Correspondence: Jun-Qi Ling, Institute of Stomatological Research, Guanghua School of Stomatology, Sun Yat-sen University, 74 Zhong Shan Er Road, Guangzhou 510080, China. Tel.: +86-20-83862621; fax: +86-20-83822807; e-mail: email@example.com
Biofilm detachment is a physiologically regulated process that facilitates the release of cells to colonize new sites and cause infections. Streptococcus mutans is one of the major inhabitants of cariogenic dental plaque biofilm. This study tested the hypothesis that S. mutans biofilm-detached cells exhibit distinct physiological properties compared with their sessile and planktonic counterparts. Biofilm-detached cells showed a longer generation time of 2.85 h compared with planktonic cells (2.06 h), but had higher phosphotransferase activity for sucrose and mannose (P < 0.05). Compared with planktonic cells, they showed higher chlorhexidine (CHX) resistance and fourfold more adherent (P < 0.05). Increased mutacin IV production in biofilm-detached cells was noted by a larger inhibition zone against Streptococcus gordonii (31.07 ± 1.62 mm vs. 25.2 ± 1.74 mm by planktonic cells; P < 0.05). The expressions of genes associated with biofilm formation (gtfC and comDE) and mutacin (nlmA) were higher compared with planktonic cells (P < 0.05). In many properties, biofilm-detached cells shared similarity with sessile cells except for a higher phosphotransferase activity for sucrose, glucose, and mannose, increased resistance to CHX, and elevated expression of gtfC-, comDE-, and acidurity-related gene aptD (P < 0.05). Based on data obtained, the S. mutans biofilm-detached cells are partially distinct in various physiological properties compared with their planktonic and sessile counterparts.
In natural and medical settings, bacteria attaching to different surfaces are able to develop communities known as biofilm (Costerton, 2004), which have been associated with many chronic infections in humans (Archer et al., 2011). The biofilm life cycle has been identified as having several phases, including attachment, microcolony formation, development and maturation, and detachment (Sauer et al., 2002). Biofilm detachment is a physiologically regulated process facilitating the release of cells from a biofilm into a planktonic state, which then contribute to bacterial survival, new sites of colonization, and infection spread (Kaplan, 2010). Detachment of Pseudomonas putida and Pseudomonas fluorescens biofilm cells in response to starvation and glucose has been reported (Wrangstadh et al., 1988; Allison et al., 1998). In oral bacteria, mature Neisseria subflava and Aggregatibacter actinomycetemcomitans biofilms can release single or clusters of cells into the surrounding medium, and biofilm-detached cells can reattach to distal surfaces (Kaplan & Fine, 2002). More recently, carbon source–induced detachment has also been noted in Candida albicans biofilms. The detached cells exhibited an enhanced ability to adhere to endothelial cells and form biofilms (Uppuluri et al., 2010). Despite the breadth of detrimental effects caused by biofilm-detached cells, the physiological properties of these cells and how they colonize new sites, especially in dental plaque biofilm, have not been adequately investigated.
Streptococcus mutans, well known for its ability to adhere to tooth surfaces and form biofilms, is one of the principal inhabitants of cariogenic plaque biofilm. Oral biofilms, regardless of their location, share several features in common with other microbial biofilms, including synthesis of adhesive extracellular matrix and their ultimate detachment (Kaplan, 2010). In S. mutans, biofilm detachment resulting from an endogenous enzyme, which releases adhesin surface proteins, has been reported (Lee et al., 1996). At present, limited information is available regarding the physiological properties of S. mutans biofilm-detached cells and whether they possess virulence factors similar to those of planktonic or sessile cells. In this study, we investigated selected physiological properties of S. mutans UA159 biofilm-detached cells and compared them with their well-characterized planktonic and sessile counterparts.
Materials and methods
Bacterial growth, biofilm formation, and agents
Streptococcus mutans UA159 was grown in brain heart infusion broth (BHI; Difco, Detroit, MI) overnight anaerobically (10% H2, 5% CO2, and 85% N2; Forma Scientific, Inc, Marietta, OH) at 37 °C. A 25 mL of S. mutans suspension in BHI with 1% sucrose (105 CFU mL−1) was placed into a 100-mm-diameter Petri dish containing four polystyrene (PLS) blocks (22 × 30 × 1 mm; VWR Scientific, Radnor, PA) for 48 h at 37 °C allowing biofilm formation on upper side of the blocks.
Planktonic, sessile, and biofilm-detached cell suspensions preparation
To collect biofilm-detached cells, PLS blocks with adherent S. mutans UA159 biofilms were transferred to a flask connected to a peristaltic pump to allow for continuous flow of BHI at a constant rate (0.5 mL min−1) (Rollet et al., 2009). The flowthrough collected for the first 2 h was thrown away to remove loosely attached cells. The biofilm-detached cells were obtained by centrifugation of the flowthrough collected over the following 6-h period. The PLS blocks with adherent biofilm were rinsed twice with sterile saline solution (0.9% NaCl), and the sessile cells were collected using a sterile disposable cell lifter (TPP; D. Dutscher, Brumath, France) and resuspended in 0.9% NaCl. Planktonic cells grown anaerobically to stationary phase in BHI at 37 °C were collected by centrifugation. All three cell suspensions were washed three times with 0.9% NaCl and dispersed by sonication on ice using a 30-s pulse at 7 W (Vibra cell™, Sonics & Materials Inc, Newtown, CT) and adjusted to 105 CFU mL−1 for further use.
For regrowth of planktonic, sessile, and biofilm-detached cells, 96-well microtiter plates containing respective cell suspension (103 CFU mL−1) and BHI were incubated at 37 °C anaerobically. Growth kinetics was monitored spectrophotometrically at A550 nm over a 28-hr period. Based on the growth curve, generation time of the three populations was calculated according to the formula: td = (t2 − t1)ln(2)/[ln(OD2) − ln(OD1)], td is generation time, t1 and t2 are time for the beginning and end of the logarithmic phase, and OD1 and OD2 are optical density at A550 nm of t1 and t2 (Khalichi et al., 2004).
Phosphoenolpyruvate: sugar phosphotransferase system (PTS) activity
All three cell populations were harvested and resuspended in 75 mM Tris–HCl and 10 mM MgSO4 (pH 7.0). To allow exogenous phosphoenolpyruvate (PEP) to cross the cell membrane, cells were permeabilized by adding 30 uL mL−1 of toluene: ethanol (1 : 9). Cell suspensions were vortexed, frozen, thawed twice at 37 °C, centrifuged, and stored at −80 °C until use (Hoelscher & Hudson, 1996). PEP phosphotransferase assay mixtures (1 mL) contained 0.1 mM NADH; 10 U rabbit muscle lactic acid dehydrogenase; 5 mM PEP; 10 mM NaF; carbohydrate substrate; and toluene–acetone-treated cell suspension. The rate of NADH oxidation was followed at A340 nm in a 1-cm light path quartz cell (Gilford 2400 spectrophotometer, Kornberg & Reeves, 1972).
Minimum biofilm eradication concentration (MBEC) and chlorhexidine (CHX) susceptibility
Measurements of the antimicrobial susceptibilities of S. mutans biofilms were performed as previously described MBEC microdilution assay with the following modifications (Ceri et al., 1999). The 48-h biofilms were grown on the pegs of a MBECTM P&G Assay 96-well tissue-culture microtiter plates and washed three times with 200 μL phosphate-buffered saline (PBS). Biofilms on the pegs were placed into the wells of a microtiter plate with serial twofold dilutions of CHX ranging from 50 to 0 ug mL−1 for 24 h at 37 °C, followed with washing with PBS and incubation in BHI at 37 °C for another 24 h. The MBEC was defined as the lowest concentration of CHX that prevented S. mutans regrowth. Susceptibility of the three cell populations to different concentrations of CHX (Sigma, St. Louis, MO) was determined with the modified method from that of Nudera (Nudera et al., 2007). Tubes containing the three populations (105 CFU mL−1) and CHX (0–1 ug mL−1) in BHI were incubated at 37 °C. After 12-hr incubation, bacterial suspension was serially diluted in PBS (50 mM, pH 6.8) and plated onto BHI agar in triplicate. All plates were incubated anaerobically at 37 °C for 48 h, and the number of colonies was then determined.
A 10 μL quantity of respective cell suspension was spotted onto pH 7.0 and pH 3.0 BHI agar plates and incubated at 37 °C anaerobically for 48 h. Viable colonies were visualized and counted under stereomicroscopy.
Bacteriocin production assay
The method of Mohammad & Indranil (2011) was used. A 20 μL each of the respective cell suspension (105 CFU mL−1) was stabbed into BHI agar plates and incubated anaerobically at 37 °C overnight. The plates were then overlaid with a 4-mL soft agar suspension mixed with 0.2 mL indicator species (Streptococcus gordonii Challis, or Enterococcus faecalis ATCC29212) and incubated anaerobically for 24 h, and zones of growth inhibition were measured.
Sucrose-dependent reattachment assay
The reattachment of the three cell populations was determined by means of a four-well chamber slide (culture area 1.8 cm2 per well; Nunc Lab-Tek, Rochester, NY). Each chamber slide contained 400-μl cell suspension (105 CFU mL−1 in BHI-1% sucrose). Following anaerobic incubation at 37 °C for 1, 2, and 4 h, the chamber slides were washed twice with 0.9% NaCl to remove unattached cells. The attached cells were stained with Live/Dead BackLight™ (Molecular Probes, Invitrogen, Carlsbad, CA) for 15 min and examined by confocal laser scanning microscopy (LSM 710; Zeiss, Jena, Germany) with a 40× water immersion objective. Images were captured at 488 and 544 nm and analyzed by Image-Pro Plus 5.1 (Media Cybernetics Inc., Bethesda, MD). The proportion of coverage area was calculated from three different images of the same sample.
Reverse transcription-quantitative PCR (RT-qPCR)
RNA of collected planktonic, sessile, and biofilm-detached cells was extracted with an RNeasy minikit (QIAGEN, Valencia, CA). Reverse transcription was performed by the 1st Strand cDNA Synthesis Kit with random hexamer primers (Invitrogen, Madison, WI). Real-time PCR was used for quantification of nlmA, nlmC, comDE, gtfC, atpD, and gtfB mRNA expression, with 16S rRNA gene as an internal control. All primers for RT-qPCR were designed with primer 3 and obtained from Sigma-Aldrich Corp. Amplification was performed with SYBR Green Master Mix (Applied Biosystems, Foster City, CA) on the iCycler iQ detection system (Applied Biosystems). Threshold cycle values (CT) were determined, and data were analyzed by StepOne software v2.0 (Applied Biosystems) with the 2−ΔΔCT method.
All experiments were performed in triplicate and repeated at least three times. Data were analyzed by spss (version 13.0 for Windows). Two-group comparison was performed using Student's t-test. Significance was set at a P value of 0.05.
Growth rates of S. mutans UA159 planktonic, sessile, and biofilm-detached cells
When grown in a batch culture, biofilm-detached cells exhibited a lag phase of approximately 8 h and plateaued at 24 h with a generation time of 2.85 h. The planktonic cells exhibited a shorter lag phase of 5 h and reached stationary growth phase at 16 h with a shorter generation time of 2.06 h. The lag phase of sessile cells was 12 h with a longer generation time of 3.98 h, compared with that of planktonic and biofilm-detached cells (P < 0.05) (Fig. 1a).
PTS activity of the three populations
Biofilm-detached cells exhibited higher PTS activities for sucrose and mannose, which was 1.31- and 2.19-fold as that in planktonic cells (P < 0.05). However, there were no significant differences in glucose transport activity between these two populations (P > 0.05). Sessile cells showed a relatively lower PTS activity for sucrose, glucose, and mannose when compared with biofilm-detached cells (P < 0.05), while no significant difference was noted for fructose (P > 0.05) (Fig. 1b).
CHX susceptibility and acidurity
The MBEC of CHX for S. mutans biofilms was 6.25 μg mL−1, and CHX inhibited growth of S. mutans planktonic and sessile cells at an MIC of 0.625 μg mL−1 and at 1 μg mL−1 for biofilm-detached cells (Fig. 2a). The planktonic cells did not grow on pH 3.0 agar plate, but the identical inoculum of sessile and biofilm-detached cells survived the low pH. All three populations grew well on pH 7.0 agar plate (Fig. 2b).
When tested for mutacin IV production against S. gordonii, the biofilm-detached cells exhibited larger inhibition zone (31.07 ± 1.62 mm) compared with that of planktonic cells (25.20 ± 1.74 mm) (P < 0.05). However, no significant differences were noted in comparison with sessile cells (P > 0.05). As for mutacin V production against E. faecalis, the biofilm-detached cells displayed smaller inhibition zone (34.1 ± 1.24 mm) than sessile cells (39.50 ± 1.36 mm) and planktonic cells (37.90 ± 1.97 mm) (P > 0.05).
Sucrose-dependent reattachment ability
When the sucrose-dependent reattachment ability of washed S. mutans cells was examined, the surface coverage portion of biofilm-detached cells was 17.0% at 1 h, 65.2% at 2 h, and 98.58% at 4 h, respectively. Planktonic cells exhibited lower reattachment ability at 2 and 4 h postattachment (13.55% and 69.53%; P < 0.05). However, no significant difference was observed between the biofilm-detached and sessile cells at all experimental time points (P > 0.05) (Fig. 3).
Expression profiles of virulence-associated genes
Compared with planktonic cells, the biofilm-detached cells exhibited up-regulated expression levels in genes associated with biofilm formation (comDE and gtfC), acidurity (atpD), and mutacin IV (nlmA) (P < 0.05). However, no significant difference was detected in the expression level of mutacin V–related gene nlmC and biofilm formation–associated gene gtfB (P > 0.05). Compared with the sessile cells, a similar up-regulatory effect was observed in biofilm-detached cells in the expression of comDE, gtfC, and aptD (P < 0.05), while no significant difference was observed in the expression of gtfB, nlmA, and nlmC (P > 0.05) (Fig. 4a).
Recent studies have reported that biofilm-detached cells display phenotypes and properties distinct from those of their planktonic counterparts. In C. albicans, the biofilm-detached cells possessed an enhanced adherence to endothelial cells and caused increased damage compared with planktonic cells (Uppuluri et al., 2010). In Pseudomonas aeruginosa, the biofilm-detached cells grew more slowly and adhere more easily to new surfaces than planktonic cells (Rollet et al., 2009). Different susceptibilities to disinfectants among planktonic, sessile, and biofilm-detached cells of Burkholderia cepacia and P. aeruginosa have also been reported (Aaron et al., 2002). Analysis of data presented in the current study demonstrated that different physiological states existed in S. mutans UA159 planktonic, sessile, and biofilm-detached cells.
It has been reported that sessile and biofilm-detached cells of P. aeruginosa exhibited longer lag phases than planktonic cells, suggesting that the biofilm-detached cells were less able to return to planktonic mode after detachment (Rollet et al., 2009). Consistent with this observation, our data also showed that slower growth rate was observed in the sessile and biofilm-detached cells of S. mutans UA159. However, under the experimental condition, no significant difference in growth rate was noted between the biofilm-detached and sessile cells.
The early stage of S. mutans biofilm formation is characterized by sucrose-dependent attachment facilitated by adherent polysaccharides produced from sucrose via cell surface-adsorbed glucosyltransferases (GTFs) (Coykendall et al., 1974). Among the three GTFs of S. mutans, GTFB and GTFC play crucial roles in the in vitro sucrose-dependent attachment to smooth surfaces (Tsumori & Kuramitsu, 1997; Fujiwara et al., 2002; Koo et al., 2010). Compared with planktonic cells, the superior ability of biofilm-detached cell to attach to new surfaces may be explained by the up-regulated expression of gtfC and the regulatory gene comDE. Similar observation has been made in P. aeruginosa by other workers, suggesting that biofilm-detached cells exhibit distinct phenotype from that of planktonic cells. The greater capacity of the detached cells to form biofilms demonstrated that this population should not be neglected (Ymele-Leki & Ross, 2007). The ability of S. mutans to transport nutrients from the environment is the premise of basic physiological activities (Colby & Russell, 1997). Moreover, study suggests that carbon flux is intricately tied to bacteria biofilm formation (Loo et al., 2003). The increase in sucrose-, glucose-, and mannose-specific PTS activities in biofilm-detached cells, noted in our study, may also have contributed to their ability to reattach to new surfaces and following biofilm formation.
Streptococcus mutans resides in dental plaque, in which fierce interspecies competition limits their proportion in the community (Kolenbrander et al., 2006, 2010; Kuramitsu et al., 2007). To outcompete other members, S. mutans secretes mutacins that display diverse inhibitory spectra. Mutacin IV, encoded by nlmA, is a major contributor to the antimicrobial spectrum and is active against other streptococcal species. Mutacin V, encoded by nlmC, is active against nonstreptococcal targets and streptococcal species of four subgroups (pyogenic, anginosus, mitis, and bovis) (Mohammad & Indranil, 2011). In this study, S. mutans UA159 biofilm-detached cells displayed a larger inhibition zone against S. gordonii compared with the planktonic cells, reflecting an increase in mutacin IV production. This was consistent with the up-regulated expression of nlmA in biofilm-detached cells. It seemed likely that, after being released from biofilm, the biofilm-detached cells were prepared to compete with other oral bacteria to survive. However, additional data are needed to support this hypothesis.
It has been reported that S. mutans cells within a biofilm are better able to survive acid challenges than planktonic counterparts or other oral streptococci (McNeill & Hamilton, 2003). The membrane-bound F1F0-ATPase system is considered the primary determinant of acid tolerance in S. mutans. This system pumps protons from cells and maintains internal pH balance (Kobayashi et al., 1986). Our data suggested that the up-regulated expression of S. mutans UA159 atpD, a subunit of the proton translocator, may potentially lead to decreased cytoplasmic acidity and increased acid adaptation in sessile and biofilm-detached cells.
As for antimicrobial resistance, in vitro studies have shown that the planktonic, sessile, and biofilm-detached cells of P. aeruginosa exhibited similar profiles of susceptibility to selected antibiotics (Rollet et al., 2009). Although antibiotic resistance was not investigated in this study, our data showed that S. mutans biofilm-detached cells were less susceptible to CHX, at sub-MIC levels, than the planktonic cells. Further in-depth studies are necessary to elucidate the antimicrobial resistance mechanism of S. mutans biofilm-detached cells.
In this study, we established the S. mutans biofilm detachment model under flow conditions, which discriminated the biofilm-detached cells from that growing planktonically in the effluent. Based on data obtained from this study, S. mutans UA159 biofilm-detached cells (1) are more aciduric and more resistant to CHX than planktonic cells; (2) use sucrose more efficiently with enhanced reattachment ability than planktonic cells; and (3) exhibit different expression profiles of selected virulence-associated genes from those of planktonic and sessile cells. Further studies are warranted to evaluate whether these observed physiological differences are biological meaningful.
This research was supported by Innovate Talent-training Research Fund for the Doctoral Program of Sun-Yat- Sen University, China, and the Department of Pediatric Dentistry, College of Dentistry, University of Illinois at Chicago. Jia Liu is the recipient of a scholarship granted by the State Scholarship Fund, the China Scholarship Council.