The objective of this study was to characterize Polish penicillin-resistant, ampicillin-susceptible Enterococcus faecalis (PRASEF), increasingly reported to the National Reference Centre for Susceptibility Testing, Poland, to elucidate the path of emergence of such strains. A total of 136 isolates were examined by antimicrobial susceptibility testing and for the β-lactamase production (cefinase test). The clonality of isolates was established by multilocus sequence typing (MLST) and the penicillin-binding protein pbp4 gene was sequenced to search for putative mutation(s). The presence of pheromone-responsive plasmids was investigated by clumping test and PCR detection of plasmid-specific genes. All Polish PRASEF were multidrug resistant and β-lactamase-negative. MLST assigned isolates mostly to high-risk enterococcal clonal complexes (HIRECCs) 6 (57.4%) and 87 (30.1%), in addition to to CC88 (12.5%). The sequencing of pbp4 revealed mutations upstream of a putative promoter region and amino acid alterations in PBP4, affecting 24 positions and resulting in 30 variants. While production of aggregation substance was observed for 17.6% of isolates, genes of pheromone plasmids were much more commonly detected. However, no conjugal transfer of penicillin resistance was observed. Penicillin resistance in E. faecalis emerges mostly in HiRECCs due to PBP4 overproduction and/or mutations. The acquisition of penicillin resistance by HiRECCs may represent the next step in the evolution of E. faecalis as human nosocomial pathogen.
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