VanA-type enterococci from humans, animals, and food: species distribution, population structure, Tn1546 typing and location, and virulence determinants

Abstract

VanA-type human (n=69), animal (n=49), and food (n=36) glycopeptide-resistant enterococci (GRE) from different geographic areas were investigated to study their possible reservoirs and transmission routes. Pulsed-field gel electrophoresis (PFGE) revealed two small genetically related clusters, M39 (n=4) and M49 (n=13), representing Enterococcus faecium isolates from animal and human feces and from clinical and fecal human samples. Multilocus sequence typing showed that both belonged to the epidemic lineage of CC17. purK allele analysis of 28 selected isolates revealed that type 1 was prevalent in human strains (8/11) and types 6 and 3 (14/15) were prevalent in poultry (animals and meat). One hundred and five of the 154 VanA GRE isolates, encompassing different species, origins, and PFGE types, were examined for Tn1546 type and location (plasmid or chromosome) and the incidence of virulence determinants. Hybridization of S1- and I-CeuI-digested total DNA revealed a plasmid location in 98% of the isolates. Human intestinal and animal E. faecium isolates bore large (>150 kb) vanA plasmids. Results of PCR-restriction fragment length polymorphism and sequencing showed the presence of prototype Tn1546 in 80% of strains and the G-to-T mutation at position 8234 in three human intestinal and two pork E. faecium isolates. There were no significant associations (P>0.5) between Tn1546 type and GRE source or enterococcal species. Virulence determinants were detected in all reservoirs but were significantly more frequent (P<0.02) among clinical strains. Multiple determinants were found in clinical and meat Enterococcus faecalis isolates. The presence of indistinguishable vanA elements (mostly plasmid borne) and virulence determinants in different species and PFGE-diverse populations in the presence of host-specific purK housekeeping genes suggested that all GRE might be potential reservoirs of resistance determinants and virulence traits transferable to human-adapted clusters.

FIG. 1.

Dendrograms showing the similarity index among the 154 isolates of E. faecium (A) and E. faecalis, E. durans, and E. gallinarum (B). Clusters sharing ≥70% similarity are shown in gray. A, animal isolate; F, food isolate; HC, human clinical isolate; HI, human intestinal isolate. CI, central Italy; NI, northern Italy; SI, southern Italy; NO, Norway; BE, Belgium. PFGE types showing a clonal spread are boxed.

FIG. 2.

Plasmid profile (A) and vanA hybridization (B) of animal (lines 1 to 9) and human (lines 10 to 19) isolates. Line 1, E. durans A-VI4; line 2, E. faecium A-PD33; line 3, E. durans A-VR5; line 4, E. faecium A-PD6; line 5, E. faecium A-VR8; line 6, E. faecium A-PD37; line 7, E. faecium A-VI9; line 8, E. faecium A-VI10; line 9, E. faecium A-VR12; line 10, E. faecium HI-MI29; line 11, E. faecium HI-MI30; line 12, E. faecium HI-MI57; line 13, E. faecium HI-MI58; line 14, E. faecium HI-MI31; line 15, E. faecium HI-MI32; line 16, E. faecium HI-MI27; line 17, E. faecium HI-MI60; line 18, E. faecium HI-MI34; line 19, E. faecium HI-MI28. M, molecular size marker (Marker II; Roche).

FIG. 3.

PFGE of S1-digested (A) and I-CeuI-digested (C) total DNA and corresponding vanA (B and E) and 16S rRNA gene (D) hybridization. Lane 1, E. faecium HI-MI28; lane 2, E. faecium HI-MI34; lane 3, E. faecium HI-MI60; lane 4, E. faecium HI-MI32; lane 5, E. faecium HI-MI31; lane 6, E. faecalis HI-MI58; lane 7, E. faecium HI-MI57; lane 8, E. faecium HI-MI30; lane 9, E. faecium HI-MI25; lane 10, E. faecium HI-MI30; lane 11, E. faecium HI-MI25; lane 12, E. faecium HI-MI31; lane 13, E. faecium HI-MI32; lane 14, E. faecium HI-MI60; lane 15, E. gallinarum A-BE48; lane 16, E. gallinarum F-PM3. M, low range marker (BioLabs).

FIG. 4.

Schematic representation of the Tn1546 prototype (A) and 11 different Tn1546-like elements (A* to D1) detected in the 101 vanA isolates carrying a single vanA element. Locations of primers, ClaI target sites, and the mutation at position 8234 are indicated. Left-side deletions (δ, deletion size) are indicated by dotted lines, insertions by gray boxes. The origin (HC, HI, A, and F) and number of isolates carrying the Tn1546 type are reported on the right. The labels of the Tn1546-like elements of Palepou et al. (43) that may correspond to those characterized in the present study are reported in parentheses: A, A* (A); B* (D); B₁ (D/M); B₂ (M); B₃ (P); C, C1 (B/C); C2 (no correspondence); C3 (H-L); D (E); and D1 (Q-S).

FIG. 5.

EcoRI restriction analysis of AGG amplicons of nine E. faecalis isolates and of E. faecalis OG1RF(pAD1, asa1) and E. faecalis OG1RF(pCF10, prgB) reference strains. Lane 1, HI-AN23; line 2, HC-VI4 (also asa373 positive); line 3, HC-AN22; line 4, HC-UD6; line 5, HC-R35; line 6, HC-AN21; line 7, F-KM6; line 8, F-KM7; line 9, F-PM25; line 10, OG1RF(pAD1; asa1); and line 11, OG1RF(pCF10; prgB). *, additional EcoRI site. M, GeneRuler 100-bp DNA Ladder Plus marker (M-Medical Genenco).