Characterization of class IIa bacteriocin resistance in Enterococcus faecium

Vancomycin-resistant enterococci, particularly resistant Enterococcus faecium, pose an escalating threat in nosocomial environments because of their innate resistance to many antibiotics, including vancomycin, a treatment of last resort. Many class IIa bacteriocins strongly target these enterococci and may offer a potential alternative for the management of this pathogen. However, E. faecium's resistance to these peptides remains relatively uncharacterized. Here, we explored the development of resistance of E. faecium to a cocktail of three class IIa bacteriocins: enterocin A, enterocin P, and hiracin JM79. We started by quantifying the frequency of resistance to these peptides in four clinical isolates of E. faecium. We then investigated the levels of resistance of E. faecium 6E6 mutants as well as their fitness in different carbon sources. In order to elucidate the mechanism of resistance of E. faecium to class IIa bacteriocins, we completed whole-genome sequencing of resistant mutants and performed reverse transcription-quantitative PCR (qRT-PCR) of a suspected target mannose phosphotransferase (ManPTS). We then verified this ManPTS's role in bacteriocin susceptibility by showing that expression of the ManPTS in Lactococcus lactis results in susceptibility to the peptide cocktail. Based on the evidence found from these studies, we conclude that, in accord with other studies in E. faecalis and Listeria monocytogenes, resistance to class IIa bacteriocins in E. faecium 6E6 is likely caused by the disruption of a particular ManPTS, which we believe we have identified.