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Infection and Immunity, January 2009, p. 485-491, Vol. 77, No. 1
0019-9567/09/$08.00+0     doi:10.1128/IAI.00863-08
Copyright © 2009, American Society for Microbiology. All Rights Reserved.

Neutrophils Are Essential for Rapid Clearance of Enterococcus faecium in Mice

Masja Leendertse,^1,2,4* Rob J. L. Willems,⁴ Ida A. J. Giebelen,^1,2 Joris J. T. H. Roelofs,³ Marc J. M. Bonten,^4,5 and Tom van der Poll^1,2

Center for Infection and Immunity Amsterdam (CINIMA),¹ Center for Experimental and Molecular Medicine,² Department of Pathology, Academic Medical Center, Amsterdam, The Netherlands,³ Department of Medical Microbiology,⁴ Julius Center for Health Studies and Primary Care, University Medical Center Utrecht, Utrecht, The Netherlands⁵

Received 14 July 2008/ Returned for modification 11 August 2008/ Accepted 29 October 2008

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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.

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Enterococci are part of the normal bacterial flora of human and animal gastrointestinal tracts. Although enterococci were once not regarded as virulent, they are now recognized as a major cause of nosocomial infections worldwide (13). They are the third most common cause of nosocomial bacteremia in the United States and the fourth most common in Europe (http://www.earss.rivm.nl/). Although enterococci rarely cause diseases in healthy individuals, they can become pathogenic in patients in intensive care units, in hospitalized patients with severe underlying diseases or an impaired immune system, and in elderly people (23). Severely ill patients with hematologic malignancies and deep neutropenia are especially at an increased risk of developing enterococcal bacteremia (6, 7, 18, 29, 43).

The emergence of infections with enterococci can largely be attributed to their multiresistant nature to various classes of antibiotics. Especially Enterococcus faecium has acquired resistance to high-dose aminoglycosides, beta-lactam antibiotics, and vancomycin (5, 20, 37). Hospital-acquired E. faecium isolates belong predominantly to a distinct genetic subpopulation currently known as clonal complex 17 (CC17), which has adapted extremely well to the hospital environment and has spread worldwide (39). CC17 is characterized by the acquisition of multiple adaptive mechanisms, including ampicillin and quinolone resistance, a putative pathogenicity island harboring the esp virulence gene, and other cell surface protein genes (16, 19-21).

Despite the clinical importance of enterococci, little is known about defense mechanisms that protect the normal host against invasive enterococcal infections. The innate immune system represents the first line of defense against bacterial infections (27, 46). In previous studies, we described the normal immune response during primary E. faecium peritonitis (22). In a nonlethal model, we found a fast and brisk peritoneal neutrophil influx and a consecutive, rapid decline in peritoneal and systemic enterococcal load. In Toll-like receptor 2 (TLR2) and myeloid differentiation protein 88 knockout mice, a significantly reduced amount of neutrophils was attracted to the peritoneal cavity, which was accompanied by a delay in enterococcal clearance (22). These data, together with the fact that neutropenic patients are more vulnerable to acquiring E. faecium infections, prompted us to investigate the role of neutrophils during nonlethal E. faecium peritonitis with subsequent bacteremia.

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Mice. Specific-pathogen-free, 10-week-old female C57BL/6 mice were purchased from Harlan Sprague-Dawley (Horst, The Netherlands). The animals were housed in rooms with a controlled temperature and a 12-h light-dark cycle. They were acclimatized for 1 week prior to usage, and received standard rodent chow and water ad libitum. The Animal Care and Use Committee of the University of Amsterdam approved all experiments.

In vivo neutrophil depletion. To characterize the role of neutrophils during E. faecium peritonitis, mice were depleted of neutrophils before the E. faecium challenge. For depletion, mice were treated intraperitoneally (i.p.) with the rat anti-mouse monoclonal antibody (MAb) RB6-8C5 directed against Ly-6G, previously known as Gr-1, an antigen on the surface of murine granulocytes (36). The antibody (Ly-6G) was produced by TSD BioServices (Germantown, NY) by i.p. injection of RB6-8C5 hybridoma into nude mice and by subsequent ascites collection. A total of 100 µg of RB6-8C5 was administered i.p. 1 day before the challenge with E. faecium. When mice were followed up until 5 days after induction of E. faecium peritonitis, mice were injected with the antibody on days 1 and 3 as well. The specificity and efficacy of RB6-8C5 have been well established (9, 35, 42). Control mice were given the equivalent amount of purified rat immunoglobulin G (rIgG) (Sigma, St. Louis, MO).

Bacterial strain. A vancomycin-resistant E. faecium strain, E155, was used in all experiments. This clinical isolate from the Cook County Hospital, Chicago, IL, belongs to a genetic subpopulation of hospital-associated E. faecium, currently labeled CC17. For all experiments, the bacteria were grown overnight on sheep blood agar plates and then grown for approximately 3.5 h in Todd-Hewitt broth (Difco, Detroit, MI) to mid-logarithmic phase at 37°C, with shaking.

Induction of peritonitis. Peritonitis was induced as described previously (22). Briefly, E. faecium was cultured in Todd-Hewitt broth at 37°C, harvested at mid-log phase, and washed twice in sterile saline to clear the bacteria of medium. Bacteria were then resuspended in sterile isotonic saline, and mice were injected i.p. with 10⁸ CFU of E. faecium in 200 µl sterile isotonic saline. This bacterial dose is gradually cleared by normal C57BL/6 mice and is not associated with lethality (22). The inoculum was plated immediately after inoculation on sheep blood agar plates to determine viable counts.

Collection of samples. Mice were anesthetized by inhalation of isoflurane (Abbott Laboratories Ltd., Kent, United Kingdom)/O₂ (2%/2 liters), and a peritoneal lavage was performed with 5 ml sterile phosphate-buffered saline using an 18-gauge needle; peritoneal lavage fluid was collected in sterile polypropylene tubes (Plastipak; Becton-Dickinson, Mountain View, CA). After collection of peritoneal fluid, blood was drawn by cardiac puncture, transferred to heparin-gel Vacutainer tubes, and immediately placed on ice. Next, the abdomen was opened and the liver and lungs were harvested.

Determination of bacterial outgrowth. The number of E. faecium CFU was determined in peritoneal lavage fluid, blood, and liver and lung homogenates. Lungs and livers were harvested and homogenized at 4°C in 4 volumes of sterile saline using a tissue homogenizer (Biospec Products, Bartlesville, OK). CFU were determined from serial dilutions; 50 µl of each dilution was plated onto blood agar plates and incubated at 37°C at 5% CO₂ for 20 h before colonies were counted and corrected for the dilution factor.

Cell counts and differentials of blood and peritoneal lavage fluid. Erythrocytes were lysed with ice-cold isotonic NH₄CL solution (155 mM NH₄CL, 10 mM KHCO₃, 0.1 mM EDTA, pH 7.4), and the remaining cells were washed with phosphate-buffered saline. These cells and cells in the peritoneal lavage samples were counted using a Coulter Counter (Beckman Coulter, Fullerton, CA). Differential cell counts for the determination of neutrophils, macrophages/monocytes, and lymphocytes were performed on Cytospin preparations, stained with a modified Giemsa stain (Diff-Quick; Dade Behring). Peritoneal fluid supernatant and plasma were stored at –20°C until determination of cytokines.

FACS analysis. As RB6-8C5 MAb also effects Gr-1-positive subpopulations of monocytes/macrophages (10), fluorescence-activated cell sorter (FACS) analysis of blood monocytes and peritoneal macrophages from RB6-8C5 and control antibody-treated mice was performed. Immunostaining for cell surface molecules was performed for 30 min at 4°C, using directly labeled antibodies against F4/80 and Gr-1. Antibodies were used in concentrations recommended by the manufacturer (BD Pharmingen). Cells were analyzed using FACSCalibur (BD Biosciences, Mountain View, CA).

Histology. Directly after sacrifice, lungs and livers were fixed in 4% formalin and embedded in paraffin for routine histology. Sections of 4-µm thickness were stained with hematoxylin and eosin. All slides were coded and scored by a pathologist without knowledge of the type of mouse or treatment.

Assays. Macrophage inflammatory protein 2 and cytokine-induced neutrophil chemoattractant (KC) were measured in peritoneal lavage fluid by enzyme-linked immunosorbent assays (ELISAs; R&D Systems, Minneapolis, MN) according to the manufacturer's instructions. Tumor necrosis factor alpha (TNF-), interleukin-6 (IL-6), IL-10, IL-12p70, gamma interferon (IFN-), and monocyte chemoattractant protein 1 (MCP-1) were measured in peritoneal lavage fluid and plasma by using a commercially available cytometric bead array multiplex assay (BD Biosciences, San Jose, CA) in accordance with the manufacturer's recommendations. Serum amyloid A (SAA) was measured by a commercially available ELISA for mouse SAA, in accordance with the manufacturer's instructions (Biosource International). Complement 3 (C3) was detected by sandwich ELISA as described previously (32). Aspartate aminotransferase, alanine aminotransferase, lactate dehydrogenase, and creatinine were determined with commercially available kits (Sigma-Aldrich, St. Louis, MO), using a Hitachi analyzer (Boehringer Mannheim, Mannheim, Germany).

Statistical analysis. All data are expressed as means ± standard error of the means (SEM). Serial data were analyzed by two-way analysis of variance followed by a post hoc Bonferroni test. Two group comparisons were done by using the Mann-Whitney U test. For all analysis, GraphPad Prism version 4 (GraphPad Software, San Diego, CA) was used. A P value of <0.05 was considered statistically significant.

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Neutropenia is associated with a strongly reduced early clearance of E. faecium. To determine the importance of neutrophils in host defense against E. faecium peritonitis, mice were depleted of neutrophils by administration of Ly-6G 1 day prior to infection and where applicable 1 and 3 days after infection. Treatment with Ly-6G was successful for inducing neutropenia (<50 neutrophils/µl) (Fig. 1A). E. faecium peritonitis was induced in neutropenic and rIgG-treated control mice, and groups of mice were sacrificed on days 1, 2, 3, and 5 after infection. An inoculum of 10⁸ CFU was chosen, as we had previously found that healthy mice are able to clear this infection in 2 to 3 days, whereas an inoculum of 10⁹ CFU resulted in 30% mortality (22). Mice injected with Ly-6G remained neutropenic during the entire 5-day observation period (<50 neutrophils/µl; P < 0.01 versus control mice) (Fig. 1A). rIgG-treated control mice showed an early rise in peripheral blood neutrophil numbers, peaking at day 1; thereafter, neutrophil counts returned to baseline values. In addition, control mice displayed a strong increase in the number of neutrophils in their peritoneal lavage fluid 1 day after infection; such an increase was not observed in neutropenic mice (P < 0.001) (Fig. 1C). Remarkably, although before infection with E. faecium neutropenic mice had unaltered macrophage numbers in their peritoneal lavage fluid, they demonstrated a reduced influx of macrophages into their peritoneal cavities relative to that in control mice after i.p. infection (P < 0.01) (Fig. 1D). Of note, monocyte counts in peripheral blood were similar in neutropenic and control mice (Fig. 1B). FACS analysis of peritoneal and blood cells obtained from mice 1 day after antibody treatment (e.g., the moment of E. faecium challenge) revealed that 0.69% (±0.83%) of peritoneal macrophages of control mice were Gr-1 positive. After RB6-8C5 MAb treatment, this amount was reduced to 0.05% (±0.03%). Of blood monocytes, 2.82% (±0.62%) were Gr-1 positive in control mice and 0.20% (±0.08%) in RB6-8C5 MAb-treated mice.

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FIG. 1. Mice injected with Ly-6G were neutropenic during the entire experiment. Mice were injected with Ly-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, Ly-6G-treated mice had <50 circulating neutrophils/µl; these mice stayed neutropenic during the entire experiment. (B) Ly-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.

One day after infection, E. faecium was cultured from all of the following organs investigated: peritoneal fluid (the primary site of infection), blood, lungs, and livers (Fig. 2A to D). At this time point, all organs from neutropenic mice contained at least 10-fold more E. faecium CFU than rIgG-treated control mice (P < 0.01 to P < 0.0001). Neutropenic mice stayed bacteremic for up to 3 days after the infection, whereas blood cultures in control mice were sterile from day 2 after infection onward (P < 0.01 for the difference between groups). Similarly, in the peritoneal fluid of neutropenic mice, 100-fold more E. faecium CFU were cultured than in the peritoneal fluid of control animals 2 and 3 days postinfection (P < 0.001 and P < 0.01, respectively). Moreover, the lungs of neutropenic mice displayed very high bacterial loads at days 2 and 3 postinfection; at these time points, E. faecium could not be recovered from lungs of control mice anymore (P < 0.001). Of note, in spite of the strongly impaired clearance during the first 3 days after infection, neutropenic mice eventually cleared E. faecium from all body compartments; i.e., cultures from all tested body sites were sterile 5 days after infection. Figure 3 displays a Cytospin preparation of peritoneal cells 24 h after infection in control mice, showing the importance of neutrophils in phagocytosing E. faecium. Of note, Ly-6G treatment per se (i.e., before infection with E. faecium) did not result in positive cultures of peritoneal fluid or blood; in addition, cultures obtained from mice infected with E. faecium grew only this pathogen.

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FIG. 2. Neutropenic mice demonstrate a severely delayed clearance of E. faecium. Mice were injected with Ly-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.

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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, x40).

Prolonged high E. faecium load associated with higher cytokine response and prolonged, elevated acute-phase protein levels. At the moment of i.p. E. faecium injection (i.e., 1 day after injection of RB6-8C5 or control antibody), mice had no signs of an inflammatory response, as reflected by unaltered cytokine levels (data not shown). In accordance with our previous study (22), control mice displayed low or undetectable levels of TNF-, IL-6, MCP-1, IL-10, IL-12p70, IFN-, KC, and macrophage inflammatory protein 2 in plasma and peritoneal fluid 1 day after infection and beyond (Fig. 4) (Table 1 and data not shown). In contrast, neutrophil-depleted mice had detectable plasma TNF- and IL-6 levels for up to 3 days after the infection (Fig. 4A and B). Moreover, neutropenic mice had detectable levels of TNF-, IL-6, KC, and MCP-1 in their peritoneal lavage fluid (Table 1). Additionally, in these mice, plasma C3 and SAA levels were persistently elevated (Fig. 5A and B).

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FIG. 4. Neutropenic mice display elevated plasma TNF- and IL-6 concentrations. Mice were injected with Ly-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- (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.

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TABLE 1. Elevated cytokine and chemokine levels in peritoneal fluid of neutropenic mice 24 h after infection with E. faecium

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FIG. 5. Neutropenic mice display a prolonged acute-phase protein response. Mice were injected with Ly-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.

Neutrophil depletion does not cause organ damage. In accordance with our earlier study (22), E. faecium peritonitis and bacteremia were not associated with organ injury, as determined by the histopathology of liver and lungs and by clinical chemistry (aspartate aminotransferase, alanine aminotransferase, lactate dehydrogenase, and creatinine) (data not shown). In spite of the prolonged enterococcal load in multiple body sites, neutrophil-depleted mice did not show any signs of organ injury either (data not shown).

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Since the 1970s, a progressive increase of enterococcal bacteremia has been described, and enterococci are currently ranked as the third most common cause of bloodstream infections (7, 34). Vancomycin-resistant enterococci are especially of main concern, as only a few antibiotics are effective against this multiresistant pathogen. The vast majority of vancomycin-resistant enterococcal infections are caused by E. faecium, which is specifically adapted to the hospital environment and belongs to a genetic subpopulation, currently labeled CC17 (20, 40, 47). CC17 is characterized by the acquisition of multiple adaptive mechanisms, including ampicillin and quinolone resistance, a putative pathogenicity island harboring the esp virulence gene, and other cell surface protein genes (16, 19-21).

Patients with hematologic malignancies and severe neutropenia display the highest risk of acquiring enterococcal bacteremia (6, 7, 18, 29, 43). Knowledge of how the intact innate immune system deals with E. faecium is limited. In an earlier study, we showed that E. faecium is recognized by immune cells through TLR2 and that interruption of TLR-dependent myeloid differentiation protein 88 signaling after i.p. administration of this pathogen is associated with a reduced early neutrophil recruitment to the primary site of the infection and a modestly delayed bacterial clearance (22). In the current study, we focused on the role of neutrophils in this model of E. faecium peritonitis. The main finding was a strongly impaired early clearance of E. faecium from all body sites examined in mice with neutropenia. Indeed, neutropenic mice had prolonged high enterococcal loads in all (cultured) organs and remained bacteremic for at least 3 days after infection, whereas control mice were no longer bacteremic after 2 days. Of note, bacteria were eventually cleared even in neutropenic mice.

Interestingly, E. faecium peritonitis and bacteremia did not convert to a lethal infection in neutrophil-depleted mice, although in previously healthy mice, i.p. infection with 10 times more bacteria of the same E. faecium strain resulted in 30% lethality (22). This discrepancy might be explained by the absence of neutrophils, as neutrophils play a dual role during infection and inflammation. On the one hand, these cells are of great importance in killing bacteria and clearing infections. On the other hand, attraction of neutrophils to the site of inflammation can cause serious tissue damage and propagate the inflammatory response by the release of neutrophil-generated oxygen free radicals and proteases (45, 46). Indeed, Walley et al. (44) and Ness et al. (28) demonstrated that in a lethal model of polymicrobial peritonitis, inhibition of neutrophil recruitment into the peritoneal cavity by elimination or inhibition of neutrophil-attracting chemokines was associated with a reduced mortality, supporting the notion that enhanced neutrophil influx can cause tissue damage during abdominal infection. One can hypothesize that the low-virulence nature of E. faecium does not cause serious organ damage without the participation of neutrophils during infection. Nonetheless, neutropenic mice did show evidence of an enhanced systemic inflammatory response, as reflected by sustained elevations of the plasma concentrations of TNF- and IL-6, as well as of the acute-phase proteins SAA and C3.

Healthy mice clear an infection with a large inoculum of 10⁸ CFU of E. faecium without showing important signs of illness, which resembles the scenario in healthy humans who are unlikely to develop infection and disease by E. faecium. Clearly, the healthy innate immune system is able to control E. faecium infections and prevent the development of severe disease. Previously, we found the recruitment of large numbers of neutrophils to the primary site of infection in the model of E. faecium peritonitis used here (22). We now firmly establish that this early reaction significantly contributes to an effective antibacterial response. Of note, although neutrophils are crucial in host defense against a wide array of pathogens, their contribution is not the same for all infections or even for different routes of infection with the same pathogen (3, 4, 8, 9, 11, 24, 25, 33, 36, 42). Neutrophils were found to impact the efficacy of therapeutic interventions (i.e., granulocyte colony-stimulating factor or IFN- given together with appropriate antibiotics) after i.p. infection with E. faecalis (30, 31). However, these studies, in which neutropenia was induced by cyclophosphamide, did not directly investigate the role of neutrophils in host defense against E. faecalis peritonitis (30, 31).

To deplete mice of neutrophils, MAb RB6-8C5 was used. This antibody recognizes an antigen, Ly-6G, present on the cell surfaces of mature neutrophils and eosinophils, and specifically depletes these granulocytes in vivo (12, 38). Additionally, Gr-1 is intermediately expressed by other cells, such as a small proportion of monocytes, dendritic cells, and CD8⁺ and CD4⁺ T cells (10, 14, 26, 41). We found a depletion of Gr-1-expressing monocytes/macrophages after treatment with the RB6-8C5 MAb. It is unlikely, however, that the depletion of this Gr-1-positive subset of monocytes/macrophages explains the reduced antibacterial defense seen in the RB6-8C5 MAb-treated mice, as the proportion of these cells (in uninfected mice) is <3%. Furthermore, during infection, the amounts of circulating monocytes did not differ between the two groups.

Previous studies that used MAb RB6-8C5 to investigate the role of neutrophils during Legionella pneumophila and Acinetobacter baumannii infections showed that its primary effect is depletion of neutrophils (36, 42). During E. faecium peritonitis, MAb RB6-8C5-treated mice attracted fewer macrophages to the peritoneal cavity than their controls, despite a greater enterococcal burden and higher cytokine and chemokine concentrations in their peritoneal fluids. These results are similar to those previously reported by LaFleur et al. (17), which showed that neutrophil depletion by MAb RB6-8C5 and subsequent i.p. thioglycolate treatment resulted in 33% reduced peritoneal macrophage recruitment in neutrophil-depleted mice despite unchanged numbers of circulating monocytes. The underlying mechanism for this phenomenon remains to be established but could be related to the release of macrophage-attracting mediators (not investigated in our study or by LaFleur et al. [17]) by neutrophils that have migrated into the peritoneal cavity in the normal situation.

Improving our knowledge of the pathogenesis of E. faecium infections is necessary in the face of the increasing prevalence of multiresistant E. faecium isolates. In this study, we demonstrate that the attraction of neutrophils to the site of E. faecium infection is an important facet in the early and rapid clearance of this bacterium. Nonetheless, in a neutrophil-deficient but otherwise healthy host, other components of the immune system, e.g., complement proteins, natural antibodies (1, 2, 15), and monocytes/macrophages, are eventually able to compensate for the deficit in neutrophils.

 
We thank J. Daalhuisen and M. ten Brink for providing expert technical assistance.

 
* Corresponding author. Mailing address: Center for Experimental and Molecular Medicine, Room G2-130, Academic Medical Center, Meibergdreef 9, 1105 AZ Amsterdam, The Netherlands. Phone: 31-20-5665910. Fax: 31-20-6977192. E-mail: m.leendertse{at}amc.uva.nl

Published ahead of print on 10 November 2008.

Editor: B. A. McCormick

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Infection and Immunity, January 2009, p. 485-491, Vol. 77, No. 1
0019-9567/09/$08.00+0     doi:10.1128/IAI.00863-08
Copyright © 2009, American Society for Microbiology. All Rights Reserved.

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