Ampicillin resistance in Enterococcus faecium is a serious concern worldwide, complicating the treatment of E. faecium infections. Penicillin-binding protein 5 (PBP5) is considered the main ampicillin resistance determinant in E. faecium. The three known E. faecium clades showed sequence variations in the pbp5 gene that are associated with their ampicillin resistance phenotype; however, these changes alone do not explain the array of resistance levels observed among E. faecium clinical strains. We aimed to determine if the levels of PBP5 are differentially regulated between the E. faecium clades, with the hypothesis that variations in PBP5 levels could help account for the spectrum of ampicillin MICs seen in E. faecium. We studied pbp5 mRNA levels and PBP5 protein levels as well as the genetic environment upstream of pbp5 in 16 E. faecium strains that belong to the different E. faecium clades and for which the ampicillin MICs covered a wide range. Our results found that pbp5 and PBP5 levels are increased in subclade A1 and A2 ampicillin-resistant strains compared to those in clade B and subclade A2 ampicillin-susceptible strains. Furthermore, we found evidence of major clade-associated rearrangements in the region upstream of pbp5, including large DNA fragment insertions, deletions, and single nucleotide polymorphisms, that may be associated with the differential regulation of PBP5 levels between the E. faecium clades. Overall, these findings highlight the contribution of the clade background to the regulation of PBP5 abundance and point to differences in the region upstream of pbp5 as likely contributors to the differential expression of ampicillin resistance.KEYWORDS ampicillin, Enterococcus faecium, penicillin-binding protein 5, protein levels, resistance A mpicillin-resistant Enterococcus faecium has emerged to be one of the leading pathogens in the hospital setting (1, 2). In the United States, the incidence of infections caused by ampicillin-resistant E. faecium strains has increased since the 1980s, and this has been accompanied by a progressive increase in the MICs over time (3, 4); currently, the majority of E. faecium isolates recovered in hospitals around the United States are resistant to this ␤-lactam antibiotic (1, 2). The decreased susceptibility of E. faecium to ampicillin and other ␤-lactams has been mainly attributed to the high-molecular-weight penicillin-binding protein 5 (PBP5) (3-5). The proposed mechanism of PBP5-mediated action is a low affinity for ␤-lactam antibiotics, allowing peptidoglycan synthesis (transpeptidation) and bacterial growth when the other PBPs are inhibited by the drug (6, 7).

While tissue injury and repair are known to involve adaptive immunity, the profile of lymphocytes involved and their contribution to dermal scarring remain unclear. We hypothesized that restoration of T cell deficiency attenuates dermal scarring. Approach: We assessed the temporal-spatial distribution of T lymphocytes and their subtypes during the physiological dermal wound repair process in mice. Also, we compared the scarring outcomes between wild-type (WT) and severe combined immunodeficient (SCID) mice, which are lymphocyte deficient. Complementary gain-of-function experiments were performed by adoptively transferring lymphocyte subsets to validate their contribution to tissue repair in wounded SCID mice. Results: CD4 + T lymphocytes were present within dermal wounds of WT mice beginning on day 1 and remained through day 30. Wounds of SCID mice exhibited accelerated closure, increased inflammation, limited neovascularization, and exacerbated scarring compared with WT mice. Conversely, transfer of either mixed B and T lymphocytes or CD4 + lymphocytes alone into SCID mice resulted in moderated healing with less inflammation, collagen deposition, and scarring than control SCID wounds. In contrast, transfer of other lymphocyte subsets, including helper T lymphocytes (CD3 + CD4 + CD25-), CD8 + T cells and B cells, or regulatory T lymphocytes (CD4 + CD25 + CD127 low), did not reduce scar. Innovation: The finding that lymphocytes delay wound healing but reduce scar is novel and provides new insights into how dermal scarring is regulated. Conclusion: Our data support a suppressive role for CD4 + T cells against inflammation and collagen deposition, with protective effects in early-stage dermal wound healing. These data implicate adaptive immunity in the regulation of scarring phenotypes.

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