# Characteristics of Clinical Isolates of _Streptococcus mutans_
## 2 Materials and Methods
### 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, BeeRS, 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. \begin{table}
\begin{tabular}{l c} \hline \hline \multicolumn{1}{c}{**AST-ST01**} \\ _Streptococcus pneumoniae_, _Beta-haemolytic _Streptococcus_, _Streptococcus viridans_ \\ \hline \hline \multicolumn{1}{c}{**Antibiotic**} & **MIC Scope** \\ \hline \hline \multicolumn{1}{c}{Ampicillin} & 0.25–16 \\ \hline \hline \multicolumn{1}{c}{Benzylpenicillin} & 0.06–8 \\ \hline \hline \multicolumn{1}{c}{Cefotaxime} & 0.12–8 \\ \hline \hline \multicolumn{1}{c}{Ceftriaxone} & 0.12–8 \\ \hline \hline \multicolumn{1}{c}{Clindamycin} & 0.25–1 \\ \hline \hline \multicolumn{1}{c}{Erythromycin} & 0.12–8 \\ \hline \hline \multicolumn{1}{c}{ICR _S. agalactiae, S. pyogenes_} & NEG/POS \\ \hline \hline \multicolumn{1}{c}{Levofloxacin} & 0.25–16 \\ \hline \hline \multicolumn{1}{c}{Linezolid} & 2–8 \\ \hline \hline \multicolumn{1}{c}{Tetracycline} & 0.25–16 \\ \hline \hline \multicolumn{1}{c}{Trimethoprim/Sulfameth} & 10 (0.5/9.5)–320 (16/304) \\ \hline \hline \multicolumn{1}{c}{Vancomycin} & 0.12–8 \\ \hline \hline \end{tabular}
\end{table} 
Table 1: Antibiotic panel of AST-ST01 card. \begin{table}
\begin{tabular}{l c c} \hline \hline
**Primers/Multiplex** & **Product Size** & **TCS Name** \\ \hline SMU\_1516 HK-F CGGCTTCTTGCCTTATCAAC & & \\ SMU\_1516 HK-R GTAAAGTCCCTATTATTAGAAAGCTTTGGA & 510 bp & VicKR \\ SMU\_1517 RR-F CCCCAAACCCTTCCAAGTA & 211 bp & \\ SMU\_1517 RR-R AAAATATTGAATCCGCAGCTG & & \\ SMU\_1128 HK-F GCTTTGACGTCCAGCCAAT & & \\ SMU\_1128 HK-R CGATTATTATCAGTGTGATGATTGT & & \\ SMU\_1129 RR-FGAGAATTAAAAAAATGCGTATTCAG & & \\ SMU\_1129 RR-R CAAAAATTGTGACTTAGGTAAAATG & & \\ SMU\_1129 RR-R CAAAAATTGTGACTTAGGTAAAATG & & \\ SMU\_1486 HK-F GCTGATTGGCTTGTTCTGA & & \\ SMU\_1487 RR-F GCTAGTTGTTGTTGTTGTTGTTGTTGTTGTTG  
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 \({}^{\circ}\)C for 15 min. The total volume of the multiplex PCR reaction mixture was 25 \(\upmu\)L: 2.0 \(\upmu\)L genomic DNA, 2.5 \(\upmu\)L TaqMan buffer with KCl, 1.5 \(\upmu\)L 25 mM MgCl2, 0.5 \(\upmu\)L 10 mM dNTP mixture, 2.5 \(\upmu\)L each primer, 0.2 \(\upmu\)L Taq DNA polymerase and 8.3 \(\upmu\)L 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 \({}^{\circ}\)C for 5 min, 95 \({}^{\circ}\)C for 30 s, 54 \({}^{\circ}\)C for 1 min, 72 \({}^{\circ}\)C for 1 min (25 cycles), final elongation of 72 \({}^{\circ}\)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 \(\upmu\)g/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).