Skip to main content
NCBI home page
NIHPA Author Manuscripts logoLink to NIHPA Author Manuscripts
. Author manuscript; available in PMC: 2013 Jul 1.
Published in final edited form as: FEMS Immunol Med Microbiol. 2012 Mar 8;65(2):270–282. doi: 10.1111/j.1574-695X.2012.00944.x

Biofilm and planktonic Enterococcus faecalis elicit different responses from host phagocytes in vitro

Kasturee Daw 1, Arto S Baghdayan 1, Shanjana Awasthi 1, Nathan Shankar 1,*
PMCID: PMC3366019  NIHMSID: NIHMS358626  PMID: 22333034

Abstract

Enterococcus faecalis is a commensal organism of the gastrointestinal tract but can also cause serious opportunistic infections. In addition to high levels of antibiotic resistance, the ability to form biofilms on abiotic surfaces and on in-dwelling devices within the host, complicate treatment strategies and successful outcomes of antibiotic therapy. Despite rapid advances made in recent years in understanding the genomics and virulence of this organism, much remains to be learned regarding the host response to enterococcal infections. In this study we investigated the interaction of RAW264.7 macrophages and JAWS II dendritic cells with biofilm and planktonic E. faecalis, in vitro. Specifically, we compared phagocytosis, intracellular survival, secretion of proinflammatory cytokines and the activation and maturation of phagocytes. Our results revealed that both macrophages and dendritic cells phagocytize biofilm mode cells at levels equal to or better than their planktonic counterparts. Internalized biofilm bacteria showed relatively greater survival at 24 hours in macrophages than in dendritic cells, and led to slightly higher expression of phagocyte activation markers. Macrophages infected with biofilm cells also secreted lower levels of proinflammatory cytokines studied. Overall these results suggest that biofilm E. faecalis may be better adapted to overcome host defenses in vivo.

Keywords: Enterococcus faecalis, biofilm, macrophage, dendritic cell

Introduction

Bacterial biofilms are microbial communities encased within a complex matrix that are capable of colonizing natural body surfaces such as the epithelium, lung and heart as well as implanted medical devices such as central venous and urinary catheters, intra-uterine devices, and prosthetic heart valves (Donlan & Costerton, 2002). The biofilm mode of growth offers many advantages to the bacteria, of which particularly important is the capability to acquire an increased resistance towards antibiotics and biocides. This leads to complications in the management of biofilm infections and thereby limits therapeutic options (Mah & O’Toole, 2001; Stewart, 2002; Davies, 2003). In addition, bacterial biofilms also pose a challenge to the host immune system (Guenther, et al., 2009).

Enterococci are commensal flora of the gastrointestinal tract of most complex metazoans. Although they rarely cause infections in healthy individuals, they can become pathogenic in patients in intensive care units, especially those with an impaired immune system, and in patients with hematologic malignancies and neutropenia. Currently Enterococci are the third leading cause of nosocomial infections accounting for about 17% of surgical site infections, 11.5% of bacteremia and 14.3% of acquired urinary tract infections (Richards, et al., 2000). The ability to form biofilm is an important aspect of the lifestyle of enterococci and is a critical factor in causing endodontic and urinary tract infections as well as endocarditis. Enterococcal biofilm most likely results in foci of infections that resist host immune defenses and subsequently continually seed the bloodstream. Recently emerging data has confirmed that a vast majority of the difficult-to-cure pathologies may be a result of biofilm type infections (Hall-Stoodley & Stoodley, 2009). Numerous studies have identified different factors contributing to the biofilm formation in E. faecalis, such as the adhesin Esp (Toledo-Arana, et al., 2001; Tendolkar, et al., 2004), the two-component quorum-sensing signal transduction system, Fsr (Hancock & Perego, 2004), the secreted metalloprotease, GelE (Qin, et al., 2001; Kristich, et al., 2004; Thomas, et al., 2008), the sugar-binding transcriptional regulator, BopD (Hufnagel, et al., 2004), an autolysin, the enterococcal polysaccharide antigen, Epa (Mohamed, et al., 2004) and pili encoded by the ebp (Nallapareddy, et al., 2006) and bee locus (Tendolkar, et al., 2006).

Despite the clinical importance of enterococci, little is known about how the host immune system responds during invasive enterococcal infections. It had been reported that neutrophils (PMNs) play an important role in the host immune response to enterococcal infection with a role for complement and specific antibodies in opsonization and killing of E. faecalis (Arduino, et al., 1994). Interactions between host immune cells and Enterococcus faecium have been studied to a greater extent and a mouse peritonitis model demonstrated that recruitment of neutrophils to the primary site of infection was essential to clear the peritonitis (Leendertse, et al., 2008). In similar studies, local concentrations of proinflammatory cytokines and chemokines and the early release of proinflammatory cytokines into the circulation were enhanced in peritoneal macrophage-depleted mice with an increase in neutrophil numbers (Leendertse, et al., 2009). These data also revealed that in addition to the TLR family, other components of the innate immune system were important in clearing enterococcal infection.

Macrophages play a major role in host defense against infection and contribute to the initial inflammatory response (Dunn, et al., 1985; Topley, et al., 1996; Broche & Tellado, 2001; Knudsen, et al., 2002). They orchestrate several functions such as phagocytosis, antigen processing and secretion of pro- and anti-inflammatory cytokines. They have also been shown to be important for the early containment of infection within the peritoneal cavity with neutrophils becoming involved in later stages of infection (Dunn, et al., 1985). Following bacterial translocation across the intestinal epithelium, survival within macrophages and dendritic cells may serve as a vehicle for the bacteria to travel to the mesenteric lymph nodes and cause systemic infection. Few studies have examined the interaction of enterococci with macrophages and a number of bacterial factors including transcriptional regulators and the production of slime appear to promote prolonged survival within macrophages (Gentry-Weeks, et al., 1999; Giard, et al., 2006; Leendertse, et al., 2009). Our recent studies in this regard point to the role for the transcriptional regulator, PerA, in promoting survival within macrophages (Coburn, et al., 2008).

Dendritic cells are phagocytic cells capable of triggering adaptive immunity by initiating phagocytic uptake and antigen processing (Sato & Fujita, 2007). This leads to maturation of dendritic cells and production of inflammatory cytokines, chemokines and up-regulation of costimulatory molecules. The maturation process also facilitates dendritic cell migration to specialized lymphoid tissues for antigen presentation to T cells (Serbina, et al., 2008). Although the population of dendritic cells in the peritoneal cavity is quite small, their role in modulating the infection process is of immense importance.

Despite the growing importance of enterococcal biofilms in infection, there is little information available regarding host immunity to enterococcal biofilms or how the biofilm bacteria affect the host defense mechanisms. Due to the complexity of the biofilm matrix, bacteria within them may be recognized differently by the host immune system when compared to their planktonic counterpart. A previous study with macrophages has shown that E. faecalis recovered from biofilm on dentin material was capable of stronger surface adherence and better survival inside monocytes in vitro (Mathew, et al., 2010). Infected monocytes produced lower amounts of proinflammatory cytokines compared to monocytes infected with planktonic bacteria. Another study using an isogenic pair of E. faecalis strains created to evaluate the role of exopolysaccharide, concluded that bacteria under biofilm promoting conditions survived better within host phagocytes (Baldassarri, et al., 2005). More recent studies with P. aeruginosa and S. aureus have directly compared the immune response to planktonic and biofilm bacteria (Ciornei, et al., 2010; Thurlow, et al., 2011). We employed a similar approach in this study to directly compare planktonic and biofilm E. faecalis for their interaction in vitro with macrophages and dendritic cells to understand how the biofilm phenotype affects phagocytosis, survival, maturation and activation of phagocytes and proinflammatory cytokine expression.

Material and Methods

Bacterial strains

Two E. faecalis strains, E99 and FA2-2, were used in this study to evaluate the influence of strain backgrounds. E99 is a clinical isolate from an UTI, is MLST type 4 and forms biofilms mediated by surface protein Esp and Bee pilus (Tendolkar, et al., 2006). Strain FA2-2(pESPF) has been described earlier (Tendolkar, et al., 2004), is an MLST type 8 strain, and forms biofilm by virtue of expression of Esp at the cell surface. Biofilm parameters such as biovolume, average and maximum thickness are different for these two strains (Tendolkar, et al., 2004; unpublished data), and they also harbor appreciable genetic differences (McBride, et al., 2009). Strains expressing green fluorescent protein were generated by introduction of plasmid pMV158GFP as reported earlier (Nieto & Espinosa, 2003). Enterococcal strains were cultivated in Trypticase soy broth (TSB) plus 0.75% glucose. Antibiotics used for selection were obtained from Sigma Chemical (St. Louis, MO, USA) and included kanamycin (25 μg mL−1), rifampicin (20 μg mL−1), fusidic acid (10 μg mL−1), and tetracycline (10 μg mL−1).

Cell culture and media

RAW264.7 macrophages (ATCC, Manassas, VA, USA) were cultivated initially in Dulbecco modified Eagle medium (DMEM; ATCC 30-2002) plus 10% fetal bovine serum (FBS; ATCC 30-2020) to confluence in T-25 flasks. The cells were reseeded into Techne stirrer flasks (Bibby Scientific, Burlington, NJ, USA) at a concentration of 75,000 cells mL−1 and grown for up to 48–72 hours to generate large amount of viable cells in suspension. Harvested cells were > 95% viable as tested by trypan blue dye exclusion and counted by TC10 Automated Cell Counter (Bio-Rad, Hercules, CA, USA). JAWS II cells are an immortalized immature myeloid dendritic cell line derived from the bone marrow of p53−/− C57BL6 mice. JAWS II cells were grown in complete Alpha MEM medium (Cellgro, Manassas, VA, USA) containing 20% FBS, 5 ng mL−1 GM-CSF (Peprotech, Rocky Hill, NJ, USA), gentamicin (50 μg mL−1), penicillin (100 U mL−1) and streptomycin (100 μg mL−1) (Awasthi & Cox, 2003; Awasthi, et al., 2005).

Establishing cells in biofilm mode

We have previous demonstrated the ability of E. faecalis to form a biofilm on polystyrene and polyvinyl chloride surfaces in vitro and adopted the same method with modifications to generate biofilms within the wells of 6- and 24- well polystyrene plates (Tendolkar, et al., 2004; Tendolkar, et al., 2005). Briefly, E. faecalis strains were grown overnight in TSB plus 0.75% glucose and supplemented with the appropriate antibiotics. Cell suspensions were centrifuged at 6,000 × g for 10 minutes, and the cell pellets were resuspended in fresh medium. The optical densities of the bacterial suspensions were measured by using a Genova 6320D spectrophotometer (Jenway, Burlington, NJ, USA), normalized to OD600 of 1.0, diluted 1:40 in fresh medium and 0.5 ml and 5 ml were dispensed into the wells of 24- and 6-well polystyrene plates respectively. After incubation at 37 °C for 24 hours, the medium was aspirated, and the wells were washed three times with sterile phosphate-buffered saline (PBS). The bacterial cells adhering to the wells represented intact biofilm mode cells. Adherent cells were dislodged gently but firmly by scraping with a cell scraper, resuspended in appropriate buffer and vortexed vigorously to disrupt any clumps as previously described to produce the dislodged biofilm mode cells (Ciornei, et al., 2010). Microscopic observation was also performed to confirm disruption of aggregates. Bacteria were grown in liquid culture to produce the planktonic mode cells.

Phagocytosis assay

To determine whether macrophages and dendritic cells were capable of phagocytizing intact biofilm and disrupted biofilm cells, E. faecalis biofilm and planktonic cells were incubated with macrophages or dendritic cells as previously described (Drevets & Campbell, 1991; Drevets, et al., 1994; Thurlow, et al., 2009). Briefly, macrophages and dendritic cells were exposed to GFP-expressing planktonic, intact biofilm and dislodged biofilm mode bacteria at an MOI of 10 and incubated at 37 °C for 45 minutes to allow bacterial uptake. Trypsin was then added at a final concentration of 0.25% for 10 minutes at 37 °C to remove any residual bacteria at the macrophage or dendritic cell surface. Cells were then washed thrice with PBS to remove remaining surface bacteria and then subjected to flow cytometry using a Accuri C6 flow cytometer (BD Accuri, Ann Arbor, MI, USA). Aliquots of cells were also spotted on glass slides and visualized using a Leica DM-4000B fluorescence microscope (Thurlow, et al., 2011).

Bacterial viability within host immune cells

Survival of E. faecalis within macrophages was assessed as described earlier (Coburn, et al., 2008). Briefly RAW264.7 macrophages were seeded onto 6-well polystyrene plates at approximately 106 cells well−1 and incubated at 37 °C under 5% CO2 for 24 hours prior to infection. E. faecalis E99 and FA2-2(pESPF) were grown in TSB containing 0.75% glucose supplemented with the appropriate antibiotics for 16 h, cells pelleted by centrifugation and resuspended in PBS. E. faecalis were also grown in a 6-well plate as described above to obtain biofilm cells. Triplicate wells of RAW264.7 cells were infected at an MOI of 10 for each bacterial strain in planktonic and dislodged biofilm mode for 45 minutes at 37 °C under 5% CO2. The cells were then washed thrice with PBS, and further incubated with DMEM plus 10% FBS containing vancomycin (16 μg mL−1) and gentamicin (150 μg mL−1) to kill all extracellular bacteria. At 3, 6, and 24 hours the macrophages were washed twice with PBS and harvested in 1 mL of PBS. The viability and cell count were assessed by trypan blue staining using a TC10 Automated Cell Counter. Macrophages were then lysed by adding one-tenth of the volume of a saponin cell lysis solution (saponin [40 mg mL−1], polypropylene glycol [P-2000; 8 mL L−1 ], sodium polyanetholsulfonate [9.6 mg mL−1]) to release intracellular bacteria. Bacteria were quantified by serial dilution and plating. The number of viable bacteria at each time point was expressed as CFU per 105 macrophages. Experiments were performed three times, and the means and standard errors were determined for each time point. The statistical significance of the results was determined by performing pairwise comparisons at each time point.

For experiments with dendritic cells, JAWS II dendritic cells were transferred to 4.5 ml tubes at a concentration of 5×105 cells mL−1 in complete alpha-MEM medium and infected at a MOI of 10 (Niedergang, et al., 2000). After 45 minutes of incubation at 37 °C in 5% CO2, the cells were washed twice with sterile PBS and once with complete alpha-MEM medium supplemented with vancomycin (16 μg mL−1) and gentamicin (150 μg mL−1), and then incubated in the latter medium for the indicated times. At each time point, cells were processed as mentioned above for macrophages.

In the case of intact biofilms, the biofilms were allowed to set for 24 hours at 37°C. The wells were gently washed three times with 1 ml of PBS, to remove loosely adherent and planktonic cells. Adherent cells were dislodged gently but firmly by scraping with a cell scraper, vortexed vigorously to disperse any clumps and appropriate dilutions plated on selective agar to estimate the number of viable bacteria. These initial experiments allowed us to estimate the average number of adherent bacteria (biofilm cells) per well. Based on these numbers, dilutions of immune cells were made in respective growth medium so that 2 ml of medium contains a tenth of the number of biofilm bacteria in the 6-well plates. When overlayed over the bacterial cells, this represented an MOI of 10. The bacteria-immune cell co-culture was incubated for 45 minutes at 37 °C under 5% CO2. Trypsin was then added at a final concentration of 0.25% for 10 minutes at 37 °C to remove any residual bacteria at the macrophage or dendritic cell surface. Dislodged cells were transferred to 4.5 ml tubes, then washed thrice with PBS to remove remaining surface adherent bacteria, supplemented with media containing vancomycin (16 μg mL−1) and gentamicin (150 μg mL−1) and incubated for the indicated times. At each time point, cells were processed as mentioned above for macrophages.

Expression of cytokines and chemokine by host cells

Cytokine and chemokine expression by infected cells were essentially assayed as described previously (Awasthi, et al., 2005). Briefly, 200 μl aliquots of supernatants were removed at 0, 3, 6 and 24 hour time points following infection. The aliquots were centrifuged to pellet cells and other debris, the supernatant filtered through a 0.22 μm filter and analyzed for IL-6, MCP-1 and TNF-α using a commercially available BD CBA Flex Set (BD Biosciences, San Jose, CA, USA) in accordance with the manufacturer’s recommendations. Data was collected using BD FACS Calibur flow cytometer and analyzed using FCAP Array Software.

Flow cytometric analysis of surface activation markers

RAW264.7 and JAWS II cells were infected for 60 minutes with E99 (dislodged biofilm and planktonic) and FA2-2(pESPF) (dislodged biofilm and planktonic) at an MOI of 10 or left uninfected as controls. Another set of cells was challenged with E. coli LPS (1 μg mL−1, 100 ng mL−1 and 1 ng mL−1 respectively) to serve as positive controls. After eliminating all non-internalized bacteria, cells were incubated for 3 and 24 hours, then harvested, washed and resuspended to a concentration of 1×106 cells in 100 μL of FACS staining buffer (Awasthi, et al., 2005). The cells were incubated with 1 μg rat anti-mouse CD16/CD32 antibody (Fc block) for 15 minutes prior to staining with the following flourochrome conjugated rat anti-mouse antibodies: FITC-CD86, PE-CD80 and APC-CD40 for RAW264.7 macrophages and FITC-CD14, PE-CD11c, APC-MHC II, FITC-CD86, PE-CD80 and APC-CD40 for the JAWS II cells. All antibodies were purchased from eBioscience, San Diego, CA, USA. The appropriate isotype matched control antibodies were used to determine the levels of background staining. All antibodies were pre-titrated in preliminary experiments and used at saturating concentrations. Cell staining was done for 30 minutes on ice and washed cells were analyzed by Accuri C6 flow cytometer. Data was collected on 35,000 cells.

Statistics

Statistical significance was analyzed using Student’s t test and Anova on Ranks test wherever applicable using SigmaPlot 11 (Systat Software, Inc., San Jose, CA, USA). Differences between experimental groups were considered significant when P values were < = 0.05.

Results

Biofilm mode E. faecalis are effectively phagocytized by RAW264.7 macrophages and JAWS II dendritic cells in vitro

To examine the phagocytosis of bacteria by the immune cells, flow cytometry was employed using GFP-labeled bacterial strains. In these experiments, RAW264.7 macrophages and JAWS II dendritic cells were added to intact biofilms, dislodged biofilm cells and planktonic cells for 45 minutes at 37 °C, then harvested and subjected to FACS analysis. Our results revealed that both intact biofilm and dislodged biofilm cells were quite capable of being phagocytized by RAW264.7 macrophages. (Fig. 1a and b). Figure 1a shows that the population of GFP+ve macrophages is significantly higher (P = 0.010) when infected with E99 dislodged biofilm cells compared to infection with planktonic E99. This observation was similar but more pronounced in the FA2-2 background, where a significantly higher (P < 0.001) population of macrophages were GFP+ve when infected with both intact biofilm and dislodged biofilm cells compared to macrophages infected with planktonic bacteria (Fig. 1b). Figure 1b also shows a significantly higher (P < 0.001) percentage of GFP+ve macrophages infected with dislodged biofilm compared to infection with intact biofilm. Figure 1c and d show the uptake of the bacteria by JAWS II dendritic cells. While there was no significant difference in the number of GFP+ve macrophages infected with either E99 intact biofilm or dislodged biofilm or planktonic cells (Fig. 1c), in case of infection with intact and dislodged biofilm cells of FA2-2, a significantly higher (P = 0.002) percentage of JAWS II dendritic cells were GFP+ve. (Fig. 1d). Further, fluorescence microscopy (Fig. 2a, b, d and e) showed more bacteria in intact and dislodged biofilm mode to be associated with macrophages than planktonic E. faecalis (Fig. 2c and f). Similar observations were made with dendritic cells infected with FA2-2(pESPF), where both intact and dislodged biofilm mode bacteria (Fig. 2j and k) were generally associated more with the immune cells compared to the planktonic counterpart (Fig. 2l). However, there was no appreciable difference in the levels of uptake of E99 by dendritic cells when infected with either the biofilm or planktonic mode (Fig. 2g. h and i).

Fig. 1.

Fig. 1

Open in a new tab

Uptake of intact biofilm, dislodged biofilm and planktonic mode bacteria by RAW264.7 macrophages and JAWS II dendritic cells. RAW264.7 macrophages were infected for 45 minutes with a) GFP-labeled E. faecalis E99 inatct biofilm, dislodged biofilm and planktonic cells and b) GFP-labeled E. faecalis FA2-2(pESPF) intact biofilm, dislodged biofilm and planktonic cells. JAWS II dendritic cells were infected for 45 minutes with c) GFP-labeled E. faecalis E99 intact biofilm, dislodged biofilm and planktonic mode and d) GFP-labeled E. faecalis FA2-2(pESPF) intact biofilm, dislodged biofilm and planktonic cells. Association of GFP-labeled bacteria was determined by FACS analysis. The bar graphs represent pooled data of triplicates from independently performed duplicate experiments. Mean and standard error of means are reported. Anova on Ranks test was performed to determine the statistical significance. Panels a, b and d show significances (*) of P = 0.010, P < 0.001 and P = 0.002, respectively.

Fig. 2.

Fig. 2

Open in a new tab

Fluorescence microscopy of RAW264.7 macrophages and JAWS II dendritic cells infected with intact biofilm, dislodged biofilm and planktonic mode bacteria. RAW264.7 macrophages at 45 minutes after infection with GFP-expressing a) E99 intact biofilm, b) E99 dislodged biofilm, c) E99 planktonic, d)FA2-2(pESPF) intact biofilm, e) FA2-2(pESPF) dislodged biofilm and f) FA2-2(pESPF)planktonic mode cells. JAWS II dendritic cells at 45 minutes after infection with GFP-labeled g) E99 intact biofilm, h) E99 dislodged biofilm, i) E99 planktonic, j)FA2-2(pESPF) intact biofilm, k) FA2-2(pESPF) dislodged biofilm and l) FA2-2(pESPF)planktonic mode cells. Macrophages and dendritic cells were stained with CellTracker Red fluorescent probe which was retained in the cytoplasm and the nuclei were stained with DAPI. A 100x magnification was used to visualize the images. The excitation and emission wavelengths of GFP, DAPI and CellTracker Red fluorescent probe are 495 and 519nm, 359 and 461nm and 577 and 602 nm respectively. The images are representative of three independent experiments.

Intracellular survival of bacteria within macrophages and dendritic cells

We evaluated if there was a difference in the intracellular survival of intact biofilm, dislodged biofilm and planktonic bacteria once they are phagocytized by host macrophages and dendritic cells. RAW264.7 macrophages were infected with intact biofilm, dislodged biofilm and planktonic E99 (Fig. 3a) and FA2-2(pESPF) (Fig. 3b) cells at an MOI of 10 and bacterial viability measured over a 24 hours time period. A statistically significant (P < 0.001) difference was observed between the survival of E99 intact biofilm and planktonic bacteria as well as between dislodged biofilm mode and planktonic bacteria 24 hours post infection, with E99 biofilm cells exhibiting higher persistence. There was around a half-log reduction in the number of bacterial CFUs recovered from the planktonic mode when compared to the intact biofilm mode and dislodged biofilm mode bacteria 24 hours post infection in the FA2-2 background. No difference in survival between intact biofilm, dislodged biofilm and planktonic mode bacteria was evident within JAWS II dendritic cells (Fig. 3c and d). Fluorescence microscopy (Figs. 4 and 5) corroborated these observations.

Fig. 3.

Fig. 3

Open in a new tab

Intracellular survival of intact biofilm, dislodged biofilm mode and planktonic E. faecalis E99 and FA2-2(pESPF) in RAW264.7 macrophages and JAWS II dendritic cells. RAW264.7 macrophages were infected with a) E99 intact biofilm, dislodged biofilm and planktonic bacteria and b) FA2-2(pESPF) intact biofilm, dislodged biofilmand planktonic cells. JAWS II dendritic cells were infected with c) E99 intact biofilm, dislodged biofilm and planktonic mode and d) FA2-2(pESPF) intact biofilm, dslodged biofilm and planktonic cells. The number of viable bacteria recovered at 3, 6 and 24 h post infection is expressed as CFU per 105 macrophages or DCs. Experiments were performed three times, and the means and standard errors are reported for each time point. Anova on ranks test was used to perform statistical analyses at each time point. Panel 3a shows a significant (P < 0.001) difference between cells infected with intact biofilm and planktonic bacteria, as well as between dislodged biofilm and planktonic bacteria at 24 h. Black circles indicate intact biofilm bacteria, grey circles indicate dislodged biofilm bacteria and open circles indicate planktonic bacteria.

Fig. 4.

Fig. 4

Open in a new tab

Survival of E99 biofilm and planktonic mode bacteria within RAW264.7 macrophages by fluorescence microscopy. Panels (a–c) show RAW264.7 macrophages infected with E99 intact biofilm mode bacteria at 3, 6 and 24 hours post-infection, respectively. Panels (d–f) show macrophages infected with E99 dislodged bacteria at 3, 6 and 24 hours post infection, respectively. Panels (g–i) show macrophages infected with E99 planktonic bacteria at 3, 6 and 24 hours post infection, respectively. Likewise, images were obtained with the FA2-2(pESPF) strain which corroborated with the results shown in Figure 3b. A 100x magnification was used to visualize the images. The images are representative of three independent experiments.

Fig. 5.

Fig. 5

Open in a new tab

Survival of E99 biofilm and planktonic mode bacteria within JAWS II dendritic cells by fluorescence microscopy. Panels (a–c) show JAWS II dendritic cells infected with E99 intact biofilm mode bacteria at 3, 6 and 24 hours post-infection respectively. Panels (d–f) show JAWS II dendritic cells infected with E99 dislodged bacteria at 3, 6 and 24 hours post infection, respectively. Panels (g–i) show JAWS II dendritic cells infected with E99 planktonic bacteria at 3, 6 and 24 hours post infection, respectively. A 100x magnification was used to visualize the images. The images are representative of three independent experiments. Similar images were obtained with the FA2-2(pESPF) strain (data not shown).

The phagocytosis and intracellular survival data above confirmed that the dislodged biofilm mode cells were a good representation of biofilm mode cells and the immune cell response to these two cell types were nearly identical. Therefore, in subsequent experiments we only compared dislodged biofilm cells and planktonic cells.

Differential regulation of host cytokine and chemokine expression upon infection

A cytometric bead array protocol was employed to investigate the cytokine response once the bacteria is phagocytized and assess if there was a difference in the cytokine expression profile between dislodged biofilm and planktonic mode infection. We assayed proinflammatory cytokines, IL-6 and TNF-α and chemokine MCP-1 because our prior experience with enterococcal infections demonstrated a significant induction of these three factors. While macrophages infected with E99 and FA2-2(pESPF) dislodged biofilm cells appeared to induce lower expression of the proinflammatory cytokines in general, the effect was significantly more pronounced in the FA2-2 background (Fig. 6). Figure 6d shows a statistically significant (P = 0.009) reduction in the induction of IL-6 by macrophages infected with dislodged biofilm mode bacteria when compared to planktonic bacteria. In case of MCP-1 and TNF-α, although the differences appeared to be appreciable, they were not found to be statistically significant. Similar assays performed on infected JAWS II cells did not reveal any appreciable differences in cytokine induction between dislodged biofilm and planktonic infection modes (data not shown).

Fig. 6.

Fig. 6

Open in a new tab

Differential induction of cytokines in RAW264.7 macrophages by E99 and FA2-2(pESPF). RAW264.7 macrophages were either left uninfected or infected for 60 minutes with dislodged biofilm and planktonic cells of E99 (Panels a–c) and FA2-2(pESPF) (Panels d–f) in antibiotic-free medium at an MOI of 10. At the end of the infection period, non-internalized bacteria were killed by the addition of gentamicin and vancomycin and macrophages were further incubated for 3, 6 and 24 hours. Cell-free culture supernatants were collected at specified time points and assayed for cytokines using a BD CBA Flex Set. Experiments were performed three times, and the means and standard errors are reported for each time point. Student’s t test was used to determine statistical significance. Panel d shows significance (*) of P = 0.009.

Expression of surface activation markers by RAW264.7 macrophages upon infection

We employed flow cytometric analysis to evaluate the ability of E99 and FA2-2(pESPF) in their dislodged biofilm and planktonic modes to influence the expression of surface activation markers CD40, CD80 (B7-1) and CD86 (B7-2) on macrophages. Stimulation of the macrophages for 3 hours by E. faecalis or LPS (data not shown) was not sufficient to obtain detectable surface molecule expression and required at least 24 hours of stimulation at 37 °C. As shown in Fig. 7, macrophages infected with dislodged biofilm mode E99 and FA2-2(pESPF) showed more pronounced expression of CD40 and CD80 than those infected with the planktonic cells as depicted by the increase in their mean fluorescence intensity (MFI). As expected, LPS stimulation increased the expression of both these markers, indicating that macrophages were not pre-activated and were primed for activation upon stimulation (data not shown). We also investigated the expression of another costimulatory molecule, CD86, which in contrast to the above proteins were found to be marginally induced by dislodged biofilm bacteria (Fig. 7).

Fig. 7.

Fig. 7

Open in a new tab

Differential expression of CD40, CD80 and CD86 on RAW264.7 macrophages after 24 hours of infection by E. faecalis. Flow cytometric histogram charts of macrophages 24 hours post infection with E99 and FA2-2(pESPF) dislodged biofilm and planktonic bacteria are depicted. The cells were stained with fluorochrome-conjugated antibodies or isotype control antibody (black area). The black lines indicate uninfected cells (U) and grey lines indicate bacteria-infected cells [E99 or FA2-2(pESPF)]. The percent number of cells positive for respective antibodies is shown within the chart itself. Values shown within parenthesis indicate MFI values and are represented as 1000x values. The percent number and MFI values of isotype control (I) stained cells in M1 region are also shown. The results presented here are representative of at least three independent experiments.

Expression of maturation and surface activation markers by JAWS II dendritic cells upon infection

With regard to activation and maturation markers on dendritic cells, there was no difference in the levels of maturation (CD11c and MHC II) markers of JAWS II cells infected with dislodged biofilm and planktonic bacteria (data not shown). Similarly, JAWS II cells did not show differences in activation markers 3 hours post infection with dislodged biofilm and planktonic bacteria when compared to infected macrophages (data not shown). However, as shown in Fig. 8., when JAWS II cells were infected with dislodged biofilm mode cells of E99 and FA2-2(pESPF) for 24 hours, there was a marginal increase in the expression of activation markers (CD80 and CD86), which could be attributed to the increase in the mean percentage of cells in the population expressing the respective markers, even though the MFI values were not appreciably increased.

Fig. 8.

Fig. 8

Open in a new tab

Flow cytometric analysis of T cell co-stimulatory molecules (CD40, CD80. CD86) on dendritic cells 24 hours post infection with E. faecalis. Flow cytometric histogram charts of dendritic cells 24 hours post infection with E99 and FA2-2(pESPF) dislodged biofilm and planktonic bacteria are shown. The cells were stained with fluorochrome-conjugated antibodies or isotype control antibody (black area). The black lines indicate uninfected cells (U) and grey lines indicate bacteria-infected cells [E99 or FA2-2(pESPF)]. The percent number of cells positive for respective antibodies is shown within the chart itself. Values shown within parenthesis indicate MFI values (represented as x1000). The percent number and MFI values of isotype control (I) stained cells in M1 region are also shown. The results presented here are representative of at least three independent experiments.

DISCUSSION

Our studies comparing the uptake of E. faecalis biofilm cells to their planktonic counterparts revealed that biofilm cells may be taken up efficiently by immune cells such as macrophages and dendritic cells and in fact at significantly higher numbers depending on the strain background. This is in contrast to the popular notion that biofilm cells resist phagocytosis by immune cells, but is in agreement with other reports that have shown leukocyte mediated uptake and killing of biofilm cells (Leid, et al., 2002; Jesaitis, et al., 2003; Meyle, et al., 2010). While we used standard methodology to examine phagocytosis, including a trypsin treatment step to remove adherent bacteria on the exterior of the phagocyte, we are mindful of the caveat that biofilm cells encased in an exopolysaccharide matrix may be somewhat resistant to such protease treatment. The observed number of phagocytosed cells may thus represent in large part those that are truly internalized but include a few adherent bacteria. To get a true sense of the fate of the internalized bacteria we therefore analyzed the survival of intracellular bacteria over time, in which experiments the extracellular bacteria are killed by antibiotics in the incubation medium.

A previous study with E. faecalis strains grown under conditions known to induce synthesis of exopolysaccharide and form biofilms, found that biofilm positive cells survived for more than 24 hours in infected rat peritoneal macrophages compared to isogenic strains that were exopolysaccharide-negative (Baldassarri, et al., 2005). Our results in this study reveal that while there is no appreciable difference in the levels of intracellular bacteria at t0 between biofilm cells and their planktonic counterparts in both strain backgrounds studied, the overall survival of biofilm mode cells is significantly higher in macrophages for the E99 strain. This difference in survival between the two strains could at least partially be attributed to the biofilm matrix itself which is influenced by exopolysaccharide and other cell surface components. As noted earlier, strains E99 and FA2-2 differ significantly in their genetic makeup (McBride, et al., 2009) and studies in our laboratory (unpublished) have shown that the architecture of their biofilms is quite distinct as well. While we were expecting to find poor survival of E. faecalis cells within dendritic cells as they are potent antigen-presenting cells and rapidly degrade ingested bacteria, it was surprising to note that the strains in this study survived quite well at least up to 24 hours. Similar findings were reported by Baldassarri et al (2005) where biofilm E. faecalis were cultivable for up to around 48 hours from infected mouse fetal skin derived dendritic cells and bone marrow derived dendritic cells.

Antigen-presenting cells like macrophages and dendritic cells are the crucial link between the innate and acquired immunity as they are adept in phagocytosis, secretion of cytokines and antigen presentation. Activation of macrophages and dendritic cells initiate T-cell mediated immune response to an invading pathogen. CD40, CD80 and CD86 are well studied T cell co-stimulatory molecules expressed on activated antigen-presenting cells. Our data with E. faecalis infected macrophages showed that upon activation with the biofilm mode bacteria for 24 hours, the macrophages showed increased expression of these molecules. Immature dendritic cells phagocytize pathogens and transform from antigen-capturing immature dendritic cells to antigen-presenting mature dendritic cells. This process is accompanied by up regulation of MHC I and II molecules and enhanced expression of cell surface costimulatory molecules CD40, CD80 and CD86 (Hu, et al., 2006). In previous studies, infection of dendritic cells with Campylobacter jejuni (Hu, et al., 2006), Salmonella enterica serovar typhimurium (Norimatsu, et al., 2004) and Helicobacter pylori (Kranzer, et al., 2004) have been shown to up regulate dendritic cell surface expression of MHC II, CD40, CD80 and CD86 molecules. We observed a consistent increase in CD80 and CD86 expression in biofilm mode E. faecalis infected dendritic cells and macrophages when MFI or percent number of cells were compared. CD80 and CD86 molecules are ligands for T cell receptor and skew the T cell responses to Th1 and Th2 type of responses, respectively (Slavik, et al., 1999). Antibody-blockade and stimulation experiments with recombinant proteins have shown that CD80 promotes Th1 response and CD86 promotes Th2 response (Kuchroo, et al., 1995; Gajewski, 1996; Freeman, et al., 1995). A comprehensive study is warranted in future to investigate the T cell skewing by dendritic cells and macrophages against E. faecalis antigens (derived from biofilm producing and planktonic E. faecalis strains).

Our analysis of cytokine and chemokine expression by infected macrophages suggest that at least for the two strain backgrounds studied here, biofilm cells seemed to invoke less proinflammatory cytokine (TNF-α, IL-6) and chemokine (MCP-1) expression relative to planktonic cells. These results are in agreement with an earlier study which evaluated the immunogenic potential of E. faecalis biofilms under simulated biofilm conditions (Mathew, et al., 2010). While earlier reports have shown robust IL-6 and TNF-α release by peripheral blood cells and peritoneal macrophages upon stimulation in vitro by E. faecalis and E. faecium (Papasian, et al., 2002; Leendertse, et al., 2008), only a weak cytokine response during peritonitis in mice was observed. While these findings cannot be fully explained at this time, it is interesting to note that biofilm mode cells elicited less of a proinflammatory response from infected cells. Since it is known that enterococcal lipoteichoic acids can stimulate the production of IL-6 and TNF-α by cultured monocytes (Bhakdi, et al., 1991), it is possible that the matrix in biofilm mode cells masks recognition of LTA by the phagocyte. In contrast, there was no significant difference in the cytokine and chemokine expression profile of the dendritic cells infected with the bacteria.

In summary, our study highlights the fact that enterococcal biofilm cells adhering to a matrix or such as those that might escape from infection foci, can be efficiently phagocytized by host macrophages and dendritic cells similar to their planktonic counterparts. A strain background-dependent survival edge may be present for biofilm cells in macrophages and it appears that overall E. faecalis biofilm cells appear to evoke a more tempered proinflammatory response in vitro. Undoubtedly the in vivo scenario is more complex with the involvement of a multitude of host cells and factors but these characteristics likely provide biofilm cells the ability to circumvent host defense strategies and allow persistence in the host.

Acknowledgments

This work was supported by U.S. Public Health Services Grant DE020928 (N.S.) from the National Institutes of Health. The technical assistance of Jim Henthorn with the microscopy and cytokine analyses is greatly appreciated.

References

  1. Arduino RC, Murray BE, Rakita RM. Roles of antibodies and complement in phagocytic killing of enterococci. Infect Immun. 1994;62:987–993. doi: 10.1128/iai.62.3.987-993.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Awasthi S, Cox RA. Transfection of murine dendritic cell line (JAWS II) by a nonviral transfection reagent. Biotechniques. 2003;35:600–602. 604. doi: 10.2144/03353dd03. [DOI] [PubMed] [Google Scholar]
  3. Awasthi S, Awasthi V, Magee DM, Coalson JJ. Efficacy of antigen 2/proline-rich antigen cDNA-transfected dendritic cells in immunization of mice against Coccidioides posadasii. J Immunol. 2005;175:3900–3906. doi: 10.4049/jimmunol.175.6.3900. [DOI] [PubMed] [Google Scholar]
  4. Baldassarri L, Bertuccini L, Creti R, et al. Glycosaminoglycans mediate invasion and survival of Enterococcus faecalis into macrophages. J Infect Dis. 2005;191:1253–1262. doi: 10.1086/428778. [DOI] [PubMed] [Google Scholar]
  5. Bhakdi S, Klonisch T, Nuber P, Fischer W. Stimulation of monokine production by lipoteichoic acids. Infect Immun. 1991;59:4614–4620. doi: 10.1128/iai.59.12.4614-4620.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Broche F, Tellado JM. Defense mechanisms of the peritoneal cavity. Curr Opin Crit Care. 2001;7:105–116. doi: 10.1097/00075198-200104000-00009. [DOI] [PubMed] [Google Scholar]
  7. Ciornei CD, Novikov A, Beloin C, et al. Biofilm-forming Pseudomonas aeruginosa bacteria undergo lipopolysaccharide structural modifications and induce enhanced inflammatory cytokine response in human monocytes. Innate Immun. 2010;16:288–301. doi: 10.1177/1753425909341807. [DOI] [PubMed] [Google Scholar]
  8. Coburn PS, Baghdayan AS, Dolan GT, Shankar N. An AraC-type transcriptional regulator encoded on the Enterococcus faecalis pathogenicity island contributes to pathogenesis and intracellular macrophage survival. Infect Immun. 2008;76:5668–5676. doi: 10.1128/IAI.00930-08. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Davies D. Understanding biofilm resistance to antibacterial agents. Nat Rev Drug Discov. 2003;2:114–122. doi: 10.1038/nrd1008. [DOI] [PubMed] [Google Scholar]
  10. Donlan RM, Costerton JW. Biofilms: survival mechanisms of clinically relevant microorganisms. Clin Microbiol Rev. 2002;15:167–193. doi: 10.1128/CMR.15.2.167-193.2002. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Drevets DA, Campbell PA. Macrophage phagocytosis: use of fluorescence microscopy to distinguish between extracellular and intracellular bacteria. J Immunol Methods. 1991;142:31–38. doi: 10.1016/0022-1759(91)90289-r. [DOI] [PubMed] [Google Scholar]
  12. Drevets DA, Canono BP, Leenen PJ, Campbell PA. Gentamicin kills intracellular Listeria monocytogenes. Infect Immun. 1994;62:2222–2228. doi: 10.1128/iai.62.6.2222-2228.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Dunn DL, Barke RA, Knight NB, Humphrey EW, Simmons RL. Role of resident macrophages, peripheral neutrophils, and translymphatic absorption in bacterial clearance from the peritoneal cavity. Infect Immun. 1985;49:257–264. doi: 10.1128/iai.49.2.257-264.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Freeman GJ, Boussiotis VA, Anumanthan A, et al. B7-1 and B7-2 do not deliver identical costimulatory signals, since B7-2 but not B7-1 preferentially costimulates the initial production of IL-4. Immunity. 1995;2:523–532. doi: 10.1016/1074-7613(95)90032-2. [DOI] [PubMed] [Google Scholar]
  15. Gajewski TF. B7-1 but not B7-2 efficiently costimulates CD8+ T lymphocytes in the P815 tumor system in vitro. J Immunol. 1996;156:465–472. [PubMed] [Google Scholar]
  16. Gentry-Weeks CR, Karkhoff-Schweizer R, Pikis A, Estay M, Keith JM. Survival of Enterococcus faecalis in mouse peritoneal macrophages. Infect Immun. 1999;67:2160–2165. doi: 10.1128/iai.67.5.2160-2165.1999. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Giard JC, Riboulet E, Verneuil N, Sanguinetti M, Auffray Y, Hartke A. Characterization of Ers, a PrfA-like regulator of Enterococcus faecalis. FEMS Immunol Med Microbiol. 2006;46:410–418. doi: 10.1111/j.1574-695X.2005.00049.x. [DOI] [PubMed] [Google Scholar]
  18. Guenther F, Stroh P, Wagner C, Obst U, Hansch GM. Phagocytosis of staphylococci biofilms by polymorphonuclear neutrophils: S. aureus and S. epidermidis differ with regard to their susceptibility towards the host defense. Int J Artif Organs. 2009;32:565–573. doi: 10.1177/039139880903200905. [DOI] [PubMed] [Google Scholar]
  19. Hall-Stoodley L, Stoodley P. Evolving concepts in biofilm infections. Cell Microbiol. 2009;11:1034–1043. doi: 10.1111/j.1462-5822.2009.01323.x. [DOI] [PubMed] [Google Scholar]
  20. Hancock LE, Perego M. The Enterococcus faecalis fsr two-component system controls biofilm development through production of gelatinase. J Bacteriol. 2004;186:5629–5639. doi: 10.1128/JB.186.17.5629-5639.2004. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Hu L, Bray MD, Osorio M, Kopecko DJ. Campylobacter jejuni induces maturation and cytokine production in human dendritic cells. Infect Immun. 2006;74:2697–2705. doi: 10.1128/IAI.74.5.2697-2705.2006. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Hufnagel M, Koch S, Creti R, Baldassarri L, Huebner J. A putative sugar-binding transcriptional regulator in a novel gene locus in Enterococcus faecalis contributes to production of biofilm and prolonged bacteremia in mice. J Infect Dis. 2004;189:420–430. doi: 10.1086/381150. [DOI] [PubMed] [Google Scholar]
  23. Jesaitis AJ, Franklin MJ, Berglund D, et al. Compromised host defense on Pseudomonas aeruginosa biofilms: characterization of neutrophil and biofilm interactions. J Immunol. 2003;171:4329–4339. doi: 10.4049/jimmunol.171.8.4329. [DOI] [PubMed] [Google Scholar]
  24. Knudsen E, Iversen PO, Van Rooijen N, Benestad HB. Macrophage-dependent regulation of neutrophil mobilization and chemotaxis during development of sterile peritonitis in the rat. Eur J Haematol. 2002;69:284–296. doi: 10.1034/j.1600-0609.2002.02657.x. [DOI] [PubMed] [Google Scholar]
  25. Kranzer K, Eckhardt A, Aigner M, et al. Induction of maturation and cytokine release of human dendritic cells by Helicobacter pylori. Infect Immun. 2004;72:4416–4423. doi: 10.1128/IAI.72.8.4416-4423.2004. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Kristich CJ, Li YH, Cvitkovitch DG, Dunny GM. Esp-independent biofilm formation by Enterococcus faecalis. J Bacteriol. 2004;186:154–163. doi: 10.1128/JB.186.1.154-163.2004. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Kuchroo VK, Das MP, Brown JA, et al. B7-1 and B7-2 costimulatory molecules activate differentially the Th1/Th2 developmental pathways: application to autoimmune disease therapy. Cell. 1995;80:707–718. doi: 10.1016/0092-8674(95)90349-6. [DOI] [PubMed] [Google Scholar]
  28. Leendertse M, Willems RJ, Giebelen IA, Roelofs JJ, van Rooijen N, Bonten MJ, van der Poll T. Peritoneal macrophages are important for the early containment of Enterococcus faecium peritonitis in mice. Innate Immun. 2009;15:3–12. doi: 10.1177/1753425908100238. [DOI] [PubMed] [Google Scholar]
  29. Leendertse M, Willems RJ, Giebelen IA, et al. TLR2-dependent MyD88 signaling contributes to early host defense in murine Enterococcus faecium peritonitis. J Immunol. 2008;180:4865–4874. doi: 10.4049/jimmunol.180.7.4865. [DOI] [PubMed] [Google Scholar]
  30. Leid JG, Shirtliff ME, Costerton JW, Stoodley P. Human leukocytes adhere to, penetrate, and respond to Staphylococcus aureus biofilms. Infect Immun. 2002;70:6339–6345. doi: 10.1128/IAI.70.11.6339-6345.2002. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Mah TF, O’Toole GA. Mechanisms of biofilm resistance to antimicrobial agents. Trends Microbiol. 2001;9:34–39. doi: 10.1016/s0966-842x(00)01913-2. [DOI] [PubMed] [Google Scholar]
  32. Mathew S, Yaw-Chyn L, Kishen A. Immunogenic potential of Enterococcus faecalis biofilm under simulated growth conditions. J Endod. 2010;36:832–836. doi: 10.1016/j.joen.2010.02.022. [DOI] [PubMed] [Google Scholar]
  33. McBride SM, Coburn PS, Baghdayan AS, Willems RJ, Grande MJ, Shankar N, Gilmore MS. Genetic variation and evolution of the pathogenicity island of Enterococcus faecalis. J Bacteriol. 2009;191:3392–3402. doi: 10.1128/JB.00031-09. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Meyle E, Stroh P, Gunther F, Hoppy-Tichy T, Wagner C, Hansch GM. Destruction of bacterial biofilms by polymorphonuclear neutrophils: relative contribution of phagocytosis, DNA release, and degranulation. Int J Artif Organs. 2010;33:608–620. doi: 10.1177/039139881003300906. [DOI] [PubMed] [Google Scholar]
  35. Mohamed JA, Huang W, Nallapareddy SR, Teng F, Murray BE. Influence of origin of isolates, especially endocarditis isolates, and various genes on biofilm formation by Enterococcus faecalis. Infect Immun. 2004;72:3658–3663. doi: 10.1128/IAI.72.6.3658-3663.2004. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Nallapareddy SR, Singh KV, Sillanpaa J, Garsin DA, Hook M, Erlandsen SL, Murray BE. Endocarditis and biofilm-associated pili of Enterococcus faecalis. J Clin Invest. 2006;116:2799–2807. doi: 10.1172/JCI29021. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Niedergang F, Sirard JC, Blanc CT, Kraehenbuhl JP. Entry and survival of Salmonella typhimurium in dendritic cells and presentation of recombinant antigens do not require macrophage-specific virulence factors. Proc Natl Acad Sci U S A. 2000;97:14650–14655. doi: 10.1073/pnas.97.26.14650. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Nieto C, Espinosa M. Construction of the mobilizable plasmid pMV158GFP, a derivative of pMV158 that carries the gene encoding the green fluorescent protein. Plasmid. 2003;49:281–285. doi: 10.1016/s0147-619x(03)00020-9. [DOI] [PubMed] [Google Scholar]
  39. Norimatsu M, Chance V, Dougan G, Howard CJ, Villarreal-Ramos B. Live Salmonella enterica serovar Typhimurium (S. Typhimurium) elicit dendritic cell responses that differ from those induced by killed S. Typhimurium. Vet Immunol Immunopathol. 2004;98:193–201. doi: 10.1016/j.vetimm.2003.12.008. [DOI] [PubMed] [Google Scholar]
  40. Papasian CJ, Silverstein R, Gao JJ, Bamberger DM, Morrison DC. Anomalous role of tumor necrosis factor alpha in experimental enterococcal infection. Infect Immun. 2002;70:6628–6637. doi: 10.1128/IAI.70.12.6628-6637.2002. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Qin X, Singh KV, Weinstock GM, Murray BE. Characterization of fsr, a regulator controlling expression of gelatinase and serine protease in Enterococcus faecalis OG1RF. J Bacteriol. 2001;183:3372–3382. doi: 10.1128/JB.183.11.3372-3382.2001. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Richards MJ, Edwards JR, Culver DH, Gaynes RP. Nosocomial infections in combined medical-surgical intensive care units in the United States. Infect Control Hosp Epidemiol. 2000;21:510–515. doi: 10.1086/501795. [DOI] [PubMed] [Google Scholar]
  43. Sato K, Fujita S. Dendritic cells: nature and classification. Allergol Int. 2007;56:183–191. doi: 10.2332/allergolint.R-06-139. [DOI] [PubMed] [Google Scholar]
  44. Serbina NV, Jia T, Hohl TM, Pamer EG. Monocyte-mediated defense against microbial pathogens. Annu Rev Immunol. 2008;26:421–452. doi: 10.1146/annurev.immunol.26.021607.090326. [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. Slavik JM, Hutchcroft JE, Bierer BE. CD80 and CD86 are not equivalent in their ability to induce the tyrosine phosphorylation of CD28. J Biol Chem. 1999;274:3116–3124. doi: 10.1074/jbc.274.5.3116. [DOI] [PubMed] [Google Scholar]
  46. Stewart PS. Mechanisms of antibiotic resistance in bacterial biofilms. Int J Med Microbiol. 2002;292:107–113. doi: 10.1078/1438-4221-00196. [DOI] [PubMed] [Google Scholar]
  47. Tendolkar PM, Baghdayan AS, Shankar N. The N-terminal domain of enterococcal surface protein, Esp, is sufficient for Esp-mediated biofilm enhancement in Enterococcus faecalis. J Bacteriol. 2005;187:6213–6222. doi: 10.1128/JB.187.17.6213-6222.2005. [DOI] [PMC free article] [PubMed] [Google Scholar]
  48. Tendolkar PM, Baghdayan AS, Shankar N. Putative surface proteins encoded within a novel transferable locus confer a high-biofilm phenotype to Enterococcus faecalis. J Bacteriol. 2006;188:2063–2072. doi: 10.1128/JB.188.6.2063-2072.2006. [DOI] [PMC free article] [PubMed] [Google Scholar]
  49. Tendolkar PM, Baghdayan AS, Gilmore MS, Shankar N. Enterococcal surface protein, Esp, enhances biofilm formation by Enterococcus faecalis. Infect Immun. 2004;72:6032–6039. doi: 10.1128/IAI.72.10.6032-6039.2004. [DOI] [PMC free article] [PubMed] [Google Scholar]
  50. Thomas VC, Thurlow LR, Boyle D, Hancock LE. Regulation of autolysis-dependent extracellular DNA release by Enterococcus faecalis extracellular proteases influences biofilm development. J Bacteriol. 2008;190:5690–5698. doi: 10.1128/JB.00314-08. [DOI] [PMC free article] [PubMed] [Google Scholar]
  51. Thurlow LR, Thomas VC, Fleming SD, Hancock LE. Enterococcus faecalis capsular polysaccharide serotypes C and D and their contributions to host innate immune evasion. Infect Immun. 2009;77:5551–5557. doi: 10.1128/IAI.00576-09. [DOI] [PMC free article] [PubMed] [Google Scholar]
  52. Thurlow LR, Hanke ML, Fritz T, et al. Staphylococcus aureus biofilms prevent macrophage phagocytosis and attenuate inflammation in vivo. J Immunol. 2011;186:6585–6596. doi: 10.4049/jimmunol.1002794. [DOI] [PMC free article] [PubMed] [Google Scholar]
  53. Toledo-Arana A, Valle J, Solano C, et al. The enterococcal surface protein, Esp, is involved in Enterococcus faecalis biofilm formation. Appl Environ Microbiol. 2001;67:4538–4545. doi: 10.1128/AEM.67.10.4538-4545.2001. [DOI] [PMC free article] [PubMed] [Google Scholar]
  54. Topley N, Mackenzie RK, Williams JD. Macrophages and mesothelial cells in bacterial peritonitis. Immunobiology. 1996;195:563–573. doi: 10.1016/S0171-2985(96)80022-2. [DOI] [PubMed] [Google Scholar]

RESOURCES