Comparative genotyping of Streptococcus mutans by repetitive extragenic palindromic polymerase chain reaction and multilocus sequence typing
Summary The genetic diversity of Streptococcus 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 mu...
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| Published in | Molecular oral microbiology Vol. 28; no. 1; pp. 18 - 27 |
|---|---|
| Main Authors | , , , , , |
| Format | Journal Article |
| Language | English |
| Published |
Denmark
Blackwell Publishing Ltd
01.02.2013
Wiley Subscription Services, Inc |
| Subjects | |
| Online Access | Get full text |
| ISSN | 2041-1006 2041-1014 2041-1014 |
| DOI | 10.1111/omi.12002 |
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| Abstract | Summary
The genetic diversity of Streptococcus 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 PubMLST database for Oral Streptococcus using the Nakano 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 GGs, 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 PubMLST. 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. |
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| AbstractList | The genetic diversity of Streptococcus 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 PubMLST database for Oral Streptococcus using the Nakano 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 GGs, 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 PubMLST. 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. Summary The genetic diversity of Streptococcus 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 PubMLST database for Oral Streptococcus using the Nakano 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 GGs, 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 PubMLST. 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. Summary The genetic diversity of Streptococcus 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 DNAWorkbench and alleles were compared with the PubMLST database for Oral Streptococcus using the Nakano 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 GGs, 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 PubMLST. 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. The genetic diversity of Streptococcus 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 PubMLST database for Oral Streptococcus using the Nakano 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 GGs, 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 PubMLST. 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.The genetic diversity of Streptococcus 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 PubMLST database for Oral Streptococcus using the Nakano 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 GGs, 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 PubMLST. 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. The genetic diversity of Streptococcus 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 PubMLST database for Oral Streptococcus using the Nakano 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 GGs, 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 PubMLST. 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. |
| Author | Ruby, J.D. Moser, S.A. Cheon, K. Momeni, S.S. Childers, N.K. Whiddon, J. |
| Author_xml | – sequence: 1 givenname: S.S. surname: Momeni fullname: Momeni, S.S. organization: Department of Pediatric Dentistry, University of Alabama School of Dentistry, AL, Birmingham, USA – sequence: 2 givenname: J. surname: Whiddon fullname: Whiddon, J. organization: Department of Pathology, University of Alabama School of Medicine, AL, Birmingham, USA – sequence: 3 givenname: S.A. surname: Moser fullname: Moser, S.A. organization: Department of Pathology, University of Alabama School of Medicine, AL, Birmingham, USA – sequence: 4 givenname: K. surname: Cheon fullname: Cheon, K. organization: Department of Pediatric Dentistry, University of Alabama School of Dentistry, AL, Birmingham, USA – sequence: 5 givenname: J.D. surname: Ruby fullname: Ruby, J.D. organization: Department of Pediatric Dentistry, University of Alabama School of Dentistry, AL, Birmingham, USA – sequence: 6 givenname: N.K. surname: Childers fullname: Childers, N.K. email: nkc@uab.edu organization: Department of Pediatric Dentistry, University of Alabama School of Dentistry, AL, Birmingham, USA |
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| Notes | Table S1. Multilocus sequence typing sequence type allele profile assignments for 22 Streptococcus mutans library strainsTable S2. Multilocus sequence typing sequence type allele profile assignments for six Streptococcus mutans genotype groups. istex:515A50A33C84FFCF0B3F3A9074A4C6666E1FE248 ark:/67375/WNG-D34WRSR8-B National Institute of Dental and Craniofacial Research National Institutes of Health ArticleID:OMI12002 ObjectType-Article-1 SourceType-Scholarly Journals-1 ObjectType-Feature-2 content type line 14 ObjectType-Article-2 ObjectType-Feature-1 content type line 23 |
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The genetic diversity of Streptococcus mutans has been extensively studied using a variety of genotyping methods. Repetitive extragenic... The genetic diversity of Streptococcus mutans has been extensively studied using a variety of genotyping methods. Repetitive extragenic palindromic-polymerase... Summary The genetic diversity of Streptococcus mutans has been extensively studied using a variety of genotyping methods. Repetitive extragenic... |
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| SubjectTerms | Alcohol Oxidoreductases - genetics Alleles Amino Acid Isomerases - genetics Bacterial Proteins - genetics Bacterial Typing Techniques - methods Child Child, Preschool Chromosome Mapping Clone Cells dental caries diversilab DNA Gyrase - genetics DNA, Concatenated - genetics Genetic diversity Genetic Variation - genetics Genotype Genotypes Glutamate Synthase - genetics Glutamate-Ammonia Ligase - genetics Guanine Humans Inverted Repeat Sequences - genetics Membrane Proteins - genetics molecular oral microbiology Multilocus Sequence Typing - methods phylogenetics Real-Time Polymerase Chain Reaction - methods Sequence Analysis, DNA - methods Serine Endopeptidases - genetics Streptococcus Streptococcus mutans Streptococcus mutans - classification Streptococcus mutans - genetics Transketolase - genetics |
| Title | Comparative genotyping of Streptococcus mutans by repetitive extragenic palindromic polymerase chain reaction and multilocus sequence typing |
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