TY - JOUR
T1 - High-resolution melting genotyping of enterococcus faecium based on multilocus sequence typing derived single nucleotide polymorphisms
AU - Tong, Steven
AU - Xie, Shirley
AU - Richardson, Leisha Jade
AU - Ballard, Susan
AU - Dakh, Farshid
AU - Grabsch, Elizabeth
AU - Grayson, Lindsay
AU - Howden, Benjamin
AU - Johnson, Paul
AU - Giffard, Philip
PY - 2011
Y1 - 2011
N2 - We have developed a single nucleotide polymorphism (SNP) nucleated high-resolution melting (HRM) technique to genotype Enterococcus faecium. Eight SNPs were derived from the E. faecium multilocus sequence typing (MLST) database and amplified fragments containing these SNPs were interrogated by HRM. We tested the HRM genotyping scheme on 85 E. faecium bloodstream isolates and compared the results with MLST, pulsed-field gel electrophoresis (PFGE) and an allele specific real-time PCR (AS kinetic PCR) SNP typing method. In silico analysis based on predicted HRM curves according to the G+C content of each fragment for all 567 sequence types (STs) in the MLST database together with empiric data from the 85 isolates demonstrated that HRM analysis resolves E. faecium into 231 "melting types" (MelTs) and provides a Simpson's Index of Diversity (D) of 0.991 with respect to MLST. This is a significant improvement on the AS kinetic PCR SNP typing scheme that resolves 61 SNP types with D of 0.95. The MelTs were concordant with the known ST of the isolates. For the 85 isolates, there were 13 PFGE patterns, 17 STs, 14 MelTs and eight SNP types. There was excellent concordance between PFGE, MLST and MelTs with Adjusted Rand Indices of PFGE to MelT 0.936 and ST to MelT 0.973. In conclusion, this HRM based method appears rapid and reproducible. The results are concordant with MLST and the MLST based population structure. � 2011 Tong et al.
AB - We have developed a single nucleotide polymorphism (SNP) nucleated high-resolution melting (HRM) technique to genotype Enterococcus faecium. Eight SNPs were derived from the E. faecium multilocus sequence typing (MLST) database and amplified fragments containing these SNPs were interrogated by HRM. We tested the HRM genotyping scheme on 85 E. faecium bloodstream isolates and compared the results with MLST, pulsed-field gel electrophoresis (PFGE) and an allele specific real-time PCR (AS kinetic PCR) SNP typing method. In silico analysis based on predicted HRM curves according to the G+C content of each fragment for all 567 sequence types (STs) in the MLST database together with empiric data from the 85 isolates demonstrated that HRM analysis resolves E. faecium into 231 "melting types" (MelTs) and provides a Simpson's Index of Diversity (D) of 0.991 with respect to MLST. This is a significant improvement on the AS kinetic PCR SNP typing scheme that resolves 61 SNP types with D of 0.95. The MelTs were concordant with the known ST of the isolates. For the 85 isolates, there were 13 PFGE patterns, 17 STs, 14 MelTs and eight SNP types. There was excellent concordance between PFGE, MLST and MelTs with Adjusted Rand Indices of PFGE to MelT 0.936 and ST to MelT 0.973. In conclusion, this HRM based method appears rapid and reproducible. The results are concordant with MLST and the MLST based population structure. � 2011 Tong et al.
KW - article
KW - bacterium isolate
KW - blood culture
KW - controlled study
KW - DNA base composition
KW - Enterococcus faecium
KW - genotype
KW - high resolution melting analysis
KW - intermethod comparison
KW - multilocus sequence typing
KW - nonhuman
KW - population structure
KW - pulsed field gel electrophoresis
KW - real time polymerase chain reaction
KW - reproducibility
KW - single nucleotide polymorphism
KW - classification
KW - DNA denaturation
KW - genetics
KW - genotyping technique
KW - isolation and purification
KW - methodology
KW - Electrophoresis, Gel, Pulsed-Field
KW - Genotyping Techniques
KW - Multilocus Sequence Typing
KW - Nucleic Acid Denaturation
KW - Polymorphism, Single Nucleotide
UR - http://www.scopus.com/inward/record.url?scp=83455182089&partnerID=8YFLogxK
U2 - 10.1371/journal.pone.0029189
DO - 10.1371/journal.pone.0029189
M3 - Article
C2 - PubMed:22195020
VL - 6
SP - 1
EP - 8
JO - PLoS One
JF - PLoS One
SN - 1932-6203
IS - 12
M1 - e29189
ER -