Plasmids containing hyl _Efm (pHyl_Efm) were previously shown to increase gastrointestinal colonization and lethality of Enterococcus faecium in experimental peritonitis. The hyl _Efm gene, predicting a glycosyl hydrolase, has been considered as a virulence determinant of hospital-associated E. faecium, although its direct contribution to virulence has not been investigated. Here, we constructed mutants of the hyl _Efm -region and we evaluated their effect on virulence using a murine peritonitis model.

Five mutants of the hyl _Efm -region of pHyl_EfmTX16 from the sequenced endocarditis strain (TX16 [DO]) were obtained using an adaptation of the PheS* system and were evaluated in a commensal strain TX1330RF to which pHyl_EfmTX16 was transferred by mating; these include i) deletion of hyl _Efm only; ii) deletion of the gene downstream of hyl _Efm (down) of unknown function; iii) deletion of hyl _Efm plus down; iv) deletion of hyl _Efm -down and two adjacent genes; and v) a 7,534 bp deletion including these four genes plus partial deletion of two others, with replacement by cat. The 7,534 bp deletion did not affect virulence of TX16 in peritonitis but, when pHyl_{EfmTX16Δ7,534} was transferred to the TX1330RF background, the transconjugant was affected in in vitro growth versus TX1330RF(pHyl_EfmTX16) and was attenuated in virulence; however, neither hyl _Efm nor hyl _Efm -down restored wild type function. We did not observe any in vivo effect on virulence of the other deletions of the hyl _Efm -region

The four genes of the hyl _Efm region (including hyl _Efm ) do not mediate the increased virulence conferred by pHyl_EfmTX16 in murine peritonitis. The use of the markerless counterselection system PheS* should facilitate the genetic manipulation of E. faecium in the future.