However, the MLST data indicated different STs due to changes in the nucleotide sequences of the analyzed housekeeping genes; ABT263 these data are consistent with the findings of Poh et al. [46]. In addition, the VREF isolates within clusters II-B1 and IV displayed identical PFGE and MLST profiles, in agreement with other authors [22, 33]. Nevertheless, pulsotypes from different wards showed similar multidrug resistance profiles, possibly due to horizontal genetic transference between these isolates. MLST is an important tool

for studying the molecular epidemiology of outbreaks of E. faecium and microbial population biology [44]. MLST analysis of VREF clinical isolates revealed four STs: ST203, ST412, ST612 and ST757. As previously reported, clonal complex 17 harbors various STs that have been involved in hospital outbreaks. Our results

revealed two allelic profiles, ST203 and ST412, belonging to clonal complex 17 STs involved in hospital outbreaks. However, clonal complex 17 has been resolved into Autophagy activity inhibition two different subgroups, one of which harbors ST17 and ST18, while the second harbors ST78 [47]. ST17, ST18 and ST203 are the major groups in the genetic lineage of E. faecium; they are distributed worldwide and have been associated with outbreaks [18, 48]. ST412 was the most frequent sequence type found in the VREF isolates from HIMFG and was genetically linked to the ST78 lineage. Interestingly, ST412 has been identified worldwide and associated with outbreaks [49]. According to the eBURST analysis, ST612 showed characteristics of the STs belonging to the 18 lineage. ST757 has not been characterized within clonal complex 17. In addition, ST757 displayed resistance markers (ampicillin and quinolones), virulence genes (esp + and/or hyl +) and the purK1 allele; however, it has not been associated with

outbreaks. Nevertheless, this community of multidrug-resistant strains is able to infect humans and might contribute to the spreading of these bacteria in the hospital, highlighting the importance of molecular typing via MLST to identify STs involved in nosocomial outbreaks. Recently, it was shown that MLST analysis of typified E. faecium based on selected alleles may generate misleading results due to the recombination of five alleles (atpA, ddl, gdh, gyd and pstS). As only the purk and adk alleles are located in RG7420 regions where there is no predicted recombination, the results must be interpreted with care [50]. The genome of E. faecium is highly plastic due to the few existing barriers to the acquisition of foreign genetic elements [51, 52]. Recent studies have provided evidence of high levels of recombination through comparative genomics analyses [51–54]. Whole-genome sequencing platforms are superior to conventional typing methods, providing an excellent tool for determining phylogenies and regions of recombination and for accurately discriminating between outbreak- and non-outbreak-causing VREF isolates [50, 55].