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These ST-78 isolates comprised 100% of all infection-associated isolates from the HCL and HNA hospitals, and 87% (28/32) of the isolates from the IPS. Although this [http://en.wikipedia.org/wiki/RVX_208 RVX-208] ST type has previously been reported in Europe, it has not been reported in South America, the USA or Spain, approximately 95% of Paraguayans are of mixed Spanish and Amerindian descent. Therefore, the origins of ST-78 in Paraguay remain unexplained, although it appears that the ST-78 strain has spread to many hospitals in Asunci��n. In total, 97% (39/40) of the isolates tested belonged to MLST clonal complex 17 (CC17), which comprises a cluster of E.?faecium genotypes previously associated with hospital infections [3,12]. ST-438 was the only non-CC17 isolate recovered. Further analysis revealed no association between the antibiotic resistance profile and PFGE or MLST genotypes (Fig.?1). Characterization of Tn1546 was performed using previously [http://www.selleckchem.com/products/Neratinib(HKI-272).html buy Neratinib] published primers [13], and a touchdown PCR thermocycling protocol [7]. PCR primer walking and sequencing were used to define insertion site-specific sequences. The results of Tn1546 characterization revealed three different variants (Fig.?2). Two of the isolates did not show any insertion or deletion compared with the classic Tn1546 (BM4147) transposon sequence [14]. In contrast, 92% (37/40) of the isolates carried a Tn1546 variant containing an insertion of IS1216V (and a resultant deletion event) in orf1, coupled to an IS1251 insertion in the intergenic region of vanS and vanH (Tn1546 type F1 described by Willems et?al. [15]). A single isolate with a new MLST genotype (ST-438) carried a novel Tn1546 variant [http://www.selleckchem.com/products/BMS-777607.html selleck chemicals llc] of type F1 containing an ISEfa5 insertion. This IS element has only previously been described in Brazilian E.?faecium isolates, where the ISEfa5 was exclusively inserted in the intergenic region of vanX and vanY [16]. In this ST-438 Paraguayan isolate, the ISEfa5 element was inserted in the left flanking region of another insertion element IS1251, which itself was situated in the intergenic region between vanS and vanH. Sequencing of the ISEfa5 flanking insertion regions showed an inverted repeat of 6?bp (AGAGCC) flanked by an 8�bp direct repeat (AGAATAAT) of the IS1251 insertion element. From current evidence, presence of the ISEfa5 element in Tn1546 appears to be specific for Enterococcus isolates originating in South America. Further, this unusual insertion event indicates that Tn1546 may still be evolving in South American E.�faecium isolates. This is the first report of molecular investigations into vancomycin-resistant E.�faecium from Paraguay. The results from 40 infection-associated VRE isolates obtained from hospitals in Asunci�n revealed that the majority of isolates were found to be MLST type ST-78, a genotype associated with MLST clonal complex 17 (hospital-associated) isolates.