4.1. Clinical Isolates and Species Identification

Clinical specimens submitted to Sapporo Mirai Laboratory, Co., Ltd. were initially cultured on Sheep Blood Agar plates (Nissui Pharmaceutical, Co., Tokyo, Japan), and occasionally on Columbia CA Sheep Blood Agar plates (Kohjin Bio, Co., Tokyo, Japan) to promote bacterial growth. Species identification was performed by MALDI-TOF mass spectrometry using MALDI Biotyper (BRUKER). All the isolates were confirmed as E. faecalis and E. faecium by the PCR targeting species-specific sequence of PBP5 genes, as described previously [69]. For some isolates that could not be identified by the PCR, the species was confirmed by the determination of the 16S rRNA gene sequence through direct sequencing with the PCR product amplified by specific primers [70]. Individual isolates were stored in Microbank (Pro-Lab Diagnostics, Richmond Hill, ON, Canada) at −80 °C, and were recovered when they were analyzed.