| Summary: | Rare information is available on clinical <i>Enterococcus faecium</i> encountered in Sardinia, Italy. This study investigated the antimicrobial susceptibility profiles and genotypic characteristics of <i>E. faecium</i> isolated at the University Hospital of Sassari, Italy, using the Vitek2 system and PCR, MLST, or WGS. Vitek2 revealed two VanB-type vancomycin-resistant <i>Enterococcus faecium</i> (VREfm) isolates (MICs mg/L = 8 and ≥32) but failed to detect vancomycin resistance in one isolate (MIC mg/L ≤ 1) despite positive genotypic confirmation of <i>vanB</i> gene, which proved to be vancomycin resistant by additional phenotypic methods (MICs mg/L = 8). This <i>vanB</i> isolate was able to increase its vancomycin MIC after exposure to vancomycin, unlike the “classic” occult <i>vanB</i>-carrying <i>E. faecium</i>, becoming detectable by Vitek 2 (MICs mg/L ≥ 32). All three <i>E. faecium</i> had highly mutated <i>vanB</i><sub>2</sub> operons, as part of a chromosomally integrated Tn<i>1549</i> transposon, with common missense mutations in VanH and VanB<sub>2</sub> resistance proteins and specific missense mutations in the VanW accessory protein. There were additional missense mutations in VanS, VanH, and VanB proteins in the <i>vanB</i><sub>2</sub>-carrying VREfm isolates compared to Vitek2. The molecular typing revealed a polyclonal hospital-associated <i>E. faecium</i> population from Clade A1, and that <i>vanB</i><sub>2</sub>-VREfm, and nearly half of vancomycin-susceptible <i>E. faecium</i> (VSEfm) analyzed, belonged to ST117. Based on core genome-MLST, ST117 strains had different clonal types (CT), excluding nosocomial transmission of specific CT. Detecting <i>vanB</i><sub>2</sub>-carrying VREfm isolates by Vitek2 may be problematic, and alternative methods are needed to prevent therapeutic failure and spread.
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