| Summary: | <p>Abstract</p> <p>Background</p> <p>Plasmids containing <it>hyl</it><sub><it>Efm </it></sub>(pHyl<sub>Efm</sub>) were previously shown to increase gastrointestinal colonization and lethality of <it>Enterococcus faecium </it>in experimental peritonitis. The <it>hyl</it><sub><it>Efm </it></sub>gene, predicting a glycosyl hydrolase, has been considered as a virulence determinant of hospital-associated <it>E. faecium</it>, although its direct contribution to virulence has not been investigated. Here, we constructed mutants of the <it>hyl</it><sub><it>Efm</it></sub>-region and we evaluated their effect on virulence using a murine peritonitis model.</p> <p>Results</p> <p>Five mutants of the <it>hyl</it><sub><it>Efm</it></sub>-region of pHyl<sub>EfmTX16 </sub>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<sub>EfmTX16 </sub>was transferred by mating; these include <it>i</it>) deletion of <it>hyl</it><sub><it>Efm </it></sub>only; <it>ii</it>) deletion of the gene downstream of <it>hyl</it><sub><it>Efm </it></sub>(<it>down</it>) of unknown function; <it>iii</it>) deletion of <it>hyl</it><sub><it>Efm </it></sub>plus <it>down</it>; <it>iv</it>) deletion of <it>hyl</it><sub><it>Efm</it></sub>-<it>down </it>and two adjacent genes; and <it>v</it>) a 7,534 bp deletion including these four genes plus partial deletion of two others, with replacement by <it>cat</it>. The 7,534 bp deletion did not affect virulence of TX16 in peritonitis but, when pHyl<sub>EfmTX16Δ7,534 </sub>was transferred to the TX1330RF background, the transconjugant was affected in <it>in vitro </it>growth versus TX1330RF(pHyl<sub>EfmTX16</sub>) and was attenuated in virulence; however, neither <it>hyl</it><sub><it>Efm </it></sub>nor <it>hyl</it><sub><it>Efm</it></sub>-<it>down </it>restored wild type function. We did not observe any <it>in vivo </it>effect on virulence of the other deletions of the <it>hyl</it><sub><it>Efm</it></sub>-region</p> <p>Conclusions</p> <p>The four genes of the <it>hyl</it><sub><it>Efm </it></sub>region (including <it>hyl</it><sub><it>Efm</it></sub>) do not mediate the increased virulence conferred by pHyl<sub>EfmTX16 </sub>in murine peritonitis. The use of the markerless counterselection system PheS* should facilitate the genetic manipulation of <it>E. faecium </it>in the future.</p>
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