Emergence of VanB phenotype-vanA genotype Enterococcus faecium clinical isolate in Bulgaria.

anA glycopeptide resistance is characterized by acquired nducible resistance to both vancomycin and teicoplanin, hereas the VanB phenotype is characterized by variable evels of resistance to vancomycin but susceptibility to eicoplanin.1 Here we report a vancomycin-resistant Enterooccus faecium (VREFm) strain with VanB phenotype-vanA enotype incongruence. In the course of antimicrobial susceptibility testing of ll Enterococcus spp. isolates, we identified a VREFm strain BG139/2013) exhibiting high-level resistance to vancomycin MIC > 256 g/mL) and susceptibility to teicoplanin (4 g/mL) ccording to the Clinical and Laboratory Standards Institute 013 recommendations, hence having the VanB phenotype. G139/2013 strain was isolated on 22 February 2013 from the lood of a male patient aged 37 years at a Department of ephrology and Dialysis in Gabrovo, Bulgaria. Species idenification was done by the VITEK 2 system (bioMérieux) and onfirmed using multiplex polymerase chain reaction (PCR) s previously described.2 Antimicrobial susceptibility testing was performed y the Etest (LIOFILCHEM). BG139/2013 showed highevel gentamicin resistance; resistance toward ampicillin MIC > 256 g/mL), erythromycin (>256), tetracycline (24), iprofloxacin (>32); and susceptibility to linezolid (1.5) and hloramphenicol (1.5). Bacterial DNA was extracted using the ISOLATE Genomic NA Mini Kit (Bioline, UK) according to the manuacturer’s instructions. The glycopeptide antibiotic resisance genes were amplified with specific primers as escribed before: A1/A2, B1/B23 and vanD-F/vanD-R.4 The rimers for the vanA and vanD genes were actualized: anAD-F 5′-GARGAYGGMWSCATMCARGGY-3′ and vanAD-R ′-MGTRAAWCCNGGCAKRGTRTT-3′. Each 25L PCR mixture onsisted of 3 L of template DNA; a 0.1 M of each primer; 2.5 L of MyTaq PCR mix (Bioline) and 7.5 L of ultrapure 8.2 M PCR water (Bioline). DNA amplification was performed sing the following protocol: initial denaturation (95 ◦C for min), followed by 35 cycles of denaturation (95 ◦C for 45 s), te in Bulgaria