Molecular Analysis of Tn 1546 in vanA -Containing Enterococcus spp. Isolated from Humans and Poultry

Abstract

The genes encoding the VanA type of vancomycin resistance in enterococci are located on elements related to Tn1546 (1). Heterogeneity of Tn1546 in Enterococcus faecium has been previously reported (3, 4, 6, 9, 10, 12, 14, 15, 16) and includes deletions, insertions, and mutations. In this study, Tn1546 elements from 43 vanA-containing isolates of different enterococcal species with diverse pulsed-field gel electrophoresis (PFGE) patterns (criteria of Tenover et al. [13]) were analyzed: 30 E. faecium isolates (18 from chicken feces or products and 12 from human fecal samples) showing 25 unrelated PFGE patterns, 7 Enterococcus durans isolates with two unrelated PFGE patterns, (chicken feces or products), 5 Enterococcus hirae isolates (4 from chicken feces or products and 1 from a human fecal sample known to have an indistinguishable or a closely related PFGE pattern), and 1 Enterococcus faecalis isolate (from a chicken product). PCR products were obtained from all isolates for the seven genes of Tn1546 (vanR, vanS, vanH, vanY [10], vanA [16], vanX [11], and vanZ [8]); IS1216V-related sequences (6) were also demonstrated by PCR for all 43 isolates. When the vanXY (9) region was amplified, the expected 1,947-bp fragment was obtained with 36 of the 43 isolates; however, in all 5 E. hirae isolates and in 2 E. faecium isolates (both from chickens, with different PFGE patterns) the fragment amplified was longer than expected. Results of hybridization of vanXY PCR products with an IS1216V probe indicated that IS1216V was located within the vanXY region in these seven isolates and outside this region in the other isolates. An IS1216V-like sequence was first described within the intergenic vanXY region (5) and later both within and outside Tn1546 (3, 6); disruption of vanS by IS1216V has been found in a clinical E. faecium isolate (2). Despite these reports for E. faecium, this is the first time that IS1216V has been reported within the vanXY region of E. hirae. The restriction of the IS1216V-vanXY association to E. hirae and two E. faecium strains isolated from chickens could suggest interspecies transmission of these transposons in animal gastrointestinal tracts. IS1251 sequences (6) were detected in 18 E. faecium isolates (13 from chickens and 5 from humans; 15 unrelated PFGE patterns) but not in the other species tested. Analysis of vanSH (9) amplicons and hybridization showed that IS1251 was not included in this region. IS1251 has been previously found in the vanSH intergenic region and at other sites in E. faecium (4, 6). IS1476, first found in the vanY gene of an E. faecium isolate (9), was not detected in our strains nor in two other studies (6, 15). Recently, a 1-bp difference in vanX was found at position 8234, with either a G (G type) or a T (T type) (6). All our vanA isolates, except one (E. faecium from ground chicken), belonged to the G type as determined by DdeI digestion of the vanX gene PCR fragment (315 bp) (6). The G type has been associated with poultry, and the T type has been associated with porcine E. faecium isolates (3, 7). Both types have been found among isolates from humans in different countries (7), although we found only the G type among vancomycin-resistant enterococci isolated from humans in Spain. In conclusion, vanA-containing E. faecium, E. faecalis, E. hirae, and E. durans strains of human and animal origins were found to contain similar genetic arrangements of the vanA gene cluster, suggesting either horizontal transfer, the existence of a common reservoir, or a predilection for insertion of certain elements at specific sites.