Comparative genotyping of S treptococcus mutans by repetitive extragenic palindromic polymerase chain reaction and multilocus sequence typing

Summary The genetic diversity of S treptococcus mutans has been extensively studied using a variety of genotyping methods. Repetitive extragenic palindromic‐polymerase chain reaction (rep‐ PCR ) is a genotyping approach used for screening large numbers of bacterial isolates. This two‐part study used multilocus sequence typing ( MLST ) analysis to evaluate genotypes previously identified as unique using rep‐ PCR . In part one, an isolate was selected from each of the 22 S . mutans rep‐ PCR genotype groups representing 8000 clinical isolates. For part two, four additional isolates were selected from the six most commonly occurring genotype groups ( GG ) for further analysis. Real‐time PCR was performed using eight housekeeping S . mutans gene loci and the amplicons were sequenced. Sequence data analysis was performed using CLC DNA Workbench and alleles were compared with the P ub MLST database for Oral S treptococcus using the N akano scheme. Concatenated sequences were evaluated with MEGA using a minimum evolution method with bootstrap. All 22 rep‐ PCR genotypes were unique by MLST analysis. Within rep‐ PCR GG s, MLST matched rep‐ PCR in three groups demonstrating clonality; three groups exhibited more diversity with MLST . The discovery of three clonal groups is unique to this study and suggests that S . mutans genotypes are shared between unrelated subjects. Furthermore, MLST defined 19 new alleles and 26 new sequence types that have been confirmed and registered with P ub MLST . Methods for processing were streamlined and a process for using MLST with rep‐ PCR is suggested. In conclusion, MLST verified that rep‐ PCR is a reliable and cost‐effective method for screening large numbers of S . mutans strains for epidemiological study.