Abstract
Clustered, Regularly Interspaced Short Palindromic Repeats and their associated Cas proteins (CRISPR-Cas) provide prokaryotes with a mechanism for defense against mobile genetic elements (MGEs). A CRISPR locus is a molecular memory of MGE encounters. It contains an array of short sequences, called spacers, that generally have sequence identity to MGEs. Three different CRISPR loci have been identified among strains of the opportunistic pathogen Enterococcus faecalis. CRISPR1 and CRISPR3 are associated with the cas genes necessary for blocking MGEs, but these loci are present in only a subset of E. faecalis strains. The orphan CRISPR2 lacks cas genes and is ubiquitous in E. faecalis, although its spacer content varies from strain to strain. Because CRISPR2 is a variable locus occurring in all E. faecalis, comparative analysis of CRISPR2 sequences may provide information about the clonality of E. faecalis strains. We examined CRISPR2 sequences from 228 E. faecalis genomes in relationship to subspecies phylogenetic lineages (sequence types; STs) determined by multilocus sequence typing (MLST), and to a genome phylogeny generated for a representative 71 genomes. We found that specific CRISPR2 sequences are associated with specific STs and with specific branches on the genome tree. To explore possible applications of CRISPR2 analysis, we evaluated 14 E. faecalis bloodstream isolates using CRISPR2 analysis and MLST. CRISPR2 analysis identified two groups of clonal strains among the 14 isolates, an assessment that was confirmed by MLST. CRISPR2 analysis was also used to accurately predict the ST of a subset of isolates. We conclude that CRISPR2 analysis, while not a replacement for MLST, is an inexpensive method to assess clonality among E. faecalis isolates, and can be used in conjunction with MLST to identify recombination events occurring between STs.
Highlights
Enterococcus faecalis is a gram-positive lactic acid bacterium and opportunistic pathogen that normally colonizes the intestinal tracts of humans and other hosts [1]
The 228 E. faecalis strains in our collection represent 62 sequence type (ST), and their isolation dates range from the early 20th century to present (S1 Dataset)
Based on limited availability of information for certain strains, it is possible that our dataset contains serial clinical isolates, for the large number of ST2 and ST6 strains in our collection
Summary
Enterococcus faecalis is a gram-positive lactic acid bacterium and opportunistic pathogen that normally colonizes the intestinal tracts of humans and other hosts [1]. It is used in the production of fermented foods and live probiotics, and as an indicator of fecal contamination in PLOS ONE | DOI:10.1371/journal.pone.0138890. Many studies seek to determine whether E. faecalis strains causing infection outbreaks are clonal. Analysis of whole genome sequences has been used to assess phylogenetic relationships among E. faecalis strains [7,8,9,10,11], the number of strains queried to date is much lower than that assessed by MLST. While whole genome sequencing is increasingly cost-effective and attractive for population studies of infection outbreaks, the technology and expertise required to generate and analyze data currently limits this approach to facilities with appropriate resources for such studies
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