Mechanism of chromosomal transfer of Enterococcus faecalis pathogenicity island, capsule, antimicrobial resistance, and other traits

Abstract

The Enterococcus faecalis pathogenicity island (PAI) encodes known virulence traits and >100 additional genes with unknown roles in enterococcal biology. Phage-related integration and excision genes, and direct repeats flanking the island, suggest it moves as an integrative conjugative element (ICE). However, transfer was observed not to require these genes. Transfer only occurred from donors possessing a pheromone responsive-type of conjugative plasmid, and was invariably accompanied by transfer of flanking donor chromosome sequences. Deletion of plasmid transfer functions, including the cis-acting origin of transfer (oriT), abolished movement. In addition to demonstrating PAI movement by a mechanism involving plasmid integration, we observed transfer of a selectable marker placed virtually anywhere on the chromosome. Transfer of this selectable marker was observed to be accompanied by chromosome-chromosome transfer of vancomycin resistance, MLST markers, and capsule genes as well. Plasmid mobilization therefore appears to be a major mechanism for horizontal gene transfer in the evolution of antibiotic resistant E. faecalis strains capable of causing human infection.

Fig. 1.

E. faecalis pathogenicity island showing tetracycline resistance marker integration within the int gene (orf EF0479), the choline bile salt hydrolase cbh gene (orf EF0521), or distal orf EF0592 encoding a surface protein of unknown function. The location and direction of primer pairs (Table S1) used to amplify the left and right PAI junctions are indicated.

Fig. 2.

SfiI macrorestriction patterns of E. faecalis PAI transconjugants. Lane 1, lambda DNA ladder standard; lane 2, recipient strain OG1RF; lanes 3–14, transconjugant strains TC1, TC2, TC7, TC4, TC6, TC8, TC9, TC10, TC3, TC12, TC5, and TC11, respectively; lane 15, donor strain V583. PFGE pattern groupings are indicated below the gel. Plasmid pTEF2, 57.7 kb in size and present in 3 of the 12 transconjugants, is indicated by arrows.

Fig. 3.

Rate of transfer of tetracycline resistance from various points in the V583 chromosome. The relative location of tetracycline resistance marker insertions within the chromosome are indicated on the horizontal axis with scale shown above. Large regions of acquired/mobile DNA that occur naturally in the V583 donor chromosome are identified by small boxes along the lower horizontal axis (putative phage shown in light gray; other mobile elements such as the pathogenicity island, black).