A clonal lineage of VanA-type Enterococcus faecalis predominates in vancomycin-resistant Enterococci isolated in New Zealand

Abstract

Avoparcin was used as a feed additive in New Zealand broiler production from 1977 until June 2000. We report here on the effects of the usage and discontinuation of avoparcin on the prevalence of vancomycin-resistant enterococci (VRE) in broilers. Eighty-two VRE isolates were recovered from poultry fecal samples between 2000 and mid-2001. VRE isolates were only obtained from broiler farms that were using, or had previously used, avoparcin as a dietary supplement. Of these VRE isolates, 73 (89%) were VanA-type Enterococcus faecalis and nine (11%) were VanA-type Enterococcus faecium. All E. faecalis isolates were found to have an identical or closely related pulsed-field gel electrophoresis (PFGE) pattern of SmaI-digested DNA and were susceptible to both ampicillin and gentamicin. The PFGE patterns of the nine E. faecium isolates were heterogeneous. All VRE contained both the vanA and ermB genes, which, regardless of species or PFGE pattern, resided on the same plasmid. Eighty-seven percent of the VRE isolates also harbored the tet(M) gene, while for 63 and 100%, respectively, of these isolates, the avilamycin and bacitracin MICs were high (>or=256 microg/ml). Five of eight vancomycin-resistant E. faecalis isolates recovered from humans in New Zealand revealed a PFGE pattern identical or closely related to that of the E. faecalis poultry VRE isolates. Molecular characterization of Tn1546-like elements from the VRE showed that identical transposons were present in isolates from poultry and humans. Based on the findings presented here, a clonal lineage of VanA-type E. faecalis dominates in VRE isolated from poultry and humans in New Zealand.

FIG. 1.

Representative SmaI PFGE patterns among VRE isolated from broilers. (A) VRE isolated in 2000. Lane 1, lambda DNA ladder standard; lane 2, PFGE pattern 2; lane 3, PFGE pattern 1a. (B) Vancomycin-resistant E. faecium isolated in 2001. Lane 1, lambda DNA ladder standard; lane 2, PFGE pattern 7; lane 3, PFGE pattern 6; lane 4, PFGE pattern 5; lane 5, PFGE pattern 4, lane 6, PFGE pattern 3. (C) Vancomycin-resistant E. faecalis isolated in 2001. Lane 1, lambda DNA ladder standard; lane 2, PFGE pattern 1a; lane 3, PFGE pattern 1b. Arrow 1 indicates the presence of an additional band, while arrow 2 denotes the absence of two bands when compared with PFGE pattern 1a. Sizes indicated are in kilobases.

FIG. 2.

Hybridization with vanA (A) and ermB (B) probes to I-CeuI-digested genomic DNA of two VRE isolates. Two lanes of genomic DNA from each isolate are shown. The first in each pair was not digested with I-CeuI, while the second was incubated with the enzyme. Lane 1, lambda DNA ladder standard; lanes 2 and 3, poultry PFGE pattern 1a; lanes 4 and 5, poultry PFGE pattern 2. Sizes indicated are in kilobases.

FIG. 3.

Conjugative transfer of vancomycin resistance. (A) SmaI macrorestriction patterns of strains involved in conjugative transfer of vancomycin resistance; (B) corresponding Southern blot hybridized with a vanA gene probe. Lane 1, recipient strain JH2-2; lane 2, transconjugant strain JH2-129; lane 3, donor strain 12-9VP; lane 4, lambda DNA ladder standard. Sizes are indicated in kilobases.

FIG. 4.

Comparison of poultry and human VRE in New Zealand. (A) PFGE of SmaI macrorestriction patterns in vancomycin-resistant E. faecalis. Lane 1, lambda DNA ladder standard; lanes 2 and 3, poultry isolates; lanes 4 to 8, human clinical isolates. (B) Long-template PCR ClaI RFLP patterns of Tn1546 elements. Lane 1, E. faecium BM4147; lanes 2 and 3, poultry VRE isolates; lanes 4 to 7, human clinical isolates; lane 8, lambda DNA cut with BstEII. ClaI fragments are 3,228, 2,968, 2,009, and 1,471 bp. Marker sizes are indicated in kilobases.