Monitoring the prevalence of viable and dead cariogenic bacteria in oral specimens and in vitro biofilms by qPCR combined with propidium monoazide
© Yasunaga et al.; licensee BioMed Central Ltd. 2013
Received: 20 December 2012
Accepted: 10 July 2013
Published: 13 July 2013
Streptococcus mutans and Streptococcus sobrinus are associated with the development of dental caries in humans. However, previous diagnostic systems are unsuitable for monitoring viable cell numbers in oral specimens. Assessing the relationship between the numbers of viable and dead bacterial cells and oral status is important for understanding oral infectious diseases. Propidium monoazide (PMA) has been reported to penetrate dead cells following membrane damage and to cross-link DNA, thereby inhibiting DNA amplification. In the present study, we established an assay for selective analysis of two viable human cariogenic pathogens, S. mutans and S. sobrinus, using PMA combined with real-time PCR (PMA-qPCR).
We designed species-specific primer sets for S. mutans and S. sobrinus, generated standard curves for measuring cell numbers, and evaluated the dynamic range of the assay. To determine the effectiveness of the assay, PMA was added to viable and autoclave-killed cell mixtures. PMA treatment effectively prevented DNA amplification from dead cells. No amplification of DNA from dead cells was observed in these organisms. In addition, we applied this assay to analyze viable cell numbers in oral specimens. A significant correlation was found between the number of viable S. mutans cells in saliva and that in plaque among caries-free patients, whereas no correlation was observed between saliva and carious dentin. The total and viable cell numbers in caries-positive saliva were significantly higher than those in caries-free saliva. Finally, we analyzed the usefulness of this assay for in vitro oral biofilm analysis. We applied PMA-qPCR for monitoring viable S. mutans cell numbers in vitro in planktonic cells and oral biofilm treated with hydrogen peroxide (H2O2). In planktonic cells, the number of viable cells decreased significantly with increasing H2O2 concentration, whereas only a small decrease was observed in biofilm cell numbers.
PMA-qPCR is potentially useful for quantifying viable cariogenic pathogens in oral specimens and is applicable to oral biofilm experiments. This assay will help to elucidate the relationship between the number of viable cells in oral specimens and the oral status.
KeywordsDental caries Dental plaque Biofilm Propidium monoazide (PMA) Quantification qPCR (Real-time PCR) Streptococcus mutans Streptococcus sobrinus Viability
Dental caries represents one of the most common infectious diseases afflicting humans . Of the mutans group of streptococci, Streptococcus mutans (serotype c, e, f, and k mutans streptococci) and Streptococcus sobrinus (serotype d and g mutans streptococci), which are Gram-positive oral commensal species, are strongly implicated as etiological agents associated with human dental caries. Previous investigations have reported that S. sobrinus has a higher acidogenic capacity compared with S. mutans, and the prevalence of S. sobrinus is more closely associated with high caries activity than is that of S. mutans[2, 3]. These studies suggest the importance of the diagnoses of infection by these organisms. Previously, several studies have reported methods for diagnosis of these organisms [4, 5]. However, DNA-based detection and quantification of specific bacteria cannot distinguish between live and dead bacteria. Bacterial DNA is degraded after the loss of cell viability; thus, the remaining DNA of already dead bacteria can still act as a template DNA for PCR. Consequently, DNA-based detection systems overestimate the cell population. However, we have not differentiated live and dead bacteria within the context of diagnosis of oral infectious diseases, including dental caries. In the present study, we successfully developed and evaluated a discriminative method between live and dead bacteria for the human cariogenic pathogens S. mutans and S. sobrinus using propidium monoazide (PMA). Previously, ethidium monoazide (EMA) was used for discriminating live from dead bacterial cells [6, 7]. EMA is a DNA/RNA intercalating substance that only enters bacterial cells with compromised cell walls and cell membranes. However, EMA was reported to possibly to penetrate viable cells of some bacterial species, resulting in underestimation of viable bacterial numbers [8–11]. Because PMA is less able to penetrate viable cells, more attention has been paid to PMA as an alternative to EMA . In the present study, we examined the population of live and dead bacteria in oral specimens. The relationships of cell viability with saliva and dental plaque or carious dentin were further analyzed. Finally, we analyzed the cell viability of S. mutans assessed by this PMA technique after treatment with hydrogen peroxide (H2O2) and proposed the usefulness of this technique for biofilm experiments. This is the first report to apply the combination of PMA plus real-time PCR (PMA-qPCR) for analysis of the prevalence of live/dead S. mutans cells in oral specimens and to reveal the relationship between cell numbers in saliva and cell numbers in dental plaque and/or carious dentin. Additionally, we applied PMA-qPCR for monitoring viable S. mutans cell numbers in vitro in planktonic cells and biofilm treated with various concentrations of H2O2 for possible application in biofilm experiments.
Specificities and sensitivities of the qPCR assay
Strains and amplification results for S. mutans and S. sobrinus
Primers used for amplification
S. mutans UA159
S. mutans Xc
S. mutans MT703R
S. mutans MT8148
S. mutans OMZ175
S. mutans NCTC10449
S. mutans Ingbritt
S. mutans GS5
S. sobrinus MT8145
S. sobrinus OU8
S. sobrinus OMZ176
S. sobrinus AHT-K
Effects of PMA and EMA on cell viability
Effect of PMA on defined ratios of viable and heat-killed bacterial suspensions
Spiking S. sobrinus cells with oral specimens
Correlation of viable S. mutans cell number assessed by PMA-qPCR and by culture
Quantitative discrimination of live/dead cariogenic bacterial cells in oral specimens
Next, we compared the number of cells in saliva from patients with and without dental caries. The mean totals of S. mutans cells (± S.D.) calculated by qPCR were 4.24 × 108 (±2.38×108) per 1 ml of saliva from patients with dental caries (n=21) and 1.02 × 108 (±5.07×107) per 1 ml of saliva from caries-free donors (n=24); viable cell numbers calculated by qPCR with PMA were 1.68 × 108 (±1.06×108) per 1 ml of saliva from patients with dental caries (n=21) and 4.45 × 107 (±3.31×107) per 1 ml of saliva from caries-free donors (n=24), representing 39.6% and 43.6% of the total cells, respectively (Figure 5B). Total cell number and viable cell number differed significantly between caries-positive and -negative saliva (p < 0.05 for each; Mann–Whitney test).
Streptococcus sobrinus was detected in only one patient with dental caries (data not shown). The total numbers of S. sobrinus cells calculated by qPCR without PMA were 8.14 × 107 CFU per 1 ml of saliva (32.5% were live cells) and 1.58 × 109 CFU per 1 mg carious dentin (7.84% were live cells).
Correlation of viable S. mutans cell number among oral specimens
Application of PMA-qPCR for monitoring live bacteria in biofilm and the planktonic phase
Streptococcus mutans and S. sobrinus are considered to be cariogenic pathogens in humans . Various studies have monitored the prevalence of caries-related organisms in oral specimens . However, attempts to differentiate between viable and dead bacteria in oral specimens in relation to dental caries have not been reported. In the present study, we initially developed a quantification method for discriminating live and dead cariogenic bacteria, specifically for S. mutans and S. sobrinus. Previous investigations have reported that EMA has a strong inhibitory effect on the amplification of genomic DNA from viable cells , and our study confirmed that EMA itself decreases cell viability. Therefore, all experiments were conducted with PMA, which penetrates a damaged cell membrane and intercalates into DNA, resulting in the inhibition of PCR, in combination with qPCR to quantitatively differentiate between viable and dead cells. We further performed a spiking experiment to evaluate whether this assay was applicable to oral specimens. In general, obtaining oral specimens that do not contain S. mutans is challenging, whereas obtaining S. sobrinus-free oral samples is relatively easy. For this reason, we performed PMA-qPCR amplification specific for S. sobrinus using S. sobrinus-free saliva and S. sobrinus-free dental plaque as an alternative in the spiking experiment. As shown in Figure 3, neither saliva nor dental plaque inhibited the PCR, indicating that this assay is applicable for measuring cariogenic bacteria in oral specimens.
We next examined the correlation between the numbers of viable S. mutans cells in oral specimens as detected by PMA-qPCR and by culture. We found a positive correlation between these quantification methods for both carious dentin and dental plaque. Compared with culture, the number of viable S. mutans cells was overestimated by PMA-qPCR. It may be that the culture method usually underestimates the cell number. The cell number determined by conventional qPCR correlated with the cell number determined by culture. Several previous investigations have reported that the cell number determined by qPCR correlated with CFU [14, 15]. However, compared with PMA-qPCR, conventional qPCR overestimated the cell number to a greater extent in both types of clinical specimens. Therefore, the cell culture count was closer to the number determined by PMA-qPCR than to that determined by conventional qPCR in the present study.
Monitoring viable bacterial cells in oral specimens provides information to help understand oral infectious diseases. When we compared the total and viable cell numbers in carious dentin from patients with dental caries and dental plaque from caries-free children, there was no significant difference between carious dentin and dental plaque in terms of either total number S. mutans cells or number of viable cells. We may not be able to simply compare the cell numbers in these specimens because the contents are not identical. Nevertheless, there was no significant difference in the percentage of viable cells between the specimens. However, there was a significant difference in total cell number and viable cell number between saliva from patients with dental caries and saliva from caries-free children. Monitoring of the viable cell number in relation to the total cell number in oral specimens has not previously been performed. To understand the variation in the viable cell number, both the viable and total cell numbers must be determined. To further understand cell viability in relation to dental caries, a greater number of specimens should be analyzed.
When the relationship between the number of viable S. mutans cells in saliva and in dental plaque from caries-free children was analyzed using PMA-qPCR, a positive correlation was found between viable S. mutans cells in saliva and in dental plaque. This result was consistent with previous reports . There was no significant correlation between the number of viable S. mutans cells in saliva and that in carious dentin from caries patients in the present study. Our data suggest that saliva reflects the number of viable cells in caries-free plaque, but not in carious dentin. Previous studies have found that the microbial biota in the dental plaque of children with severe early-childhood caries was significantly less diverse and less complex than that in caries-free children . Therefore, the viability of cariogenic bacteria in saliva may differ between caries-active and caries-free patients. This possibility should be explored in future studies.
Finally, we evaluated the number of viable of S. mutans cells in the planktonic phase and in biofilm. In the planktonic phase, the ratio of viable cells to total bacteria decreased with an increase in H2O2 concentration (34.7% at 0.0003% H2O2 and 10.0% at 0.003% H2O2). There was a significant difference in the viable/total bacterial ratio between 0% and 0.0003 and between 0% and 0.003% H2O2. However, the decreases in the viable/total cell ratio in biofilm at these concentrations were smaller (88.6% at 0.0003% H2O2 and 58.9% at 0.003% H2O2), and there was no significant difference between 0% and 0.0003 or 0.003% H2O2. These results suggest that PMA-qPCR is applicable for monitoring the numbers of viable and dead cells in biofilm. In biofilm experiments, a live/dead stain is sometimes used to distinguish visually between live and dead bacteria . Although PMA-qPCR is advantageous for quantifying viable cells, it does not provide the visualization obtained with live/dead staining. PMA-qPCR may be a powerful tool for monitoring the number of viable cells in oral biofilms.
We developed a discriminative quantification method for viable and dead S. mutans and S. sobrinus cells. We evaluated the potential of this assay and applied it to analyze the prevalence of live/dead cariogenic bacteria in oral specimens and to monitor live/dead cells in biofilm experiments. The ability to discrimination between live and dead bacterial cells in biofilm is essential for studying biofilm, and this assay will be helpful for oral biofilm research. Our assay will contribute to elucidating the role of viable bacteria in oral biofilm and saliva in relation to disease activities.
The 52 reference strains used in the present study were S. mutans UA159, S. mutans Xc, S. mutans MT703R, S. mutans MT8148, S. mutans OMZ175, S. mutans NCTC10449, S. mutans Ingbritt, S. mutans GS5, S. sobrinus MT8145, S. sobrinus OU8, S. sobrinus OMZ176, S. sobrinus AHT-K, Streptococcus S. downei Mfe28, S. downei S28, Streptococcus ratti BHT, S. ratti FA1, Streptococcus cricentus HS1, S. cricentus E49, Streptococcus mitis 903, Streptococcus sanguinis ATCC 10556, S. sanguinis ATCC 10557, S. sanguinis OMZ9, Streptococcus gordonii DL1, Streptococcus oralis ATCC 557, Streptococcus salivarius HHT, Streptococcus anginosus FW73, Streptococcus milleri NCTC10707, Lactobacillus rhamnosus JCM1136, L. rhamnosus JCM1561, L. rhamnosus JCM1563, L. rhamnosus JCM8134, L. rhamnosus JCM8135, L. rhamnosus JCM8135, Lactobacillus casei JCM8132, Porphyromonas gingivalis W83, P. gingivalis 33277, Treponema denticola ATCC 35405, Treponema medium ATCC 700293, Treponema vincenti ATCC 35580, Treponema pectinovorum ATCC 33768, Aggregatibacter actinomycetemcomitans Y4, A. actinomycetemcomitans JCM8577, A. actinomycetemcomitans SUNYaB67, A. actinomycetemcomitans SUNYaB75, Aggregatibacter naeslundii JCM8350, Prevotella loescheii JCM8530, Prevotella denticola JCM8525, Prevotella bivia JCM6331, Prevotella pallens JCM1140, Prevotella veroralis JCM6290, and Prevotella oralis ATCC 33322.
All patients were treated in accordance with the Helsinki Declaration regarding the participation of human subjects in medical research. Ethics clearance for the study was obtained from the Ethics Committee of Kyushu Dental University Hospital (reference number 11–40). The parents of participants were fully informed about the study and signed informed consent forms.
Study subjects and oral specimen sampling
Twenty-one subjects ranging in age from 3 to 10 years and who had dental caries were included in the caries group (mean age ± S.D. = 7.86 ± 0.43 years; 11 males and 10 females). A healthy (completely caries-free) control group consisted of 24 subjects (ages 3 to 12 years; mean age ± S.D. = 7.29 ± 0.56 years; 13 males and 11 females). The carious dentin was excavated from cavitated lesions. Before excavation of the carious dentin, the plaque on the surfaces of cavitated lesions was swiped. The dental plaque samples from healthy subjects were collected from the buccal or lingual surface of the second primary molar. Collected carious dentin and dental plaque were placed in 200 μl of PBS in a sterile 1.5-ml microcentrifuge tube. These samples were washed and placed in PBS solution adjusted to 1 mg per 100 μl. Saliva was collected from both the caries and healthy control groups. Fifty microliters of saliva was washed with PBS and used for analysis.
Bacterial counting from oral specimens on an agar plate
Serially diluted carious dentin or dental plaque was plated on a Mitis-Salivarius agar plate (Becton Dickinson, Franklin Lakes, NJ) supplemented with 150 g/l sucrose and 200 U/l bacitracin for selection of mutans streptococci (MSB agar). Bacterial counting was performed using a magnifying loupe.
Propidium monoazide treatment
For only viable cell quantification, PMA (3-amino-8-azido-5-[3-(diethylmethylammonio)propyl]-6-phenyl dichloride; Wako Pure Chemical, Osaka, Japan) treatment was performed for bacterial cells prior to DNA extraction, as previously described . Briefly, PMA was dissolved in 20% DMSO to produce a 25-mM stock solution. Following incubation with the dye for 5 min in the dark, similarly prepared cells were exposed for 5 min to a 500-W halogen light placed 15 cm above 500-μl samples in open microcentrifuge tubes on ice. The toxicity of PMA at 2.5–250 μM to S. mutans and S. sobrinus was analyzed at 37°C. In the present study, 25 μM PMA was employed for the analysis. All data presented are from triplicate independent cultures and/or biofilms.
Routine molecular biology techniques were performed as described by Sambrook et al. . Chromosomal DNA was isolated from the bacteria using a Puregene DNA isolation kit (Gentra Systems, Minneapolis, MN). Bacterial chromosomal DNA from oral specimens was isolated using MORA-extract (Cosmo Bio, Tokyo, Japan). Next, 150 μl of lysis buffer was added to the pellet. The lysed bacteria were transferred to a tube with glass beads and heated at 90°C for 10 min. The bacterial mixture was then disrupted using a Mini-Bead Beater (BioSpec Products, Bartlesville, OK) with 0.1-mm-diameter glass beads at 4,800 rpm for 2 min. Thereafter, 200 μl of SDS solution was added and heated at 90°C for 10 min. Next, 400 μl of phenol solution was added and mixed for 1 min. After centrifugation, the aliquot was subjected to ethanol precipitation and dissolved in 20 μl of TE buffer.
Preparation of biofilms and planktonic cells
To examine S. mutans strains for the ability to form biofilm under various H2O2 concentrations (serially diluted from 0–3%), the biofilm assay was performed. Bacterial cells were precultured overnight in chemically defined medium (CDM) supplemented with 0.5% sucrose, inoculated into 1 ml of 0.5% sucrose CDM (culture:CDM ratio, 1:50), and then incubated for 24 h under anaerobic conditions at 37°C in polystyrene 24-well plates (Corning, Inc., Corning, NY) with final H2O2 concentrations of 0–0.03% . The viable cell/total cell ratio in 0% H2O2 was considered to be 100%.
The Mann–Whitney test and Bonferroni’s test were used to determine statistical significance. A difference was deemed significant at P < 0.05.
quantitative polymerase chain reaction.
Support for the present study was provided by Grants-in-Aid (C) 25463257 (A.Y.), (B) 22390403 (T.A.), and (B) (Overseas Academic Research) 24406035 (T.A.) from the Ministry of Education, Culture, Sports, Science and Technology of Japan.
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