Characteristics of Clinical Isolates of Streptococcus mutans

Abstract

Dental caries is an infectious disease which remains a significant health problem all over the world. The purpose of the study was to characterise a collection of 60 clinical isolates of S. mutans from adults’ and children’s dental plaque (natural biofilm). The paper describes the process of isolation, identification, analysis of biofilm formation and collection testing for the presence of 13 two-component systems (TCS) identified earlier in reference strain ATCC 700610 (UA159). In the case of S. mutans strains, plaque formation is specifically influenced by binary systems. All isolated strains of S. mutans form biofilm at high levels and possess a set of 26 genes forming TSC binary systems, which have an important role in plaque formation.

1. Introduction

In 1 mL of human saliva, there are approximately 20 to 400 million microorganisms belonging to more than 700 species, including a particularly important one discovered by Clerke in 1924, Streptococcus mutans [1,2,3]. In the oral cavity, certain species occupy specific places called ecological niches, which is due to the differences in aerobic and nutritional conditions prevailing in its particular parts. The major Gram-positive, non-haemolytic (ɣ-haemolytic) species of oral streptococcal bacteraemia include S. mutans. It is a facultative anaerobe closely related to the etiopathogenesis of dental caries, and it has the ability to produce plaque (i.e., naturally occurring bacterial biofilm), which in turn is an important factor in its virulence. Importantly, microbiota of the oral cavity, along with the immune system, is an essential element of the body’s defence against infections. Among the bacteria that make up the microbiota, there are opportunistic pathogens which are controlled by a well-functioning immune system. Oral cavity microbiota reaches a certain level of stability in adulthood; however, its state can be easily disrupted, for example, by insufficient oral hygiene and tooth loss [4,5,6]. It is noteworthy that during clinical research, S. mutans strains were found in both healthy people (without carious defects) and patients with dental caries. Despite tremendous progress in civilization, dental caries continues to pose a challenge in dentistry. The availability of modern research methods provides an opportunity to understand complex processes occurring during caries development. From the point of view of clinical control of this disease, elimination of the etiological agent of caries, i.e., dental plaque, seems to be the most important factor [7,8,9]. It turns out that just a few minutes after brushing, the surface of tooth enamel is covered with pellicle containing glycoproteins from saliva, which plays an important role in protecting and remineralising the enamel. Importantly, the pellicle does not contain bacteria and the mechanism of its formation is based on physical and chemical interactions at the molecular level between the components of saliva and tooth surface Van der Waals forces, hydrophobic interaction and others. The next step in dental plaque formation is the adhesion of pioneer bacteria in saliva to the pellicle. At this stage, adhesion is still reversible. From the moment the bacteria begin to secrete extracellular polymeric substances (EPS), bacterial adhesion to the pellicle and to themselves becomes stronger. Dental plaque pioneer bacteria include: Actinomyces spp., Streptococcus sanguinis, S. oralis, S. mitis, Haemophilus spp. and Neiseria spp. [10,11]. The next step in the formation of bacterial plaque is its maturation. Once the pioneer bacteria attach to the pellicle, specific places of connection are created for subsequent species that have the ability to identify polysaccharides and receptor proteins on the surface of the pioneer bacteria. As a result of this process, they begin to form co-aggregates. Later colonising bacteria include: Fusobacterium nucleatum, Treponema spp., Tannerella forsythensis, Porphyromonas gingivalis and others. During plaque maturation, its composition changes as the number of bacteria belonging to Streptococcus and Neiseria types decreases and the number of bacteria of Actinomyces, Fusobacterium, Veillonella or Corynebacterium types increases [12,13,14]. A mature biofilm consists of many porous layers containing water channels, through which nutrients from saliva and waste products are delivered to the bacteria. In a mature biofilm, bacteria can detach individually and in whole plaque fragments as a result of the erosive force of saliva, and this phenomenon is called biofilm dispersion. Bacteria can also actively detach from the biofilm, seeking nutrient sources and new places of colonisation. A mature plaque has a complex bacterial structure with numerous interactions. These interactions can be cooperative, for instance, when bacteria influence each other in positive ways during metabolic communication, but they can also compete for nutrients and places of connection. However, the benefits to the bacteria that are elements of dental plaque outweigh the possible losses, as they are less sensitive to environmental conditions (changing oxygen and nutrients level) and exposure to oral hygiene or medical products, including antibiotics. The cause of dental caries is usually supragingival biofilm; subgingival biofilm causes the development of gingivitis and periodontal disease. Strains of S. mutans usually colonise hard surfaces; therefore, tooth loss correlates with a decrease in the presence of this species of bacteria. It can also colonise dentures and fillings. The micro-gaps between the tooth tissue and the filling can be colonised by S. mutans and the porous plaque can lead to the development of secondary caries. More than half of all interventions in restorative dentistry involve dealing with that problem. By understanding the interactions occurring within the dental plaque and the mechanisms responsible for its formation, it is possible to develop effective treatments for oral diseases, which are often the result of an imbalance in the naturally occurring microbiota [10,11,15].

The main purpose of the presented study was to create a collection of clinical S. mutans strains and characterise them. S. mutans strains were collected from adults’ and children’s dental plaque; the next step was strain isolation and identification, analysis of biofilm formation and checking the presence of 13 two-component systems (TCS) reported in the literature.

2. Materials and Methods

2.1. Isolation and Identification of S. mutans Strains

Dental plaque samples were collected from 30 adults (permanent teeth) and from 30 children (deciduous teeth) in cooperation with the Department of Conservative Dentistry and Endodontics of the Medical University of Warsaw. Isolation of the strains consisted of taking a swab from the dental plaque of volunteers registered in the Department of Conservative Dentistry and Endodontics. On the day of taking swabs, patients were asked not to brush their teeth, and white dental plaque was collected and then cultivated on Mitis Salivarius Agar (Difco™ Mitis Salivarius Agar, BD) supplemented with sucrose, bacitracin and potassium telluride.

Strains were cultured under conditions of reduced oxygen, 5% CO₂ at temperature 37 °C. They were then subjected to identification based on biochemical traits using the Vitek 2 Compact automated system with GP cards (bioMerieux).

In order to further analyse the accumulated collection of isolates, their antibiotic resistance was tested using the Vitek 2 Compact automated system (AST-ST01 cards). The card is aimed at determining the antibiotic susceptibility of bacteria of the species Streptococcus pneumoniae, beta-haemolytic Streptococcus and Streptococcus viridans for all clinical materials. As for the present study, the following antibiotic panel was used to determine the antibiotic susceptibility of S. mutans (

Table 1. Antibiotic panel of AST-ST01 card.

AST-ST01 Streptococcus pneumoniae, Beta-haemolytic Streptococcus, Streptococcus viridans
Antibiotic	MIC Scope
Ampicillin	0.25–16
Benzylpenicillin	0.06–8
Cefotaxime	0.12–8
Ceftriaxone	0.12–8
Clindamycin	0.25–1
Erythromycin	0.12–8
ICR S. agalactiae, S. pyogenes	NEG/POS
Levofloxacin	0.25–16
Linezolid	2–8
Tetracycline	0.25–16
Trimethoprim/Sulfameth	10 (0.5/9.5)–320 (16/304)
Vancomycin	0.12–8

).

2.2. Biofilm Formation of Clinical Isolates of S. mutans

The study began by testing a biofilm culture method for clinical isolates of S. mutans. BHI medium with 5% sucrose was used to obtain bacterial cultures. Isolated colonies were suspended in 0.8% NaCl solution to obtain an inoculum of a density of 10⁹ CFU ml⁻¹. The suspension was diluted with liquid BHI medium supplemented with 5% sucrose. Biofilm was cultured on polystyrene, sterile 96-well microtiter plates (Medlab Products). Incubation was carried out for 12 h at 36.6 °C in a reduced oxygen presence. After rinsing off the cells not bound to the polystyrene surface, the biofilm formed in the wells of the titration plates was stained with crystal violet. This compound binds to negatively charged molecules such as acidic polysaccharides and nucleic acids, making it possible to assess the entire mass of the biofilm formed [1,16]. After rinsing, 96% ethanol was added to the wells and absorbance was measured at the wavelength of 540 nm. The negative sample was a non-biofilm-forming bacterial strain that was selected from the clinical strain collection of the Department of Pharmaceutical Microbiology (ZMF-01). S. mutans reference strain UA159 (ATCC 700610) was used as a positive control. The determination of the biofilm formation level for each strain was performed in three replications.

2.3. Binary Regulatory Systems of S. mutans Clinical Isolates

All 60 clinical isolates were tested for the presence of 13 binary systems (TCS VicKR, CiaHR, LiaRS, ComDE, HKRR5, NsrRS, HKRR7, BceRS, HKRR9, LcrRS, HKRR11, HKRR12 and HKRR13) [2]. Based on the nucleotide sequence of S. mutans UA159 (ATCC 700610), appropriate primers were designed using the Primer 3 system (Primer3 Input (version 0.4.0)) to identify both genes encoding the sensor and regulatory domains of 13 TCS (

Table 2. TCS identification primers.

Primers/Multiplex	Product Size	TCS Name
SMU_1516 HK-F CGGCTTCTTGCCTTATCAAC SMU_1516 HK-R GTAAAGTCCTATTTAGAAGCTTTGGA SMU_1517 RR-F CCCCAAACCGTTTCAAGTAA SMU_1517 RR-R AAAATATTGAATCCGCAGTGG	510 bp 211 bp	VicKR
SMU_1128 HK-F GTCTTGACGTCCAGCCAAAT SMU_1128 HK-R CGATTATTATCAGTGTGATGATTGTTT SMU_1129 RR-FGAAGAATTAAAAATGCGTATTCAGG SMU_1129 RR-R CAAAAATTTGTGACTTAGGTAAAATGA	529 bp 211 bp	CiaHR
SMU_486 HK-F GCTGATTGGCTTGTTCTTGA SMU_486 HK-R TGAAAGTGTCTTTCCTTCTAATTCTG SMU_487 RR-F ATCGGTGAGGCTAGCAATG SMU_487 RR-R TAGAATTTTGGCTTCTTTCCAA	520 bp 150 bp	LiaRS
SMU_1916 HK-F TCTTTGGTGGAATTCTGAATGA SMU_1916 HK-R AATGAGATAATGGCACAAAAGGA SMU_1917 RR-FATTGACCATTCTTCTGGCTGTT SMU_1917 RR-R TGAGTTTATGCCCCTCACTT	500 bp 140 bp	ComDE
SMU_577 HK-FACCAGACGGTTGTTCCTTGA SMU_577 HK-R TGATGCCAACAAAGCTCGAT SMU_576 RR-F CTGCAGGAAATAATTGGTCTTG SMU_576 RR-R CAGCTACGACAGAAAAGAAAGG	500 bp 200 bp	HKRR5
SMU_660 HK-F AAAAACCTGCAGCAACAAGC SMU_660 HK-R AGCAGTTCCGTATTCCCTTT SMU_659 RR-F AGTTTTTGTCGGGACATTCG SMU_659 RR-R CCAGACTAGCATGGTGCTCA	500 bp 199 bp	NsrRS
SMU_928 HK-FAAGGAGGTAGGAAATCGAGGA SMU_928 HK-R TGTTTCGCCAGTCATTAATTCTT SMU_927 RR-F ATGGACAAGATGCTATCGAAAAA SMU_927 RR-R TAATCATCAGCACCTGCCTCT	501 bp 199 bp	HKRR7
SMU_1009 HK-F CATTTTATACTGGCGGTTCCA SMU_1009 HK-R CCATCAATTGTCAAAGAAAGGTC SMU_1008 RR-F GCCCTATTTCAATGGCTTTT SMU_1008 RR-R TGCTTAGTGAACTCGTTAGCAC	459 bp 210 bp	BceRS
SMU_1037 HK-F TCTCAGGATCTGTCTCAAATGG SMU_1037 HK-R CTCTGAGTCAACAGATTGAAGAAAA SMU_1038 RR-F GCTCTTTCCAAACCGATTCA SMU_1038 RR-R AAGCCACAATCCAGCAACTA	495 bp 200 bp	HKRR9
SMU_1145 HK-F TGGCATCACCCTTTACCAAT SMU_1145 HK-R TGTTCTTTTTAGTCATTCAAAGCTG SMU_1146 RR-F TGCAGACCCCAAACTTTTTC SMU_1146 RR-R TTTAAAAAGAGCCTATCCTGAAAA	500 bp 200 bp	LcrRS
SMU_1548 HK-F CCCCACGTTTGATCGTAATC SMU_1548 HK-R GAACAGTATTGCTGTCTTTTTGATG SMU_1547 RR-F CACTAAGCGGATTGCTGTCA SMU_1547 RR-R GGATTCGTCAGCACCAAGAT	551 bp 219 bp	HKRR11
SMU_1814 HK-F CAAGCCCATACCGCTTCTT SMU_1814 HK-R CCTACTCTGGTTGAAAGTCACTACA SMU_1815 RR-F GCTTTGAGGAGTTTTTCTCGAT SMU_1815 RR-R CTGACCCCAACAGAAATTCA	450 bp 175 bp	HKRR12
SMU_1965 HK-F ATCATAATGCAGACTGACCTGTAGC SMU_1965 HK-R TCGCTCAATATCATTGTTTTTCT SMU_1964 RR-F AATCGCTAATTGCGTTCGAT SMU_1964 RR-R TCACAAATGGCGCAGTCTAA	450 bp 216 bp	HKRR13

). The presence of all the above two-component regulatory systems was found in the genome of the S. mutans reference strain UA159 and is considered to be linked to the biofilm formation process [17]. In this study, a multiplex PCR technique was developed and applied for this purpose.

The PCR template was prepared by collecting a small number of cells from the colony, resuspending in NaCl solution (0.9%) and heating at 100 °C for 15 min. The total volume of the multiplex PCR reaction mixture was 25 µL: 2.0 µL genomic DNA, 2.5 µL Taq buffer with KCl, 1.5 µL 25 mM MgCl₂, 0.5 µL 10 mM dNTP mixture, 2.5 µL each primer, 0.2 µL Taq DNA polymerase and 8.3 µL water. The positive control was a reaction mixture containing DNA from S. mutans reference strain, and the negative control was a reaction mixture without DNA added. Positive and negative controls were performed at each PCR amplification. Amplifications for all genes were carried out under the following conditions: 95 °C for 5 min, 95 °C for 30 s, 54 °C for 1 min, 72 °C for 1 min (25 cycles), final elongation of 72 °C for 5 min. Electrophoresis of PCR products was performed in a 1.8% agarose gel in TAE buffer at room temperature at a constant voltage of 80 V. A solution of bromophenol blue with glycerol was used for sample loading and as an indicator of DNA migration rate. For visualisation of DNA under UV light, gels were stained with an aqueous solution of ethidium bromide at a final concentration of 0.5 µg/mL and analysed using a fluorescently labelled gel documentation system /Syngene/ model: G BOX F3.

The study procedure was approved by the Bioethical Committee of the Medical University of Warsaw (Opinion no. KB/62/2012).

Written informed consent was obtained from all patients prior to enrolment.

3. Results and Discussion

Sixty clinical S. mutans strains were isolated from 60 different individuals, 30 from adults and 30 from children with the symptoms of caries. The isolated strains grew only on sucrose medium. The strains were identified using the automated Vitek GP card system (bioMerieux).

No significant differences were observed in the biochemical properties of strains from adults and children. The biochemical panel is shown in

Table 3. Illustrative biochemical profile of S. mutans (GP card) of a randomly selected strain. The biochemical profile was identical for all strains.

AMY	+	PIPLC	−	dXYL	−	ADH1	−	BGAL	−	AGLU	(−)
APPA	−	CDEX	−	AspA	−	BGAR	−	AMAN	−	PHOS	−
LeuA	(−)	ProA	−	BGURr	−	AGAL	−	Pyr A	−	BGUR	−
AlaA	+	TyrA	−	dSOR	+	URE	−	POLYB	+	dGAL	+
dRIB	−	ILATk	−	Lac	+	NAG	−	dMAL	+	BACI	+
NOVO	+	NC6.5	−	dMAN	+	dMNE	+	MBdG	+	PUL	−
dRAF	+	O129R	−	SAL	+	SAC	+	dTRE	+	ADH2s	−
OPTO	+

AMY (d-amygdalin), PIPLC (phosphatidylinositol phospholipase c), dXYL (d-xylose), ADH1 (arginine dihydrolase), BGAL (beta-galactosidase), AGLU (alpha-glucosidase), APPA (ala-phe-pro arylamidase), CDEX (cyclodextrin), AspA (l-aspartate arylamidase), BGAR (beta galactopyranosidase), AMAN (alpha-mannosidase), PHOS (phosphatase), LeuA (leucine arylamidase), Pro A (l-proline arylamidase), BGURr (beta glucuronidase),AGAL(alpha-galactosidase), PyrA (l-pyrrolidonyl-arylamidase), BGUR (beta-glucuronidase), AlaA (alanine arylamidase), TyrA (tyrosine arylamidase), dSOR (d-sorbitol), URE (urease), POLYB (polymixin b resistance), dGAL (d-galactose), dRIB (d-ribose), ILATk (l-lactate alkalinisation), LAC (lactose), NAG (n-acetyl-d-glucosamine), dMAL (d-maltose), BACI (bacitracin resistance), NOVO (novobiocin resistance), NC6.5 (growth in 6,5% NaCl), dMAN (d-mannitol), dMNE (d-mannose), MBdG (methyl-b-d-glucopyranoside), PUL (pullulan), dRAF (d-raffinose), O129R (O/129 resistance), SAL (salicin), SAC (saccharose/sucrose), dTRE (d-trehalose), ADHR2s (arginine dihydrolase 2), OPTO (optochin resistance).

. The method of isolation and identification of strains was found to be effective and it provided reproducible results. The biochemical panel of the strain is typical for S. mutans strains isolated from the oral cavity of Polish patients [18].

The drug resistance of each of the S. mutans strains was determined. The system classified all tested isolates as sensitive (S) to the antibiotics listed in

Table 4. Drug susceptibility panel—AST-ST01 Card (bioMerieux).

Antimicrobial	MIC	Interpretation
Benzylpenicillin	<=0.06	Sensitive
Ampicillin	<=0.25	Sensitive
Cefotaxime	<=0.12	Sensitive
Ceftriaxone	<=0.12	Sensitive
Erythromycin	<=0.12	Insufficient evidence that species is good target for therapy
Clindamycin	<=0.25	Sensitive
Vancomycin	1	Sensitive

.

Another element in the characterisation of the strains was the assessment of biofilm production capacity.

The year 1986 marks the beginning of research into signal transduction phenomena in bacteria. At that time, Nixon et al. first used the term two-component system (TCS) [3]. The model two-component regulatory system consists of a membrane sensor protein, which is histidine kinase (HK), which receives a signal from the environment, and a cytoplasmic regulatory protein—the response regulator (RR), which, after a conformational change resulting from signal reception, regulates the expression of the target gene. As indicated in the literature, binary regulatory systems are involved in many S. mutans life processes, including biofilm formation, which is the main virulence factor of this species and plays a fundamental role in plaque formation. Compared to S. mutans reference strain UA159 ATCC 700610, strains with mutations in both vicK and vicR genes form a biofilm that differs from that typical of this species [19,20,21,22].

Determination of the biofilm level was performed using the crystal violet staining method. The applied method gave reproducible results and showed that all tested strains formed biofilm in vitro at the expected high level, comparable to the reference strain S. mutans UA 159 (Scheme 1, bar 2).

In preparing the multiplex PCR analysis, all amplifications were performed for the S. mutans reference strain UA159, in each case obtaining a product of the expected size. Analysis of 26 TCS genes was performed for all 60 strains using multiplex PCR. The results of TCS VicKR and TCS CiaHR analysis for selected isolates are presented (Figure 1 and Figure 2). Amplification was obtained for all 26 genes analysed in all 60 strains.

Based on the present study, TCS are present in the clinical isolates of S. mutans tested, as 26 TCS genes were confirmed in all of them, which may indicate that there is a good place for drugs and other anti-caries precisely because of their prevalence. However, it should be borne in mind that the determination of the presence of genes does not provide information on the level of their expression in individual strains; there may be fundamental differences. The acid-forming activity is essential to the process of the formation of caries, which leads to damage of the enamel and initiates the tooth decay process. The expression level of TCS and the activity of genes that encode proteins related to the fermentation of sugars should be assessed in separate studies.

The presented results give insight into the biochemical and genetic characteristics of the collected set of isolates, and the results are fully reproducible. The existence of common features of S. mutans strains obtained from mothers and their children has been documented in the literature on the subject [23]. Based on the research performed for this study, it can be concluded that there are no significant differences in biochemical and genetic characteristics and there are no differences in the biofilm formation capacity between the strains derived from children and adults, and among those in children or adults alone. The S. mutans strains, which were the subject of the study, are characterised by high similarity. It should be noted that the strains were isolated from different, unrelated individuals who did not live together.

Efforts have been made to identify S. mutans strains of special risk [18]. In the case of such strains, dentists should take preventive measures to protect patients from developing caries. Strains of S. mutans are also involved in the colonisation of dentures and implants. The colonisation of implants can lead to the development of peri-implantitis. The risk of poor denture hygiene is particularly high in the case of elderly people. The presence of biofilm on medical products can lead not only to oral infections, such as denture stomatitis, but also to aspiration pneumonia and chronic obstructive pulmonary disease. Factors such as the presence of TCS systems and the level of biofilm formation seemed to be important elements in the characterisation of S. mutans, which would allow one to identify the most dangerous strains. Based on the obtained results, all S. mutans strains isolated from the oral cavity have TCS systems and produce high levels of biofilm. The collected data indicate that those characteristics are of key importance for the biology of S. mutans and do not allow those features to be used for strain differentiation.

4. Conclusions

Caries is an infectious disease which, despite the progress in dentistry and hygiene, remains a significant health problem in Polish society. Therefore, it seems important to broaden the knowledge in that field, even more so because, based on the data in the literature on the subject, S. mutans may appear in the oral cavity of a child even before the appearance of deciduous teeth, settling on the surface of the tongue [10].

The extensive research on the phenomenon of biofilm confirms that approximately 95% of microorganisms that live in the natural environment form biofilms [6,8,9]. Bacterial cells of such a community are characterised by high viability and resistance to many antibiotics and disinfectants.

The research for this study was conducted on 60 S. mutans strains obtained from 30 adult patients and 30 children with various types of carious lesions. Those were unrelated individuals who did not live together.

Drug resistance of all strains was determined with the use of the standard AST-ST01 card (bioMerieux). The sensitivity of all tested strains to all analysed antibiotics was demonstrated. A previously published study on Polish strains indicated their high drug susceptibility [18]. The results obtained in other countries confirm higher drug resistance of the strains [24].

The next element of the study was to determine the level of biofilm produced by each strain.

In the literature, a biofilm is defined as an organised cluster of bacteria, made up of cells permanently attached to a solid surface [25]. Dental plaque in the mouth is a particular type of biofilm, for the formation of which glycoproteins from saliva are necessary. The cause of caries is the so-called supragingival plaque biofilm, dominated mainly by Gram-positive bacteria, including S. mutans [7,10]. The bacteria responsible for the development of caries, as a result of the fermentation of sugars to organic acids (mainly lactic), lowers the pH of the plaque, which promotes the demineralisation of the enamel and allows various bacteria to process the destruction of tooth tissue.

Determination of the biofilm level was performed using the crystal violet staining method. All analysed strains produced a large amount of biofilm, similar to that of the reference strain.

Another element of the analysis was to identify the genes that constitute the two-component TCS system. Based on the data in the literature on the subject, in the case of S. mutans strains, those genes influence plaque formation. Therefore, a detailed understanding of the occurrence and function of TCS involved in the formation of that type of biofilm on the tooth surface seems important.

All TCS described for S. mutans were analysed as part of the study: VicKR, CiaHR, LiaRS, ComDE, HKRR5, NsrRS, HKRR7, BceRS, HKRR9, LcrRS, HKRR11, HKRR12 and HKRR1 [17,19,22]. As part of the research, the multiplex PCR procedure was performed for 26 genes included in the abovementioned systems using the S. mutans ATCC 700610 (UA159) reference strain. The presence of all sought genes in all 60 tested strains was demonstrated.

Based on the obtained data, there is a significant biochemical and genetic similarity and a comparable level of biofilm production capacity in the 60 S. mutans strains under study. Caries is very common in children and adults worldwide; therefore, the search for anti-caries agents is a very important and urgent task. Due to the widespread presence of TCS in S. mutans strains, they seem to be a very promising target for potential new drugs, so basic research on those genes is important. This is even more the case because the literature data indicate that it is the TCSs that are involved in the formation of dental plaque, the main etiological cause of caries.