A-032. Heterogeneity of Tn1546 among VanA-type VRE Isolates of Enterococcus faecium
Genetic heterogeneity of Tn1546 elements was investigated among seventeen vanA-type clinical VRE isolates of E. faecium. These were characterized by individual and overlapping PCR, PFGE, plasmid DNA analysis, Southern hybridization and sequence analysis. Most of these multidrug-resistant strains harbored plasmids of 1.5- >300 kb in size. PFGE analysis of SalI and SmaI-digested plasmid and chromosomal DNA indicated that 5/17 isolates had identical profiles. The vancomycin resistance marker vanA was present on 130-210 kb plasmids and 11-80 kb SalI and SmaI restriction fragments of their plasmid and chromosomal DNA, respectively. Overlapping PCR of the vanSH region indicated larger than expected size (2.3 kb) amplicon in all the isolates. Twelve isolates yielded 4.0 kb vanSH amplicons and four yielded 5.5 kb amplicons. Most of the amplicons contained sequences between orf1-vanH genes instead of vanSH region due to a point mutation in the vanS gene and a 7 bp homology of the vanS forward primer in the orf1 region. Presence of 1.5 kb IS1251 sequence between the vanSH region resulted in 5.5 kb vanSH amplicons in the four isolates mentioned above. One of the seventeen isolates failed to amplify the orf1, orf2, vanR, and vanS genes but yielded a larger than expected (4.4 vs. 2.3 kb) vanSH amplicon. Sequence analysis of the vanSH amplicon from this isolate indicated the presence of vanH and pRUM-like plasmid sequences. The observed amplicon (4.4 kb) was obtained due to amplification between the vanH gene and an inverted repeat of the vanH reverse primer present at the junction of the plasmid’s right end (24873) and the left end (1-73). Discovery of the point mutations in the vanS gene in sixteen isolates and a loss of the orf1, orf2, vanR and vanS elements in one of the isolates is new and has never been reported. These findings also suggest a unique structural arrangement of the vanA elements that is hitherto unknown. The presence of vancomycin resistance (MIC >256 µg/ml) in the absence of regulatory/sensor elements vanR and vanS from one of the isolates suggests an alternative regulatory mechanism for the vancomycin resistance determinant region vanHAX in Tn1546.