Multidrug-resistant enterococci lack CRISPR-cas

Abstract

Clustered, regularly interspaced short palindromic repeats (CRISPR) provide bacteria and archaea with sequence-specific, acquired defense against plasmids and phage. Because mobile elements constitute up to 25% of the genome of multidrug-resistant (MDR) enterococci, it was of interest to examine the codistribution of CRISPR and acquired antibiotic resistance in enterococcal lineages. A database was built from 16 Enterococcus faecalis draft genome sequences to identify commonalities and polymorphisms in the location and content of CRISPR loci. With this data set, we were able to detect identities between CRISPR spacers and sequences from mobile elements, including pheromone-responsive plasmids and phage, suggesting that CRISPR regulates the flux of these elements through the E. faecalis species. Based on conserved locations of CRISPR and CRISPR-cas loci and the discovery of a new CRISPR locus with associated functional genes, CRISPR3-cas, we screened additional E. faecalis strains for CRISPR content, including isolates predating the use of antibiotics. We found a highly significant inverse correlation between the presence of a CRISPR-cas locus and acquired antibiotic resistance in E. faecalis, and examination of an additional eight E. faecium genomes yielded similar results for that species. A mechanism for CRISPR-cas loss in E. faecalis was identified. The inverse relationship between CRISPR-cas and antibiotic resistance suggests that antibiotic use inadvertently selects for enterococcal strains with compromised genome defense.

FIG 1

CRISPR loci in E. faecalis draft genomes. Strains are organized by date of isolation, from oldest to most recent. Homologues of E. faecalis V583 genes are shown as grey arrows and with V583 ORF assignments. CRISPR locus-specific genes are shown as white arrows, and CRISPR spacers are represented by black diamonds. CRISPR spacers with identity to mobile elements are starred. Note that CRISPR repeats are not shown. Red rectangles denote deletions in the X98, E1Sol, and DS5 CRISPR1-cas loci. An “X” denotes a frameshift mutation in the D6 CRISPR1-cas region corresponding to OG1RF_0022. The E. faecalis V583 CRISPR2 locus and the OG1RF CRISPR1-cas and CRISPR2 loci were previously reported (17, 25). Red bars and text denote novel DNA sequences present in V583. Primer sets used in CRISPR profiling are labeled with uppercase letters: A, CRISPR1-cas flanking primers; B, CRISPR1-cas csn1 screening primers; C, CRISPR2 screening primers; D, CRISPR3-cas flanking primers; E, CRISPR3-cas csn1 screening primers. The figure is not drawn to scale.

FIG 2

CRISPR-cas and acquired antibiotic resistance in a historical collection of E. faecalis strains. E. faecalis strains are listed by date of isolation, from oldest to most recent. Acquired antibiotic resistance is shown in red, and CRISPR-cas presence is shown in green. Antibiotic resistance (tetracycline [tetL and tetM], erythromycin [ermB], gentamicin [aac6′-aph2′′], chloramphenicol [cat], ampicillin [blaZ], and vancomycin [vanA and vanB]) was previously profiled (red squares) (10). A single asterisk indicates that gentamicin resistance is conferred by a 3′-5′′-aminoglycoside phosphotransferase in this strain; double asterisks denote E. faecalis strains for which draft or complete genome sequences are available.

FIG 3

CRISPR-cas distribution across the E. faecalis MLST dendrogram. An MLST-based dendrogram of the 48 strains utilized in CRISPR profiling was generated using the E. faecalis MLST database (see Materials and Methods). Strains indicated in red possess CRISPR1-cas, and those in blue possess CRISPR3-cas. Stars denote strains for which draft or complete genome sequences are available. ST, sequence type; CC, clonal complex.

FIG 4

EfmCRISPR1-cas loci in E. faecium draft genomes. Homologues of E. faecium DO genes are shown as grey arrows and with DO ORF assignments. EfmCRISPR1-cas locus-specific genes are shown as white arrows, and CRISPR spacers are represented by black diamonds. CRISPR spacers with identity to mobile elements are starred. Note that CRISPR repeats are not shown. The figure is not drawn to scale.

FIG 5

MLST, EfmCRISPR1-cas, and acquired antibiotic resistance in E. faecium draft genomes. Acquired antibiotic resistance is shown in red, and CRISPR-cas presence is shown in green. Antibiotic resistance (tetracycline [tetM], erythromycin [ermB], gentamicin [aac6′-aph2′′], and vancomycin [vanA]), EfmCRISPR1-cas, and ST profiles were generated by genomic analysis.