IADR Abstract Archives

Comparison of Streptococcus mutans genotypes using rep-PCR and MLST

Streptococcus mutans, a human dental caries pathogen, has been extensively studied. However, a longitudinal view into its genetic diversity is needed to better understand the pathogenicity of this key cariogenic organism. Objectives: Multilocus sequence typing (MLST) was used to evaluate the genetic diversity of individual S. mutans genotypes (currently 24) previously identified as unique by repetitive extragenic palindromic-polymerase chain reaction (rep-PCR). Methods: Isolates were obtained from a longitudinal study of the natural colonization of S. mutans in children with high caries risk in the Black Belt of Alabama. DNA from two subjects (2 isolates/subject) from each of the six most commonly occurring rep-PCR genotypes (n=24) was extracted and used for real-time PCR using eight S. mutans housekeeping loci primer sets (tkt, glnA, gltA, glk, gyrA, aroE, murI, lepC) to obtain 16 amplicons/isolate. Amplification parameters: 10-min at 95oC; 30 cycles of 30-sec at 95oC, 30-sec at 55oC, 45-sec at 72oC; followed by 7-min at 72oC. The sequence data of purified amplicons from each primer set were matched, trimmed and aligned through comparative analysis with the UA159 S. mutans reference strain, using CLC DNA Workbench software, version 5.1. Tree alignments were generated using the un-weighted pair group method with arithmetic averages (UPGMA). Isolates were assigned arbitrary allele numbers and analyzed with START2 Software to create the final MLST UPGMA dendogram. Results: The 6 genotypes previously identified using rep-PCR were similarly identified as unique by MLST analysis (sensitivity=100%). MLST genotypes remained consistent for 3 rep-PCR genotypes; 3 rep-PCR genotypes were further distinguished with MLST analysis (specificity=50%). Conclusion: MLST analysis confirms the usefulness of rep-PCR's discriminatory power for rapid throughput genotyping for large-scale population and transmission studies. These findings will aid in providing a better understanding of S. mutans strain diversity and the pathogenesis of dental caries.

Supported by NIDCR grant #DE016684


AADR/CADR Annual Meeting
2010 AADR/CADR Annual Meeting (Washington, D.C.)
Washington, D.C.
2010
47
Microbiology / Immunology and Infection Control
  • Momeni, Stephanie  ( University of Alabama at Birmingham, Birmingham, AL, USA )
  • Atkins, Colm  ( University of Alabama at Birmingham, Birmingham, AL, USA )
  • Osgood, Robert C.  ( Rochester Institute of Technology, Rochester, NY, USA )
  • Whiddon, Jennifer  ( University of Alabama at Birmingham, Birmingham, AL, USA )
  • Moser, Stephen  ( University of Alabama at Birmingham, Birmingham, AL, USA )
  • Ruby, John D.  ( University of Alabama at Birmingham, Birmingham, AL, USA )
  • Childers, Noel K.  ( University of Alabama at Birmingham, Birmingham, AL, USA )
    • Oral Session
    Streptococci
    03/03/2010