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Article

Characteristics of Clinical Isolates of Streptococcus mutans

by
Aleksandra Pisarska
1,*,
Renata Wolinowska
1,*,
Joanna Rudnicka
2 and
Ewa Iwanicka-Grzegorek
2
1
Department of Pharmaceutical Microbiology, Centre for Preclinical Research, Medical University of Warsaw, Banacha 1b, 02-097 Warsaw, Poland
2
Department of Conservative Dentistry and Endodontics, Medical University of Warsaw, Binieckiego 6, 02-006 Warsaw, Poland
*
Authors to whom correspondence should be addressed.
Appl. Sci. 2022, 12(9), 4579; https://doi.org/10.3390/app12094579
Submission received: 13 February 2022 / Revised: 24 April 2022 / Accepted: 25 April 2022 / Published: 30 April 2022
(This article belongs to the Special Issue Oral Microbial Communities and Oral Health: Volume II)

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).

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). 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. 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.
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.

Author Contributions

Conceptualization, A.P.; methodology, A.P. and R.W.; software, A.P.; validation, A.P. and R.W.; formal analysis, A.P.; investigation, A.P.; resources, J.R. and E.I.-G.; data curation, A.P; writing—original draft preparation, A.P.; writing—review and editing, A.P., R.W., J.R. and E.I.-G.; visualization, A.P.; supervision, R.W.; project administration, A.P.; funding acquisition, A.P. All authors have read and agreed to the published version of the manuscript.

Funding

The project was funded by the National Science Centre under decision number DEC-2012/07/N/NZ7/02019.

Institutional Review Board Statement

The study was conducted in accordance with the Declaration of Helsinki, and approved by the Bioethical Committee of the Medical University of Warsaw (Opinion no. KB/62/2012, 12 April 2012).

Informed Consent Statement

Informed consent was obtained from all subjects involved in the study.

Acknowledgments

A special thanks for help and support during the project for Stefan Tyski.

Conflicts of Interest

The authors declare no conflict of interest.

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Scheme 1. Level of biofilm formation by 10 randomly selected clinical isolates of S. mutans. 1—non-biofilm forming strain, 2—reference strain S. mutans UA159, 3–7—clinical isolates from adults, 7–12—clinical isolates from children. Bars represent mean values ± standard deviation.
Scheme 1. Level of biofilm formation by 10 randomly selected clinical isolates of S. mutans. 1—non-biofilm forming strain, 2—reference strain S. mutans UA159, 3–7—clinical isolates from adults, 7–12—clinical isolates from children. Bars represent mean values ± standard deviation.
Applsci 12 04579 sch001
Figure 1. Multiplex PCR of vicK and vir R genes (TCS VicKR) for randomly selected S. mutans clinical isolates. Size of the products: vicK-510 bp, vir R-211 bp. 1, size standard; 2, S. mutans UA 159 positive control; 3–11, clinical isolates from adults; 12–20, isolates from children.
Figure 1. Multiplex PCR of vicK and vir R genes (TCS VicKR) for randomly selected S. mutans clinical isolates. Size of the products: vicK-510 bp, vir R-211 bp. 1, size standard; 2, S. mutans UA 159 positive control; 3–11, clinical isolates from adults; 12–20, isolates from children.
Applsci 12 04579 g001
Figure 2. Multiplex PCR of ciaH and cia R genes (TCS CiaHR) for randomly selected clinical isolates. Size of the products: ciaH-529 bp, cia R-211 bp. 1, size standard; 2, positive control, S. mutans UA 159; 3–11, clinical isolates from adults; 12-20, isolates from children.
Figure 2. Multiplex PCR of ciaH and cia R genes (TCS CiaHR) for randomly selected clinical isolates. Size of the products: ciaH-529 bp, cia R-211 bp. 1, size standard; 2, positive control, S. mutans UA 159; 3–11, clinical isolates from adults; 12-20, isolates from children.
Applsci 12 04579 g002
Table 1. Antibiotic panel of AST-ST01 card.
Table 1. Antibiotic panel of AST-ST01 card.
AST-ST01
Streptococcus pneumoniae, Beta-haemolytic Streptococcus, Streptococcus viridans
AntibioticMIC Scope
Ampicillin0.25–16
Benzylpenicillin0.06–8
Cefotaxime0.12–8
Ceftriaxone0.12–8
Clindamycin0.25–1
Erythromycin0.12–8
ICR S. agalactiae, S. pyogenesNEG/POS
Levofloxacin0.25–16
Linezolid2–8
Tetracycline0.25–16
Trimethoprim/Sulfameth10 (0.5/9.5)–320 (16/304)
Vancomycin0.12–8
Table 2. TCS identification primers.
Table 2. TCS identification primers.
Primers/MultiplexProduct SizeTCS 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
Table 3. Illustrative biochemical profile of S. mutans (GP card) of a randomly selected strain. The biochemical profile was identical for all strains.
Table 3. Illustrative biochemical profile of S. mutans (GP card) of a randomly selected strain. The biochemical profile was identical for all strains.
AMY+PIPLCdXYLADH1BGALAGLU(−)
APPACDEXAspABGARAMANPHOS
LeuA(−)ProABGURrAGALPyr ABGUR
AlaA+TyrAdSOR+UREPOLYB+dGAL+
dRIBILATkLac+NAGdMAL+BACI+
NOVO+NC6.5dMAN+dMNE+MBdG+PUL
dRAF+O129RSAL+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).
Table 4. Drug susceptibility panel—AST-ST01 Card (bioMerieux).
Table 4. Drug susceptibility panel—AST-ST01 Card (bioMerieux).
AntimicrobialMICInterpretation
Benzylpenicillin<=0.06Sensitive
Ampicillin<=0.25Sensitive
Cefotaxime<=0.12Sensitive
Ceftriaxone<=0.12Sensitive
Erythromycin<=0.12Insufficient evidence that species is good target for therapy
Clindamycin<=0.25Sensitive
Vancomycin1Sensitive
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Pisarska, A.; Wolinowska, R.; Rudnicka, J.; Iwanicka-Grzegorek, E. Characteristics of Clinical Isolates of Streptococcus mutans. Appl. Sci. 2022, 12, 4579. https://doi.org/10.3390/app12094579

AMA Style

Pisarska A, Wolinowska R, Rudnicka J, Iwanicka-Grzegorek E. Characteristics of Clinical Isolates of Streptococcus mutans. Applied Sciences. 2022; 12(9):4579. https://doi.org/10.3390/app12094579

Chicago/Turabian Style

Pisarska, Aleksandra, Renata Wolinowska, Joanna Rudnicka, and Ewa Iwanicka-Grzegorek. 2022. "Characteristics of Clinical Isolates of Streptococcus mutans" Applied Sciences 12, no. 9: 4579. https://doi.org/10.3390/app12094579

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