Detection of Streptococcus mutans in symptomatic and asymptomatic infected root canals

Abstract

Objectives

To investigate the presence of Streptococcus mutans in root canals of symptomatic necrotic teeth (SNT) and their associated acute apical abscesses (AAA) and in the root canals of asymptomatic necrotic teeth (ANT). It also aimed to investigate the presence of the cnm and cbm genes in specimens that harbored S. mutans.

Materials and methods

DNA was extracted from samples collected from 10 patients presenting pulpal necrosis associated with radiographic evidence of apical periodontitis (ANT) and from 10 patients in need of endodontic therapy due to the presence of pulpal necrosis (SNT) and AAA. The control group consisted of 10 patients with teeth with normal vital pulp and requiring endodontic treatment for prosthetic reasons. The presence of S. mutans was detected by quantitative real-time-PCR (qPCR) using species-specific primers. Samples harboring S. mutans were further evaluated for the presence of CBP genes by qPCR as well.

Results

All studied sites showed a high prevalence of S. mutans, except the control group. Specifically, 60% of ANT and 70% of AAA/SNT paired samples were positive for S. mutans. The cnm gene was detected positive for S. mutans only in ANT samples (66.6%). The cbm gene was not detected in any of the investigated sites.

Conclusions

S. mutans was found in high prevalence in both asymptomatic and symptomatic endodontic infections, including in abscesses, but it was not detected in the root canals of teeth with normal vital pulp. Interestingly, cnm⁺ S. mutans was only detected in asymptomatic/chronic primary endodontic infections associated with apical lesion. Therefore, it appears that cnm, and possibly other CBPs, may play an underestimated role in chronic endodontic infections.

Clinical relevance

A high prevalence of Streptococcus mutans cnm⁺ gene was detected only in asymptomatic primary endodontic infections associated with apical lesion. Therefore, it appears that this collagen-binding protein gene plays an underestimated role in asymptomatic/chronic endodontic infections.

Introduction

There are about 10¹⁰ microorganisms in the mouth, but under normal conditions, they do not have access to the pulpal tissues, as the enamel and cementum protect the dentin against the bacterial invasion [1]. If these protective layers of enamel and/or cementum are lost, the pulpo-dentin complex is exposed to the oral environment and placed at risk of infection [1].

Caries is the most common cause of pulp exposure and infection, resulting from a complex interaction of diet, the resident microbiota, and the host [1]. Dental caries affects millions of people [2] and it is a chronic infectious disease that involves multiple microorganisms, including Streptococcus mutans [3]. It is a cascade reaction which starts with dental caries and then progresses to pulpal disease, pulpitis, pulpal necrosis, and periapical disease (apical periodontitis), if the microbial stimuli are not removed [4]. The acute apical abscess, which is considered a late stage of apical periodontitis, is an inflammatory response of the apical area of the root canal to the pulpal infection. The infection can spread from the root canal to the surrounding tissues, resulting in cellulitis [5, 6]. Reports on dental caries suggest that severe tooth decay may induce a systemic immune response [3]. This occurs mainly when caries progresses into pulpal inflammation and necrosis, resulting in abscess or fistula formation.

The microorganisms of infected root canals and acute apical abscesses have usually been investigated by culture techniques. Recently, the use of more sensitive molecular approaches, including real-time PCR, 16S DNA sequencing, and next-generation sequencing, provides a better overview of the diversity of microbial population in healthy and diseased sites, detecting not only cultivable but also non-cultivable microorganisms [7, 8]. Even though Gram-negative anaerobes such as Prevotella, Porphyromonas, and Fusobacterium predominate in these sites, facultative Gram-positive Streptococcus, including S. mutans, have also been found [4, 7].

The detection of S. mutans in the oral sites has been subject of interest, not only due to its primary role in caries onset but also due to its association with extra-oral infections such as infective endocarditis (IE) [9–13]. The virulence of S. mutans as a dental pathogen mostly resides in its ability to (i) adhere and form biofilms on tooth surfaces, (ii) produce large quantities of organic acids from a wide range of carbohydrates, and (iii) tolerate adverse environmental conditions such low pH and oxidative stresses [13–15].

Recently, it has been demonstrated that a subset of S. mutans strains (approximately 15%) have the ability to invade and survive in the cytoplasm of non-professional phagocytic human endothelial and epithelial cells [16–18], due to the presence of bona fide collagen-binding proteins (CBPs), Cnm and Cbm. Their invasive behavior can facilitate persistence in the oral cavity through mitigation of environmental challenges such as host defenses, administered antibiotics, competition with bacterial commensals, and mechanical/chemical removal [18]. Of note, Cnm and Cbm are unique to S. mutans [18, 19] and Cnm-positive strains are more often isolated than Cbm-positive ones [10, 20]. Nevertheless, S. mutans strains that do not possess Cnm and Cbm, like the common laboratory strain UA159, cannot invade human cells and only marginally bind to collagen due to the presence of certain surface adhesins that have low affinity for collagen such as WapA and SpaP [19].

Interestingly, several works have demonstrated that S. mutans strains expressing CBPs are frequently associated with extra-oral infections such as infective endocarditis, hemorrhagic stroke, cerebral microbleeds, non-alcoholic steatohepatitis, and IgA nephropathy [21–24]. Recently, Nomura et al. [25] reported that S. mutans strains were present in inflamed pulp (pulpitis) at a high frequency (50%) of which about 20% harbored the cnm gene. The presence of invasive CBP-positive S. mutans in pulp tissues could increase the risk for bacteremia and other extra-oral pathologies, especially during root canal treatment, in which there is a possibility of extrusion of organic and inorganic debris [26].

Thus, the objective of this work was to investigate the presence of S. mutans in root canals of symptomatic necrotic teeth (SNT) and their associated acute apical abscesses (AAA) and in the root canals of asymptomatic necrotic teeth (ANT). It also aimed to investigate the presence of the cnm and cbm genes in specimens that harbored S. mutans.

Material and methods

Clinical and sampling procedures were based on previous studies [8, 27–29].

Ethical aspects and patients’ selection

The Human Research Ethics Committee of the Piracicaba Dental School, State University of Campinas-UNICAMP, Piracicaba, SP, Brazil, approved this work (CAAE 86140218.0.0000.5418) describing the methods for sampling collection. Patients signed an informed consent form prior to their participation in the study.

Patients requiring endodontic therapy with pulpal necrosis associated with acute apical abscess (n = 10) and patients with pulpal necrosis with radiographic evidence of apical periodontitis (chronic infection, n = 10) were selected. The control group consisted of 10 patients with caries-free teeth with normal vital pulp that have undergone endodontic treatment for prosthetic reasons. A total of 30 patients were enrolled in this study. A sample size of 10 patients per group was calculated based on the means and standard deviations of the pilot study and according to previous studies [8, 27–29].

Acute apical abscess was determined according to the criteria reported by the American Association of Endodontists Consensus Conference on diagnostic terminology [30]. Patients presented with spontaneous pain, extreme tenderness of the tooth to pressure, and swelling of associated tissues. They reported that over-the-counter analgesics were ineffective. Most of them experienced malaise, fever, and lymphadenopathy.

Inclusion and exclusion criteria

Inclusion criteria were as follows: healthy patients with no significant medical history; teeth with necrotic pulp tissues; no response to cold testing with Endo-Ice (Hygenic Corp., Akron, OH, USA); teeth that could be appropriately isolated and restored; mature root apexes on radiographs. Only asymptomatic patients should present periapical radiolucency on the X-ray.

Exclusion criteria were as follows: (a) patients who had received antibiotic treatment within the previous 3 months; (b) patients with general disease that fit in the American Society of Anesthesiology criteria 3 (ASA 3) or above; (c) teeth that could not be isolated with a rubber dam; (d) root canals exposed to the oral cavity; (e) presence of caries in the root surface; (f) teeth with periodontal pockets deeper than 3 mm; (g) history of recent dental trauma; (h) immature teeth; (i) teeth with intracanal medication or previous endodontic treatment.

Clinical and sampling procedures

Samples were collected from the following: (a) acute apical abscesses (AAA) and from the root canals in symptomatic patients; (b) from the root canals of asymptomatic patients; and (c) from the root canals of normal vital pulp teeth submitted to endodontic treatment due to prosthetic reasons.

Acute apical abscesses (AAA) from symptomatic subjects

Sampling from the purulent abscess was performed according to Montagner et al. [8] and Sousa et al. [31]. Briefly, after disinfection of the oral mucosa with 2% chlorhexidine (CHX) gel, CHX was inactivated with a gauze immersed in 5% Tween-80 and 0.07% (w/v) lecithin solution for 1 min, which was then cleaned with another gauze containing saline solution for the same period of time. Next, the acute apical abscess (AAA) samples from the intact swollen mucosa were taken by aspiration with a sterile syringe before surgical drainage, with 100 μl being immediately placed into a test tube containing sterile 900 μl of Tris buffer.

Root canals

Sampling from the root canals was performed according to Gomes et al. [32, 33]. In all three root canals sites (SNT, ANT, and healthy control-normal vital pulp), aseptic techniques were used throughout the acquisition of endodontic samples. Briefly, the tooth was isolated from the oral cavity with a rubber dam and the operatory field disinfected with 30% hydrogen peroxide followed by 2.5% sodium hypochlorite, which was inactivated with 5% sodium thiosulfate. The disinfection of the tooth surface was monitored by taking a swab sample from both external and internal surfaces of the crown and from its surrounding structure area and (a) streaking it on 5% defibrinated sheep blood Fastidious Anaerobe Agar ((FAA) LAB M, Heywood, Lancashire, UK) plate, which were incubated anaerobically and aerobically, respectively, for up to 14 days, and (b) extracting the DNA from the swab and running a PCR using universal bacterial primers. If any positive culture or presence of bands on the agarose gel was detected, the patient would be excluded from the research. The root canals were exposed by using sterile burs under manual irrigation with sterile saline solution. Samples were taken from both external tooth surface and operative field after disinfection to monitor the disinfection protocol. Sampling included only one root canal for each tooth. If the tooth was multi-rooted, either the largest canal in the root or the one presenting the greatest periradicular radiolucency was sampled in order to confine the microbial evaluation to a single site. A size #15 Hedstrom file was used with the aim of scraping the root canal walls and then a sterile paper point was introduced into the full length of the root canal, as determined in a preoperative radiograph, and kept in place for 1 min. In the case of a dry root canal, a second paper point moistened in sterile saline solution was used to ensure adequate sample acquisition. In the case of a wet root canal, multiple paper points (as needed) were used to absorb all fluid inside the canal. The paper points were immediately transferred to a test tube containing 1 ml of sterile Tris buffer.

DNA extraction and detection of bacterial DNA

DNA was extracted from samples using the MasterPure^™ Gram-Positive DNA Purification Kit (Epicentre, Illumina Inc, Madison, WI, USA) using the protocol provided by the supplier. To confirm the presence of bacterial DNA in all clinical samples, polymerase chain reaction (PCR) amplification was performed by using universal primers for bacteria as described by [34]. The presence of S. mutans in the specimens was determined by quantitative real-time PCR (qPCR) using S. mutans–specific primers (Table 1) [36]. Briefly, for each qPCR reaction performed, standard curves containing serial dilutions of S. mutans genomic DNA and negative controls (water instead of DNA) were included. qReactions were considered successful when no amplification of the negative controls was observed and the standard curve efficiency was ~ 100% (± 10%), correlation coefficient was above 0.90, and the slope was − 3.5 (± 0.3). Of note, primer specificity for S. mutans has been demonstrated previously [36].

Table 1.

PCR primers were used in this study

Name	Purpose	Sequence (5’−3’)	Product size	References
Prbac1-F	Presence of bacterial DNA	ACT ACG TGC CAG CAG CC GGA CTA CCA GGG TATCTA ATC	296–300	Rupf et al. [35]
Prbac2-R
Smut3368-F	Detection of S. mutans	GCC TAC AGC TCA GAG ATG CTA TTCT	114 bp	Yoshida et al. [36]
Smut3481-R		GCC ATA CAC CAC TCA TGA ATT GA
cbm-F	Detection of cbm	GAT GGT ACC TAT GTTGAT TTG	99 bp	Aviles-Reyes et al. [37]
cbm-R		CCG GTA ACG TTA TGG AGA TTA TTG
cnm-CF	Detection of cnm	CTG AGG TTA CTG TCGTTA AA	137 bp	Nomura et al. [38]
cnm-CR		CAC TGT CTA CAT AAGCAT TC

Samples that tested positive for S. mutans were further evaluated for the presence of CBP-encoding genes by qPCR using cnm- and cbm-specific primers and protocols from previous studies (Table 1) [37, 38]. UA159 (cnm⁻ and cbm⁻), OMZ175 (cnm⁺ and cbm⁻), and LAR01 (cnm⁻ and cbm⁺) were used as reference strains and also as positive and negative controls when properly indicated. Nuclease-free water was used as negative control in all qPCR reactions. In specimens where amplification of cnm or cbm was detected by qPCR, the amplicons were purified using a PCR purification kit (Zymogen, Irvine, CA, USA), sequenced using gene-specific primers, and the sequences subjected to BLAST search analysis at NCBI.

Statistical analysis

The data collected were typed onto a spreadsheet and analyzed by using SPSS for Windows (SPSS Inc., Chicago, IL, USA). Absolute and relative frequencies and the 95% confidence interval were calculated.

Results

The disinfection protocol was proven effective as there was no microbial growth on the control plates after 14 days and no DNA band in the agarose gels.

With the exception of the control group with normal vital pulp, where no bacterial DNA was detected, all symptomatic and asymptomatic patients’ samples contained bacterial DNA (Table 2).

Table 2.

Prevalence of S. mutans and of the genes coding for the CBPs, cnm and cbm, in root canals of teeth with normal vital pulp (control), in root canals of asymptomatic necrotic teeth (ANT) and in root canals of symptomatic necrotic teeth (SNT) and their associated acute apical abscesses (AAA)

Sample sites	n	Bacterial DNA (number of positive cases/total)	S. mutans+ (number of positive cases/total)	cnm+ (number of positive cases/total)	cbm+ (number of positive cases/total)
Control	10	0% (0/10)	-	-	-
ANT	10	100% (10/10)	60% (6/10)	66.6% (4/6)	0% (0/6)
SNT	10	100% (10/10)	70% (7/10)	0% (0/7)	0% (0/7)
AAA	10	100% (10/10)	70% (7/10)	0% (0/7)	0% (0/7)

There was as higher prevalence of S. mutans in AAA/SNT paired samples (70%) than in ANT (60%).

Among the S. mutans positive specimens, 66.6% of the ANT samples were cnm⁺ whereas the cnm gene was not detected in any of the AAA or SNT samples. All cnm amplicons were subjected to Sanger sequencing to confirm the specificity of the primers. In addition, the cbm gene was not detected in any of the investigated sites.

Discussion

The validity of the results in clinical trials is influenced by sample sizes. Despite appearing to be a small sample size, factors such as the stringent exclusion/inclusion criteria for the selection of patients in each group, the paired samples from the symptomatic group, and the inclusion of patients with normal vital pulp still allowed a valid and robust statistical analysis. Bacterial DNA was detected in all collected samples from ANT and SNC root canals and their associated abscesses, but not in the caries-free teeth with normal vital pulp, confirming the well-established contribution of microorganisms in the onset of endodontic infections [8, 29, 39, 40].

In our study, using a highly sensitive and specific approach (qPCR), we found a high prevalence (60–70%) of S. mutans in the three investigated sites (ANT, SNC, and AAA). Previous studies in symptomatic primary endodontic infection, using culture technique without the use of selective media/growth conditions for S. mutans, reported their presence in 10% of the cases [29]. On the other hand, Nomura et al. [25] detected S. mutans in inflamed pulp at a much higher frequency (~ 50%) than that observed in dental plaque of the general population [25]. Other studies have also found a high prevalence of Streptococcus sp. using non-specific molecular methods, such as checkerboard DNA-DNA hybridization (45%) [7] and clonal analysis (over 30%) [37].

An earlier study investigated the bacterial profile of purulent exudate from AAA and from root canals of teeth with asymptomatic apical periodontitis using a closed-ended semi-quantitative reverse-capture checkerboard approach targeting 40 bacterial species/phylotypes [40]. They found the presence of Streptococcus species in AAA (45%) and in the root canals of asymptomatic patients (~ 33%). In the present study, only S. mutans was investigated and they were present in 70% of the cases with AAA (70%) and in 60% of ANT (60%).

It is certain that due to the polymicrobial nature of endodontic infections, other bacteria, like Enterorococcus faecalis, possess collagen-binding proteins (CBPs). However, Cnm and Cbm are the only bona fide CBPs that are uniquely found in S. mutans [18, 19]. Due to the association of the presence and expression of these CBPs with bacterial endocarditis, we decided to study the role of S. mutans in endodontic infections. Further studies are necessary to establish any trend linking specific host factors to expression of these genes under pathogenic conditions.

Cnm is required for S. mutans adherence to and intracellular invasion of endothelial and epithelial cells, being an important virulence factor of this invasive bacterium [18, 23, 41]. Approximately 15% of S. mutans strains isolated from dental plaque or saliva of subjects from Asia and Europe carry the cnm gene [10, 42]. Our results revealed an unexpected elevated frequency of cnm⁺ S. mutans in the ANT group (66.6%), a much higher frequency than that found for another clinical endodontic condition, inflamed pulp, where approximately 20% of these samples tested positive for cnm through conventional PCR methods [25].

It can be speculated that the high frequency of cnm⁺ S. mutans in asymptomatic/chronic endodontic infections (ANT) detected in the present study may be explained by the slow progression of this type of infection, which ultimately results in the formation of an apical lesion. In asymptomatic chronic infections, bacteria utilize mechanisms to subvert the immune system to avoid clearance, which is markedly different in acute symptomatic cases where the infection process is fast and results in a hyperinflammatory state. Notably, cnm has been shown to inhibit activation of the classical complement pathway through direct binding to the collagen-like domain of component C1q [43], hindering activation of the complement cascade. In addition, cnm enables S. mutans to invade and persist in the cytoplasm of non-professional phagocytic cells, a feature that has been attributed to chronic and systemic infections [17–19, 41, 42, 44]. Thus, it appears that cnm-positive S. mutans can be correlated with chronic endodontic infections that, if left untreated, could ultimately lead to extra-oral pathologies, especially in patients with pre-existing cardiovascular conditions or compromised immune system [41, 45–47].

One of the major factors to achieve success in the endodontic treatment is related to the microbial reduction from root canals [45]. During chemo-mechanical preparation, endodontic files and root canal irrigants are used to eliminate organic/inorganic tissues, which may harbor bacteria [45]. As a consequence, organic and inorganic debris, bacteria, and irrigants may extrude into the periapical tissues, causing pain, and a possible transient bacteremia, which happens regardless of the instrumentation limit, with a higher probability of occurrence beyond the apex [46, 47]. Some systemic diseases have their cause related to oral bacteremia, including infective endocarditis, which is classified as one of the most serious diseases [44]. Knowing that some microorganisms, especially S. mutans can be hypervirulent, dental surgeons should always verify the risk of each patient for systemic diseases. During root canal treatment, measures should be taken to decrease the amount of extruded debris, including cleaning the coronal third first before the apical instrumentation; the use of an effective irrigation/ aspiration system; and use of auxiliary chemical substance with antimicrobial properties, among others [1].

The present work is pioneer in the detection of genes cnm and cbm in symptomatic and asymptomatic endodontic infections, which makes it original, relevant, and a precursor of studies on S. mutans virulence factors in endodontic infections. Further studies with a higher number of cases are necessary to conclusively establish an association of cnm⁺ S. mutans with chronic endodontic infections and increased risk for systemic complications.

In conclusion, S. mutans was found in high prevalence in both asymptomatic and symptomatic endodontic infections, including in abscesses, but it was not detected in the root canals of teeth with normal vital pulp. Interestingly, cnm⁺ S. mutans was only detected in asymptomatic/chronic primary endodontic infections associated with apical lesion. Therefore, it appears that cnm, and possibly other CBPs, may play an underestimated role in chronic endodontic infections.