Neutrophils are essential for rapid clearance of Enterococcus faecium in mice

Abstract

A progressive increase in infections with multiresistant Enterococcus faecium has been reported, especially in cancer patients and neutropenic patients. Despite its increasing importance as a nosocomial pathogen, knowledge of the pathogenesis of E. faecium infections is highly limited. In this study, we investigated the role of neutrophils during peritonitis with subsequent bacteremia caused by E. faecium. Therefore, we depleted neutrophils by intraperitoneal injections of monoclonal antibody RB6-8C5. Mice were followed for 5 days, and the enterococcal outgrowth and inflammatory response were compared between neutropenic mice and immunoglobulin G-injected control mice. Neutropenic mice demonstrated a severe delay in enterococcal clearance from all cultured organs (peritoneal fluid, blood, and lung and liver tissue). In particular, neutropenic mice remained bacteremic for up to 3 days, whereas all nonneutropenic mice had cleared the bacteria from circulation by 2 days. Furthermore, neutropenic mice displayed elevated peritoneal cytokine and chemokine levels 1 day after the infection and attracted fewer macrophages into the peritoneal cavity. In the circulation, a prolonged elevation of tumor necrosis factor alpha, interleukin-6, and the acute-phase proteins serum amyloid A and complement 3 were measured in neutropenic mice. In conclusion, attraction of neutrophils to the primary site of E. faecium infection is important for a rapid clearance of this bacterium, thereby attenuating a systemic inflammatory response.

FIG. 1.

Mice injected with αLy-6G were neutropenic during the entire experiment. Mice were injected with αLy-6G (open symbols) or rIgG (closed symbols) 24 h before and 1 and 3 days after i.p. injection of 10⁸ CFU of E. faecium. (A) At the moment of infection, αLy-6G-treated mice had <50 circulating neutrophils/μl; these mice stayed neutropenic during the entire experiment. (B) αLy-6G did not influence blood monocyte counts. Neutropenic mice did not attract neutrophils (C) and attracted fewer macrophages to the peritoneal cavity than control mice (D). Data are means ± SEM (eight mice per group at each time point). P values in the figures represent the overall difference between groups. **, P < 0.01; ***, P < 0.001 versus control mice at the time points indicated.

FIG. 2.

Neutropenic mice demonstrate a severely delayed clearance of E. faecium. Mice were injected with αLy-6G (open symbols) or rIgG (closed symbols) 24 h before and 1 and 3 days after i.p. injection of 10⁸ CFU of E. faecium. Bacterial loads were cultured in peritoneal lavage fluid (PLF) (A), blood (B), or lung (C) and liver (D) tissue at 1, 2, 3 and 5 days after inoculation. Data are means ± SEM (n = 8 per group at each time point). P values in the figures represent the overall difference between groups. P values of <0.01 (**) and <0.001 (***) versus control mice were recorded at the time points indicated.

FIG. 3.

E. faecium phagocytosed by peritoneal neutrophils. Shown is a representative Cytospin preparation of peritoneal lavage cells stained with Giemsa 24 h after control mice were infected with 10⁸ CFU of E. faecium (magnification, ×40).

FIG. 4.

Neutropenic mice display elevated plasma TNF-α and IL-6 concentrations. Mice were injected with αLy-6G (open symbols) or rIgG (closed symbols) 24 h before and 1 and 3 days after i.p. injection of 10⁸ CFU of E. faecium. Plasma TNF-α (A) and IL-6 (B) levels were measured at the time points indicated. Data are means ± SEM (eight mice per group at each time point). P values in the figures represent the overall difference between groups. P values of <0.05 (*) and <0.001 (***) versus control mice were recorded at the time points indicated.

FIG. 5.

Neutropenic mice display a prolonged acute-phase protein response. Mice were injected with αLy-6G (open symbols) or rIgG (closed symbols) 24 h before and 1 and 3 days after i.p. injection of 10⁸ CFU of E. faecium. Plasma C3 (A) and SAA (B) levels were measured at the time points indicated. Data are means ± SEM (eight mice per group at each time point). P values in the figures represent the overall difference between groups. ***, P < 0.001 versus control mice at the time points indicated.