Relevance of hot spots in the evolution and transmission of Tn1546 in glycopeptide-resistant Enterococcus faecium (GREF) from broiler origin

Objectives: Glycopeptide-resistant enterococci are still present within the broiler sector, despite the EU ban of avoparcin more than a decade ago. In the present study, we have developed a rapid method for screening the flanking regions at the integration point of Tn1546 in glycopeptide-resistant Enterococcus faecium isolated from broiler farms. Methods: Total DNA was digested, ligated and amplified using primers from inside Tn1546. The resulting amplicons were purified and sequenced. Two new primers were designed based on obtained sequences. Results: Two main insertion points have been repeatedly found in isolates from the UK (n = 150). The first insertion point revealed that 25 isolates harboured Tn1546 positioned in a sequence with 96% homology to a streptomycin adenyltransferase gene (AY604739) from a Staphylococcus intermedius plasmid. At this insertion point, a direct repeat (GTCCT) was duplicated as previously described, indicating transposition at the target site. Furthermore, this 'hot spot' was also detected in isolates from Norway (2/8) and Denmark (17/20). The second insertion point detected in 45 isolates from the UK revealed integration into an Inc18-like plasmid, most likely by a process of target site recombination. Conclusions: The presence of a common insertion point for isolates from different geographical areas could suggest the insertion of Tn1546 by transposition in a plasmid-specific site, followed by genetic rearrangement both inside the transposon and in the flanking regions.