. dentium decreases culture medium pH to values lower than enamel critical pH when exposed to glucose or lactose. Nakajo et al. [20] also demon- strated the ability of bifidobacteria (B. dentium and Bifidobacterium longum) to decrease the pH culture below 5.0 at an initial pH of 5.0–7.0, indicating that these bacteria are able of creating an acidic environment in dental plaque and caries lesions. The acidogenic profile of bifidobacteria reaffirms their role in the acidification of the oral environment, probably contributing to dental caries development. Carbon sources used in the present study were chosen based on their presence in the diet. Glucose, lactose, and sucrose are either naturally present in fruits, vegetables, or milk or added at high concentrations to baked products, snacks, and sweets [21]. Raffinose is naturally present in beans, cabbage, brussels sprouts, broccoli, asparagus, and whole grains [21]. Xylitol is a natural sweetener that adds texture to foods and is not metabolized by most oral bac- teria, including S. mutans [22]. The results of the present study support the evidence that bifidobacteria species present in carious lesions are able to metabolize all carbon sources included in the present study at different rates. Bifidobacteria demonstrated that they are able not only to produce significant amount of acids but also to accentuate biofilm acidogenicity in combination with S. mutans. A possible explanation for the significant pH drop for the association between S. mutans and bifidobacteria is that S. mutansmetabolizes carbon sources at a higher rate, produc- ing acids more quickly than bifidobacteria and lactobacilli. Both lactobacilli and bifidobacteria prefer lower pH to pro- duce acids, so acid production by S. mutans promotes a favor- able environment to these species. Bifidobacteria are able to metabolize raffinose to a higher extent than S. mutans and L. acidophilus, which reflected in a higher pH drop. This is an important finding because bifidobacteria do not require the consumption of snacks or sweets to produce acid, since raffinose is naturally present in healthy foods consumed on a daily basis. This can indicate the cariogenicity of bifidobacteria, which should be clinically evaluated. Moreover, a synergistic effect between S. mutans and P. denticolens or S. inopinata promoted a higher pH drop than these species alone. These results show that the presence of both species in dental biofilm may indicate a higher cariogenic potential than if bifidobacteria are absent. This is of particular interest since some bifidobacteria are used in probiotic foods. The use of probiotics on a daily basis is suggested to modulate oral and intestinal microbiota. However, at the moment there are no clinical trials that proved the beneficial use of bifidobacteria on caries prevention [23]. The results of the pres- ent study suggest that the use of these species in probiotics may increase pH drop and acid production in dental biofilm. The clinical outcome of these findings should be further evaluated. Our results for planktonic cultures demonstrated that S. inopinata was not able to ferment glucose and lactose effi- ciently, which is in disaccord with the literature [24]. Perhaps it simply ferments these two sugars slowly, in comparison to the rates of fermentation of raffinose and sucrose. So, further studies on the metabolism of carbon sources should be performed not only on the species investigated in this study, but also on other bifidobacteria such as Scardovia wiggsiae, which has recently been recognized as a member of the oral microbiota [10, 25]. Recently, higher prevalence of Table 5 Colony forming units (CFU/disk; average ± sd) for dual-species biofilms and specific species (lactobacilli and bifidobacteria) and percentage of these species in relation to total count (%TM) (n = 12) Biofilm Total microorganism Lactobacilli Bifidobacteria CFU/disk % TM CFU/disk %TM S. mutans 2.77 × 107 ± 1.44 × 107 – – – – S. mutans + L. acidophilus 7.09 × 107 ± 2.83 × 107 4.20 × 105 ± 2.03 × 105 0.59 – – S. mutans + B. dentium 5.15 × 106 ± 2.18 × 106 – – 4.66 × 106 ± 2.19 × 106 90.49 S. mutans + P. denticolens 2.83 × 108 ± 3.74 × 107 – – 3.84 × 106 ± 1.67 × 106 1.36 S. mutans + S. inopinata 2.52 × 108 ± 7.12 × 107 – – 3.68 × 106 ± 1.50 × 106 1.46 1774 Clin Oral Invest (2017) 21:1769–1776 S. wiggsiae was found in caries lesions than in controls [26] and this finding reinforce the need of deeper investigation on other species. Differences in pH drops and acid production observed in the control group might be related to inefficient carbohydrate depletion. So, the production of acids might be explained by the metabolism of residual endogenous polysaccharides. In this study, pH drop was evaluated by measuring the pH at baseline and after 3 h. Multiple measurements of pH over time may also generate interesting results and should be per- formed in future studies. Overall, a higher amount of acetate was produced by bifidobacteria when cultured planktonically, which is in agree- ment with findings reported by Crociani et al. [24]. This is also the first time that the fermentative profile of bifidobacteria in the presence of lactose, raffinose, and sucrose was studied in sus- pension and biofilms grown in the presence of S. mutans. Although acetate production is beneficial in the intestinal envi- ronment, it may have detrimental effects in the oral cavity. Together with other oral species, acetate productionmay contrib- ute to environmental changes that shift healthy oral microbiota to a more cariogenic one. More importantly, when cultured with S. mutans, bifidobacteria seem to contribute to a rise in lactate production, an important feature in caries etiology. Also, other virulence factors, such as aciduricity, antimicrobial resistance, and metabolic activity should be evaluated in the future. Based on the findings of the present study, it is concluded that B. dentium, P. denticolens, and S. inopinata are as acidogenic as S. mutans. Moreover, dual-species biofilms of S. mutans and oral bifidobacteria produced a significantly greater pH drop than those produced by individual species. Acknowledgments The authors thankCoordination for the Improvement of Higher Education Personnel–CAPES/Brazil (Grant #3755/10-0) and São Paulo Research Foundation – FAPESP/Brazil (Grant #10/02063-1), and the staff of the Laboratory of Oral Microbiology – Academic Centre for Dentistry Amsterdam (ACTA) – Vrije Universiteit Amsterdam – Netherlands for its contribution (laboratory facilities and consumables). The funders had no role in study design, data collection and analysis, deci- sion to publish, or preparation of the manuscript. Compliance with ethical standards Conflict of interest The authors declare that they have no conflict of interest. Funding The work was supported by the Coordination for the Improvement of Higher Education Personnel – CAPES/Brazil (Grant #3755/10–0) and São Paulo Research Foundation – FAPESP/Brazil (Grant #2010/02,063–1). 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