Molecular epidemiology of vancomycin-resistant Enterococcus faecium: a prospective, multicenter study in South American hospitals

Abstract

Enterococcus faecium has emerged as an important nosocomial pathogen worldwide, and this trend has been associated with the dissemination of a genetic lineage designated clonal cluster 17 (CC17). Enterococcal isolates were collected prospectively (2006 to 2008) from 32 hospitals in Colombia, Ecuador, Perú, and Venezuela and subjected to antimicrobial susceptibility testing. Genotyping was performed with all vancomycin-resistant E. faecium (VREfm) isolates by pulsed-field gel electrophoresis (PFGE) and multilocus sequence typing. All VREfm isolates were evaluated for the presence of 16 putative virulence genes (14 fms genes, the esp gene of E. faecium [espEfm], and the hyl gene of E. faecium [hylEfm]) and plasmids carrying the fms20-fms21 (pilA), hylEfm, and vanA genes. Of 723 enterococcal isolates recovered, E. faecalis was the most common (78%). Vancomycin resistance was detected in 6% of the isolates (74% of which were E. faecium). Eleven distinct PFGE types were found among the VREfm isolates, with most belonging to sequence types 412 and 18. The ebpAEfm-ebpBEfm-ebpCEfm (pilB) and fms11-fms19-fms16 clusters were detected in all VREfm isolates from the region, whereas espEfm and hylEfm were detected in 69% and 23% of the isolates, respectively. The fms20-fms21 (pilA) cluster, which encodes a putative pilus-like protein, was found on plasmids from almost all VREfm isolates and was sometimes found to coexist with hylEfm and the vanA gene cluster. The population genetics of VREfm in South America appear to resemble those of such strains in the United States in the early years of the CC17 epidemic. The overwhelming presence of plasmids encoding putative virulence factors and vanA genes suggests that E. faecium from the CC17 genogroup may disseminate in the region in the coming years.

FIG. 1.

Molecular typing of VR E. faecium from the Andean region of South America. The phylogenetic tree was constructed by use of the Dice coefficient and UPGMA clustering; the band tolerance was set at 1.5%, and the threshold cutoff value was set at 85%. ST, sequence type; CC17, clonal cluster 17.

FIG. 2.

Frequency of detection of putative virulence genes in 35 VR E. faecium isolates from the Andean region of South America. The numbers on the left-hand side indicate the four clusters of genes encoding characterized putative proteins of the enterococcal pili (30).

FIG. 3.

Plasmids containing the fms20 gene or the fms21 gene (pilA), or both genes, in representative E. faecium isolates from the Andean region of South America. (A) Genetic organization of fms20-fms21 (pilA) in E. faecium TX0016 (strain DO). This locus also contains a predicted class C sortase-encoding gene (srtC4) and a class A sortase-encoding gene (srtA) (30). (B) S1 nuclease digestion of total DNA of E. faecium strains, followed by PFGE (left panel) and hybridization with an fms20 probe (right panel). Lane 1, bacteriophage lambda ladder (molecular sizes [in kilobases] are shown to the left); lanes 2 and 14, TX0016 (strain DO); lanes 3, ERV-99 (4); lanes 4, P1985 (Perú); lanes 5, P1123 (Perú); lanes 6, P2022 (Perú); lanes 7, E417 strain (Ecuador); lanes 8, P1139 (Perú); lanes 9, P575 (Perú); lanes 10, P2074 (Perú); lanes 11, E422 (Ecuador); lanes 12, P1986 (Perú); lanes 13, C1688 (Colombia); lanes 15, V2216 (Venezuela). Plasmid bands are shown as linearized fragments on the gel; the white arrows indicate the plasmid bands hybridizing with the fms20 probe. (C) S1 nuclease digestion of total DNA of E. faecium strains, followed by PFGE (left panel) and hybridization with an fms21 (pilA) probe (right panel). Lane 1, bacteriophage lambda ladder (molecular sizes [in kilobases] are shown to the left); lanes 2 and 14, TX0016 (strain DO); lanes 3, ERV-99 (4); lanes 4, P1985 (Perú); lanes 5, P1123 (Perú); lanes 6, P2022 (Perú); lanes 7, E417 (Ecuador); lanes 8, P1139 (Perú); lanes 9, P575 (Perú); lanes 10, P2074 (Perú); lanes 11, E422 (Ecuador); lanes 12, P1986 (Perú); lanes 13, C1688 (Colombia); lanes 15, V2216 (Venezuela). The white arrows indicate the plasmid bands hybridizing with the fms21 (pilA) probe.