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.2002 Mar;40(3):868-76.
doi: 10.1128/JCM.40.3.868-876.2002.

Molecular typing of selected Enterococcus faecalis isolates: pilot study using multilocus sequence typing and pulsed-field gel electrophoresis

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Molecular typing of selected Enterococcus faecalis isolates: pilot study using multilocus sequence typing and pulsed-field gel electrophoresis

Sreedhar R Nallapareddy et al. J Clin Microbiol.2002 Mar.

Abstract

The present study compared the recently developed multilocus sequence typing (MLST) approach with a well-established molecular typing technique, pulsed-field gel electrophoresis (PFGE), for subspecies differentiation of Enterococcus faecalis isolates. We sequenced intragenic regions of three E. faecalis antigen-encoding genes (ace, encoding a collagen and laminin adhesin; efaA, encoding an endocarditis antigen; and salA, encoding a cell wall associated antigen) and one housekeeping gene (pyrC) of 22 E. faecalis isolates chosen largely for their temporal and geographical diversity, but also including some outbreak isolates. MLST analysis of polymorphic regions of these four genes identified 13 distinct sequence types (STs) with different allelic profiles; the composite sequences generated from the four sequenced gene fragments of individual isolates showed 98.3 to 100% identity among the 22 isolates. We also found that the allelic profiles from two sequences, ace and salA, were sufficient to distinguish all 13 STs of this study. The 13 STs corresponded to 12 different PFGE types, with one previously designated PFGE clone (a widespread U.S. clone of beta-lactamase-producing isolates) being classified into two highly related STs which differed at 2 of 2,894 bases, both in the same allele. MLST also confirmed the clonal relationships among the isolates of two other PFGE clonal groups, including vancomycin resistant isolates. Thus, this pilot study with representative E. faecalis isolates suggests that, similar to PFGE, the sequence-based typing method may be useful for differentiating isolates of E. faecalis to the subspecies level in addition to identifying outbreak isolates.

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Figures

FIG. 1.
FIG. 1.
Chromosomal locations of the sequenced loci. The locations are marked on a constructed physical map ofE. faecalis V583 using the complete genome sequence (available athttp://www.tigr.org/ [The Institute for Genomic Research]). The 3,218-kb genome is divided into 16 segments, with each segment representing 201,125 bases. The ORF coding for chromosomal replication factor (dnaA) was identified based on identity withdnaA sequences ofS. pyogenes andS. aureus and positioned at nucleotide one. The arrowheads inside the circle represent the ORF orientations of the four loci and are marked at the respective positions.
FIG. 2.
FIG. 2.
Allelic variation of the four sequenced loci. (A) Variable sites identified in all the four gene fragments. The nucleotide sites that are identical in all the alleles are not shown. The nucleotides present in each of the variable sites of allele A (E. faecalis OG1RF) are shown. Only those sites that differ are shown for all the other alleles. The position of each variable site within the sequenced fragment is shown in the numbers above the nucleotide, read vertically. The consensus sequence is shown on the bottom. The variations that are synonymous (S) and nonsynonymous (N) are also shown. (B) Cladograms of the four loci sequenced. Phylogenetic trees were based on the matrix of pairwise sequence divergence in the sequences (generated by the Jotun Hein method of the DNASTAR software package). The length of each pair of branches represents the distance between sequence pairs, while the units at the bottom of the tree indicate the number of substitution events.
FIG. 2.
FIG. 2.
Allelic variation of the four sequenced loci. (A) Variable sites identified in all the four gene fragments. The nucleotide sites that are identical in all the alleles are not shown. The nucleotides present in each of the variable sites of allele A (E. faecalis OG1RF) are shown. Only those sites that differ are shown for all the other alleles. The position of each variable site within the sequenced fragment is shown in the numbers above the nucleotide, read vertically. The consensus sequence is shown on the bottom. The variations that are synonymous (S) and nonsynonymous (N) are also shown. (B) Cladograms of the four loci sequenced. Phylogenetic trees were based on the matrix of pairwise sequence divergence in the sequences (generated by the Jotun Hein method of the DNASTAR software package). The length of each pair of branches represents the distance between sequence pairs, while the units at the bottom of the tree indicate the number of substitution events.
FIG. 3.
FIG. 3.
Phylogenetic trees of genetic relationships among 22E. faecalis strains based on sequences of four gene fragments. (a) The dendrogram is based on the matrix of pairwise differences in the allelic sequences as determined by the UPGMA method. Linkage distances are indicated by scale at the bottom. (b) The cladogram is based on the matrix of pairwise sequence divergence in the concatenated composite sequences (generated by the Jotun Hein method of the DNASTAR software package). The length of each pair of branches represents the distance between sequence pairs, while the units at the bottom of the tree indicate the number of substitution events.
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