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Journal of Bacteriology, November 2007, p. 8233-8240, Vol. 189, No. 22
0021-9193/07/$08.00+0     doi:10.1128/JB.01205-07
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

Enterococcal Surface Protein Esp Is Important for Biofilm Formation of Enterococcus faecium E1162

Esther Heikens,^* Marc J. M. Bonten, and Rob J. L. Willems

Department of Medical Microbiology, University Medical Center Utrecht, Utrecht, The Netherlands

Received 27 July 2007/ Accepted 31 August 2007

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Enterococci have emerged as important nosocomial pathogens with resistance to multiple antibiotics. Adhesion to abiotic materials and biofilm formation on medical devices are considered important virulence properties. A single clonal lineage of Enterococcus faecium, complex 17 (CC17), appears to be a successful nosocomial pathogen, and most CC17 isolates harbor the enterococcal surface protein gene, esp. In this study, we constructed an esp insertion-deletion mutant in a clinical E. faecium CC17 isolate. In addition, initial adherence and biofilm assays were performed. Compared to the wild-type strain, the esp insertion-deletion mutant no longer produced Esp on the cell surface and had significantly lower initial adherence to polystyrene and significantly less biofilm formation, resulting in levels of biofilm comparable to those of an esp-negative isolate. Capacities for initial adherence and biofilm formation were restored in the insertion-deletion mutant by in trans complementation with esp. These results identify Esp as the first documented determinant in E. faecium CC17 with an important role in biofilm formation, which is an essential factor in infection pathogenesis.

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Enterococci are considered normal inhabitants of the gastrointestinal tracts of humans and animals. In the last 2 decades, though, enterococci have emerged as important nosocomial pathogens, with high-level resistance to antibiotics, such as ampicillin, aminoglycosides, and vancomycin (22). Enterococci are currently the third most frequent nosocomial pathogen isolated from intensive care unit patients in the United States (25). Since the turn of the century, the prevalence of enterococci has been rising in European hospitals, too. The majority of enterococcal infections are caused by Enterococcus faecalis (http://www.earss.rivm.nl). However, in parallel with the increase in nosocomial enterococcal infections, a partial replacement of E. faecalis by Enterococcus faecium took place in European and U.S. hospitals (12, 37, 38) (http://www.earss.rivm.nl). It is unlikely that these ecological changes result exclusively from increased resistance to antibiotics. A better understanding of the virulence of enterococci, therefore, is necessary to control further spread and to develop new treatment strategies.

The ability to form biofilms on abiotic surfaces is considered to be an important virulence property of enterococci (5, 7). A biofilm is an assemblage of microbial cells associated with a surface and enclosed in a matrix of primarily polysaccharide material. The defined architecture of the biofilm provides an optimal environment for the exchange of genetic material between bacteria and increases the innate resistance of the bacterium to antibiotics and activities of the host immune response (6, 7). Enterococci have been associated with biofilms in endocarditis, urinary tract infections, root canal infections, and ocular infections (4, 7, 21, 46) and in a variety of device-related infections in which biofilms were found on artificial hip prostheses, intrauterine devices, prosthetic heart valves, catheters, and stents (2, 7, 18, 29).

Compared to E. faecalis, relatively little is known about the virulence and pathogenesis of E. faecium. Previously, we described the evolutionary descent among E. faecium isolates obtained from human sources (from community, as well as hospital, reservoirs) and nonhuman sources in five continents with multilocus sequence typing. Most hospital outbreak and invasive E. faecium isolates belong to a single clonal lineage, complex 17 (CC17) (45). Furthermore, by using a mixed whole-genome microarray, we recently identified a specific E. faecium clade largely overlapping with CC17, highly specific for nosocomial outbreaks and infections, and containing more than 100 clade-specific genes (17). The ecological success of CC17 in the hospital environment is not understood. It seems to be partly related to resistance to penicillins and quinolone antibiotics (16, 45). Apart from antibiotic resistance and the clade-specific genes, CC17 is correlated with the presence of a putative pathogenicity island, which carries the enterococcal surface protein gene, esp (15, 45). In E. faecalis, Esp is also located on a pathogenicity island, is expressed on the surface of the bacterium (30, 31), and is thought to be an adhesin contributing to colonization of urinary tract epithelial cells and biofilm formation (21, 31, 32, 35). Esp of E. faecium shares up to 90% homology with Esp of E. faecalis, but its function is unknown. Interestingly, it is predominantly present in isolates associated with infections and hospital outbreaks (13, 15, 42). Furthermore, expression of Esp varies between strains, is growth condition dependent, and is quantitatively correlated with initial adherence to polystyrene and biofilm formation (41). Based on these findings, Esp may be an important determinant of adhesion and biofilm formation of E. faecium. However, the definitive role of Esp in these processes could not be determined, as an isogenic esp deletion mutant was lacking and an esp mutant in an E. faecium clinical isolate has not been successfully constructed. In general, it has been extremely difficult to inactivate genes in E. faecium by allelic exchange due to poor transformation capacity, plasmid incompatibility, and the lack of selective markers because of multiple antibiotic resistances in clinically relevant strains. Recently, Nallapareddy et al. were the first to construct an insertion-deletion mutation, in the adhesion-encoding acm gene, in a clinical E. faecium isolate by using an improved temperature-sensitive vector (24). We used the same approach to generate an esp insertion-deletion mutant in a clinical isolate of E. faecium in order to assess the role of Esp in biofilm formation.

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Bacterial strains, plasmids, and growth conditions. The bacterial strains and plasmids used in this study are listed in Table 1. Escherichia coli strains were grown in Luria-Bertani broth or agar. Enterococci were grown in either brain heart infusion (BHI), Todd-Hewitt, or Trypticase soy (TS) broth or agar or sheep red blood agar containing tryptic soy agar with 5% sheep red blood cells (BD, Alphen aan den Rijn, The Netherlands) at 37°C, unless a different growth temperature is specified. For enterococci, the antibiotics chloramphenicol, gentamicin, and erythromycin were used in concentrations of 10 µg/ml, 125 µg/ml, and 10 µg/ml, respectively. For E. coli, the antibiotic chloramphenicol was used in a concentration of 10 µg/ml. All antibiotics were obtained from Sigma-Aldrich (Saint Louis, MO). Growth was determined by measurements of the optical density at 660 nm (OD₆₆₀).

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TABLE 1. Bacterial strains and plasmids used in this study

An improved temperature-sensitive vector (pTEX5500ts), designed by Nallapareddy et al. (24), was used to introduce an insertion-deletion mutation in the esp gene of a clinical E. faecium isolate, E1162, which was isolated from the blood of a patient. E1162 is a strain from CC17. The pAT18 vector (39) was used for complementation studies.

Standard molecular techniques. Chromosomal DNA from E. faecium was prepared as described elsewhere (43, 44). The primers used in this study were purchased from Isogen Life Science (IJsselstijn, The Netherlands) and are listed in Table 2. PCRs were performed with a 9800 Fast Thermal Cycler (Applied Biosystems, Foster City, CA), and the PCR amplification conditions were as follows: initial denaturation at 95°C for 15 min, followed by 10 touchdown cycles starting at 94°C for 30 s, 60°C for 30 s, and 72°C (the time depended on the size of the PCR product) with the annealing temperature decreasing by 1°C per cycle, followed by 25 cycles with an annealing temperature of 52°C. The PCRs were, unless otherwise specified, performed in 25-µl volumes with HotStarTaq Master Mix (QIAGEN Inc., Valencia, CA.). PCR products were purified using the QIAquick PCR purification kit (QIAGEN Inc.) according to the manufacturer's instructions. Restriction enzymes were purchased from New England Biolabs (Ipswich, MA). Ligation was performed by using T4 DNA ligase (Invitrogen, Carlsbad, CA) as recommended by the manufacturer. Plasmid DNA from E. coli was purified by using the QIAprep Spin Miniprep Kit (QIAGEN Inc.) according to the manufacturer's instructions. Plasmids were transformed into E. faecium by electroporation using a Gene Pulser unit (Bio-Rad Laboratories, Richmond, CA) as described elsewhere (23). For Southern hybridization, chromosomal DNA was digested with TaqI, separated by agarose gel electrophoresis (0.7% agarose gels), and transferred onto a Hybond-N⁺ nylon membrane (GE Healthcare, Little Chalfont, Buckinghamshire, United Kingdom). A 916-bp PCR fragment obtained with primers EspUpF2 and EspDnR2 was used as a DNA probe. Labeling of the probe and DNA hybridization were performed according to the protocol supplied with the ECL Direct Nucleic Acid labeling and detection System (GE Healthcare).

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TABLE 2. Primers used in this study

Forward and reverse DNA sequencing were performed by using the forward primers pRIE298.6F, EspUpDelF, EspUpF2, CMF, and EspDnDelF; the reverse primers Esp.11R, EspDnR2, CMR, and EspUpDelR; and the BigDye Terminator reaction kit and an ABI PRISM 3700 DNA analyzer (both from Applied Biosystems).

Construction of an insertion-deletion mutation in the esp gene. To introduce an insertion-deletion mutation in the esp gene, the same protocol described by Nallapareddy et al. (24) was used with some minor modifications. In brief, an 850-bp-long internal esp fragment designated EspUp, coding for a region at the beginning of the N-terminal domain of esp, was amplified from genomic DNA of E1162 by using the primers EspUpdelF and EspUpdelR, including the restriction sites NheI and HindIII, respectively (Table 2). The PCR product was digested with NheI and HindIII and ligated to similarly digested pTEX5500ts, resulting in pEF1. In a similar way, an 830-bp-long fragment designated EspDn, coding for a region encompassing the end of the N-terminal domain of esp, was amplified by using the primers EspDndelF and EspDndelR, including the restriction sites EcoRI and SmaI, respectively. This PCR product was digested with EcoRI and SmaI and ligated to similarly digested pEF1, resulting in pEF2, pTEX5500ts with cloned esp gene fragments flanking the chloramphenicol acetyltransferase (cat) gene. The recombinant plasmids pEF1 and pEF2 were transferred into E. coli DH5 cells (Invitrogen) for propagation and plasmid purification. The recombinant plasmid pEF2 was introduced into E1162 by electroporation to generate an insertion-deletion mutation in the esp gene. After transformation, the cells were allowed to recover for 4 h at the permissive temperature of 28°C, after which the cells were plated on Todd-Hewitt agar plates with 20% sucrose and gentamicin at 28°C to select for transformants. Gentamicin-resistant colonies were picked and grown overnight in BHI broth supplemented with gentamicin at an elevated temperature (42°C) to cure the plasmid. The cells were plated on BHI agar plates with chloramphenicol at 37°C. Single-crossover integration into EspUp and EspDn regions was tested by PCR with the primers pRIE298.6F and CmR, and CmF and Esp.11R, respectively. Single-crossover mutants were saved and grown for six serial overnight passages in chloramphenicol-BHI culture at 42°C to completely cure free recombinant plasmid. The cultures were serially diluted, plated on chloramphenicol-BHI agar plates, and replica plated on gentamicin-BHI agar plates to select for double-crossover recombination. Double crossovers were colonies that retained the cat gene but lost the (aph2'''-Id gene, which encodes an aminoglycoside phosphotransferase that mediates high-level resistance to gentamicin, by plasmid excision, resulting in an insertion-deletion mutation of the esp gene. Correct generation of the insertion-deletion mutation in the esp gene was checked by PCR with the primers pRIE298.6F and Esp.11R, by Southern hybridization, and by DNA sequencing (as described above).

Complementation studies. To complement the esp mutant strain with wild-type esp, the esp gene of E1162 was amplified from genomic DNA by using the Expand Long Template PCR system (Roche Diagnostics, Mannheim, Germany) with the forward primer EspcompF and the reverse primer EspcompR. The forward primer EspcompF included the –35 and –10 promoter regions and the ribosome binding site of the constitutive promoter of the bacA gene of E. faecalis (9, 36), as well as an EcoRI restriction site, facilitating cloning of this fragment. The reverse primer also included an EcoRI restriction site. The resulting PCR product containing the esp gene and bacA promoter sequences was digested with EcoRI and ligated to similarly digested pAT18 (39), resulting in pEF3 (pAT18:esp). The recombinant plasmid pEF3 was introduced into the esp mutant strain by electroporation.

Flow cytometry and electron microscopy. Flow cytometry and electron microscopy were performed as described previously (41). Flow cytometry experiments were repeated twice independently.

Whole-cell ELISA. Plate-grown bacteria were resuspended in PBS to an OD₆₆₀ of 0.1 (1 x 10⁸ CFU/ml). From each bacterial suspension, 100 µl was added to wells of a 96-well polystyrene microtiter plate (Corning Inc., Corning, NY). The bacteria were allowed to bind overnight at 4°C. The wells were washed three times with PBS containing 0.05% Tween 20. After being washed, the wells were blocked with 4% bovine serum albumin (BSA) in phosphate-buffered saline (PBS) with 0.05% Tween 20 for 1 h at 37°C. Esp was assayed by incubation for 1 h at 37°C with rabbit anti-Esp immunoglobulin Gs (IgGs) (collected using a protein-G column; GE Healthcare) (41) in a dilution range from 10 µg/ml to 0.01 µg/ml. Bound antibodies were detected by incubation with a peroxidase-conjugated goat anti-rabbit IgG (1:5,000; Santa Cruz Biotechnology, Santa Cruz, CA.) for 1 h at 37°C. Both antibodies were diluted in PBS with 1% BSA and 0.05% Tween 20. To each well, 50 µl of 0.11 M acetate buffer with 1.6% 3,3',5,5'-tetramethylbenzidine and 0.8% ureumperoxide was added, and the reaction was stopped after 10 min with 50 µl 0.5 M sulfuric acid. The absorbance at 450 nm was measured with an enzyme-linked immunosorbent assay (ELISA) reader. The whole-cell ELISA was performed twice.

Western blotting. Plate-grown bacteria were resuspended in PBS to an OD₆₆₀ of 1.0 (1 x 10⁹ CFU/ml). Cells were harvested by centrifugation (1,560 x g; 5 min) and resuspended in 50 µl PBS. Electrophoresis sample buffer (1x) supplemented with 50 mg/ml dithiothreitol was added. Samples were boiled for 5 min and loaded onto a 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis gel. The bacterial proteins were transferred to nitrocellulose using a Bio-Rad Trans-Blot Cell tank transfer unit at 12 V overnight in 20 mM Tris, 0.15 M glycine, and 20% methanol at pH 8.3. Nonspecific sites in the blot were blocked by incubation for 1 h at 37°C with 4% skim milk powder in PBS with 0.1% Tween 20. Esp was assayed by incubation for 1 h at 37°C with rabbit anti-Esp immune serum (1:5,000) (41) as the primary antibody, followed by incubation for 1 h at 37°C with horseradish peroxidase-conjugated goat anti-rabbit (1:5,000; Santa Cruz Biotechnology) as the secondary antibody. Both antibodies were diluted in PBS with 1% Tween 20 and 1% BSA. Esp was detected by using light-emitting ECL Western Blotting Detection Reagents (GE Healthcare).

Initial polystyrene adherence assay. The initial adherence assay was performed as described previously (1). In brief, plate-grown bacteria were resuspended in TS broth to an OD₆₆₀ of 0.5 (5 x 10⁸ CFU/ml). To each well of a 96-well polystyrene microtiter plate (Corning Inc.) 100 µl bacterial suspension (5 x 10⁷ CFU) was added in triplicate and incubated for 2 h at 37°C. After incubation, the bacteria were removed and the wells were washed with 200 µl PBS. The plates were dried for 1 h at room temperature. After 1 h, 100 µl 0.2% Gram's crystal violet solution (Merck, Darmstadt, Germany) was added to each well. After 15 min, the stain was removed and the plates were washed three times with 200 µl PBS. The plates were dried for 15 min at room temperature, and the absorbance at 595 nm was measured directly with an ELISA reader. The experiment was repeated two times.

Biofilm formation assay. The biofilm assay was performed similarly to the initial adherence assay, except that the assay was performed in TS broth supplemented with 0.25% glucose and 1 x 10⁵ CFU of bacteria were incubated for 24 h at 37°C in a 96-well polystyrene microtiter plate (Corning Inc.).

Confocal laser scanning microscopy (CLSM). Bacteria were grown in TS broth supplemented with 0.25% glucose to mid-log phase. Nitrocellulose membranes (0.45 µm; diameter, 25 mm; Bio-Rad) were put on TS agar plates with 0.25% glucose, and 200 µl bacterial suspension (5 x 10⁶ CFU) was inoculated onto the nitrocellulose membranes and grown for 24 h at 37°C. After 24 h, the nitrocellulose membranes were washed three times in PBS, and the biofilms were chemically fixed in 3.7% formaldehyde (Merck) in PBS for 15 min. Nonspecific sites were blocked by incubation for 1 h at 37°C with 10% skim milk powder in PBS. The biofilms were stained by incubating the nitrocellulose membranes (Bio-Rad) for 15 min at room temperature in 0.1% acridine orange (Merck) in PBS. After incubation, the nitrocellulose membranes were washed three times with PBS and transferred to glass microscope slides and covered with glass coverslips (Marienfeld, Lauda-Königshofen, Germany). The biofilms were examined by using an inverted fluorescence microscope (Leica DMRXA2) equipped with an oil plan-neofluor x100/1.4 objective, and confocal images (scans) were developed with an MRCF-1000 laser (488 nm) scanning confocal imaging system (Bio-Rad). The acquired image stacks were viewed by using Leica Confocal Software (version 6.1). The maximum thickness of the biofilms was measured at five randomly chosen positions with the software.

Statistical analysis. For analysis of cell surface expression of Esp, initial adherence, and biofilm formation, a two-tailed Student's t test was applied.

Top ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Construction of an insertion-deletion mutation in the esp gene. esp gene fragments were cloned into pTEX5500ts flanking the cat gene, resulting in pEF2, and this recombinant plasmid was used to introduce an insertion-deletion mutation in the esp gene of a clinical E. faecium isolate (E1162). The esp mutant strain (E1162esp) was constructed in two steps. First, single-crossover mutants (E1162EspUp/EspDn: pEF2), in which pEF2 was integrated, were selected. Only left single-crossover integration was found. This was followed by selection of double-crossover events, in which the wild-type esp gene was replaced by the mutated esp gene and plasmid sequences were lost. Double-crossover mutants were expected to be chloramphenicol resistant and gentamicin susceptible. In total, 600 colonies were screened by replica plating. Of these colonies, eight were putative double-crossover mutants. In two of the eight colonies, PCR indicated correct insertion-deletion mutation (data not shown). DNA-sequencing results confirmed that a 287-bp-long fragment of esp (positions 4105 to 4392 based on the E. faecium PAI sequence deposited in GenBank under accession number AY322150) was replaced by a 993-bp-long cat gene (data not shown). Southern blot analysis results confirmed correct insertion-deletion mutation in the esp gene in these two colonies (Fig. 1).

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FIG. 1. Confirmation of correct insertion-deletion mutation in the esp gene by Southern blot analysis. (A) Schematic representation of the 5' end encoding the N-terminal domain of the wild-type esp gene (1), the esp single-crossover insertion (2), and the esp double-crossover insertion-deletion (3). The box with squares represents the cat gene coding for chloramphenicol resistance, the striped box represents the aph2'''-Id gene coding for gentamicin resistance, the hatched box represents the EspUp fragment used for recombination, the stippled box represents the EspDn fragment used for recombination, and the black line represents the DNA probe. a, TaqI restriction site; b, fragments obtained after digestion with TaqI; c, nucleotide reference positions relative to the E. faecium PAI sequence (GenBank accession no. AY322150). (B) Hybridization results of Southern blot analysis of TaqI-digested genomic DNA of the esp wild-type strain (lane 1), esp single-crossover mutant strain (lane 2), and esp double-crossover mutant strain (lane 3).

E1162esp colonies appeared to have the same size as those of the wild-type strain (E1162) when grown overnight on sheep red blood agar plates. To further characterize the behavior of E1162esp, growth was monitored by the OD₆₆₀. No difference in growth rates was observed between E1162 and E1162esp (data not shown).

Cell surface expression of Esp. Cell surface expression of Esp was analyzed by flow cytometry using rabbit anti-Esp immune serum (Fig. 2). Esp expression in E1162esp was significantly reduced (P < 0.001) compared to E1162 and was close to the background levels found in a community surveillance strain (E135) not carrying the esp gene. Whole-cell ELISA (Fig. 3) and electron microscopy (Fig. 4) confirmed the lack of cell surface Esp in E1162esp. Western blot analysis indicated that intracellular Esp expression was also abolished in E1162esp (data not shown).

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FIG. 2. Cell surface expression of Esp by flow cytometry. Shown is analysis of cell surface expression of Esp by flow cytometry using rabbit anti-Esp immune serum for the esp wild-type strain (E1162), esp mutant strain (E1162esp), esp-negative strain (E135), and esp-complemented strain. Mean values and standard deviations are shown. *, P < 0.001; **, P < 0.0005.

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FIG. 3. Cell surface expression of Esp by whole-cell ELISA. Shown is analysis of cell surface expression of Esp by whole-cell ELISA using rabbit anti-Esp IgGs in different dilutions for the esp wild-type strain (E1162) (squares), esp mutant strain (E1162esp) (triangles), esp- negative strain (E135) (diamonds), and esp-complemented strain (E1162esp:pEF3) (circles). Mean values and standard deviations are shown.

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FIG. 4. Electron microscopy. Shown are electron micrographs at a magnification of x60,000. The esp wild-type strain (E1162) (A) and the esp mutant strain (E1162esp) (B) were incubated with rabbit anti-Esp immune serum, followed by protein-A-Gold. Bars, 200 nm.

Initial polystyrene adherence assay and biofilm formation. E1162, E1162esp, and E135 were investigated for the ability to adhere to polystyrene and for biofilm formation. Strain E1162 exhibited high adherence to polystyrene and high levels of biofilm formation, while the esp-negative strain, E135, showed only low-level binding and biofilm formation (Fig. 5). Both initial adherence to polystyrene (P < 0.0005) and biofilm formation (P < 0.001) were significantly reduced in E1162esp relative to E1162 and dropped to levels seen in E135.

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FIG. 5. Initial adherence and biofilm formation. Shown are the abilities to adhere to polystyrene (A) and to form biofilm (B) of the esp wild-type strain (E1162), esp mutant strain (E1162esp), esp-negative strain (E135), and esp-complemented strain (E1162esp:pEF3). The horizontal lines represent background OD levels when wells possessing no bacteria were stained with crystal violet. Mean values and standard deviations are shown. **, P < 0.0005, and ***, P < 0.0001 (A); *, P < 0.001, and **, P < 0.0005 (B).

CLSM was used to examine biofilms formed by E1162 and E1162esp on nitrocellulose membranes. Consistent with the biofilm assay on polystyrene, biofilm formation was highly reduced in E1162esp compared to E1162 (Fig. 6). The mean maximum thickness of biofilms formed by E1162esp was significantly (P < 0.0001) lower than that of E1162.

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FIG. 6. CLSM images of the esp wild-type strain (E1162) (A) and the esp mutant strain (E1162esp) (B) grown on nitrocellulose for 24 h. The images represent the layer in a Z-stack that has the maximum bacterial coverage. The maximum thickness of biofilms was measured at five randomly chosen positions, resulting in a mean maximum thickness of 11.01 (± 0.91) µm for E1162 and 6.43 (± 0.81) µm for E1162esp. This difference was significant (P < 0.0001).

Complementation studies. Complementation experiments were performed to determine whether in trans expression of Esp from a plasmid was able to restore initial adherence and biofilm formation in the esp mutant strain. Because the promoter of esp has not been mapped, esp was expressed under the control of a constitutive promoter of the bacA gene of E. faecalis (9), as described in Materials and Methods. After transformation of E. coli DH5 cells with recombinant plasmid pEF3, the cells were highly unstable, suggesting that Esp expression had a toxic effect on the E. coli cells. Therefore, the ligation mixture was transferred directly to the E. faecium esp mutant by electroporation, resulting in an esp-complemented strain (E1162esp:pEF3). Cell surface expression of Esp was analyzed by flow cytometry using rabbit anti-Esp immune serum. Esp expression was significantly enhanced (P < 0.0005) in E1162esp:pEF3 compared to E1162esp and was comparable to, though slightly less than, amounts found in E1162 (Fig. 2). Whole-cell ELISA using rabbit anti-Esp IgGs confirmed significantly enhanced Esp expression in E1162esp:pEF3 compared to E1162esp (Fig. 3), as did Western blot analysis (data not shown). Additionally, initial adherence to polystyrene (P < 0.0001) and biofilm formation (P < 0.0005) were significantly enhanced in E1162esp:pEF3 compared to E1162esp and were comparable to the levels found in E1162 (Fig. 5).

Top ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Successful insertion-deletion mutation of esp in a clinical CC17 E. faecium isolate resulted in abolished cell surface Esp expression, significantly lower initial adherence to polystyrene, and reduced biofilm formation. The capacities for initial adherence and biofilm formation were restored in the insertion-deletion mutant by in trans complementation with esp. Esp is the first documented CC17-specific E. faecium determinant implicated in biofilm formation. Our findings suggest that Esp has played an important role in the evolutionary development of CC17 E. faecium from an avirulent commensal to an important globally spread nosocomial pathogen.

Initial adherence and biofilm formation are both considered important pathogenic properties of enterococci (5, 7). The relevance of biofilms in enterococcal infections has been demonstrated for a variety of infections (2, 4, 7, 18, 21, 29, 46). In E. faecalis, different bacterial surface proteins and genes, such as GelE (10, 14, 27), BopD (11), the fsr locus (10), the bee locus (34), and Esp (21, 32, 33, 35), are involved in this process. For instance, biofilm formation in E. faecalis was reduced in isogenic Esp-deficient strains (32). Furthermore, the N-terminal domain of Esp appeared to be sufficient to enhance biofilm formation (33). However, in another study, the effect of insertional mutagenesis in E. faecalis was strain dependent, ranging from a complete loss of the biofilm formation phenotype to no apparent effect, indicating contribution of additional cell surface proteins (35). Moreover, no correlations were found between the presence or absence of the esp gene in clinical E. faecalis isolates and biofilm formation in other studies (3, 10, 14, 28).

Both initial adherence and biofilm formation were significantly reduced in the esp mutant E. faecium strain and restored in the esp-complemented strain, indicating that Esp is important for initial adherence of E. faecium to polystyrene and subsequent development of a biofilm. The esp mutant strain, comparable with the esp-negative strain, exhibited a low but measurable degree of initial adherence and biofilm formation, which indicates that other factors besides Esp play minor roles in these processes. The reduced initial adherence in the esp mutant strain suggests that Esp is important in the primary attachment to abiotic surfaces in order to initiate biofilm formation. Whether Esp is also involved in adhesion to biotic components, like epithelial cells and extracellular matrix molecules necessary for gut colonization and infection, remains to be determined. In one study, bloodstream isolates of E. faecium enriched with esp had increased adhesion to Caco-2 human colon cancer cells (20), suggesting a role of Esp in gut colonization. In contrast, adherence of E. faecium to Caco-2 cell lines was not associated with the presence of esp in another study (8). The restored biofilm formation in the esp-complemented strain indicates that the biofilm-reduced phenotype of the esp mutant is due to the mutated esp gene and not to a polar effect on genes located downstream. The esp-complemented strain had slightly less expression of Esp than the wild-type esp strain. Nevertheless, both initial adherence to polystyrene and biofilm formation abilities were similar. Perhaps a specific amount of Esp at the surface of the bacterium is already sufficient to induce these processes.

The presence of esp in E. faecium has been associated with higher conjugation frequencies than in esp-negative isolates (19). This suggests that either Esp plays a direct role in cell-cell interaction or Esp may serve as a marker for strains with enhanced potential to acquire new genetic elements. Furthermore, conjugative transfer of the esp gene among E. faecium isolates has been described in vitro by integration of esp into a conjugative plasmid (26), suggesting that the esp gene can be transferred horizontally and spread among E. faecium isolates. Previously, we have shown that Esp expression was elevated under conditions permissive for lumen gut colonization, such as 37°C and anaerobiosis, while expression was reduced under aerobic conditions and at 20°C, mimicking environmental conditions (41). All these data suggest that Esp plays an important role in the pathogenesis of E. faecium infections. Because of the specific linkage of esp to CC17, we hypothesize that esp is one of the important determinants that explains the ecological success of this clonal complex in the hospital environment.

In conclusion, establishing an isogenic esp mutant, as performed in the present study, represents only the second successful insertion-deletion mutation experiment in E. faecium. Inactivation of esp resulted in completely abolished Esp expression on the cell surface and significantly reduced initial adherence to polystyrene and biofilm formation. Esp, therefore, plays an important role in these processes, which are considered important factors in infection pathogenesis. Esp could be a promising therapeutic target for preventing CC17 E. faecium infections.

 
This work was supported by the European Union Sixth Framework Programme "Approaches to Control Multiresistant Enterococci (ACE): Studies on molecular ecology, horizontal gene transfer, fitness and prevention" under contract LSHE-CT-2007-037410 and ZonMW "Vaccine-development to combat the emergence of vancomycin-resistant Enterococcus faecium" project number 0.6100.0008.

We thank W. van Schaik for critical reading of the manuscript and R. Broekhuizen for technical assistance with the CLSM.

 
* Corresponding author. Mailing address: Department of Medical Microbiology, University Medical Center Utrecht, Heidelberglaan 100, 3584 CX Utrecht, The Netherlands. Phone: 31.30.2505006. Fax: 31.30.2541770. E-mail: E.heikens{at}umcutrecht.nl

Published ahead of print on 7 September 2007.

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1
1. Baldassarri, L., R. Cecchini, L. Bertuccini, M. G. Ammendolia, F. Iosi, C. R. Arciola, L. Montanaro, R. R. Di, G. Gherardi, G. Dicuonzo, G. Orefici, and R. Creti. 2001. Enterococcus spp. produces slime and survives in rat peritoneal macrophages. Med. Microbiol. Immunol. 190:113-120.[Medline]
2
2. Dautle, M. P., R. L. Ulrich, and T. A. Hughes. 2002. Typing and subtyping of 83 clinical isolates purified from surgically implanted silicone feeding tubes by random amplified polymorphic DNA amplification. J. Clin. Microbiol. 40:414-421.[Abstract/Free Full Text]
3
3. Di, R. R., R. Creti, M. Venditti, R. D'Amelio, C. R. Arciola, L. Montanaro, and L. Baldassarri. 2006. Relationship between biofilm formation, the enterococcal surface protein (Esp) and gelatinase in clinical isolates of Enterococcus faecalis and Enterococcus faecium. FEMS Microbiol. Lett. 256:145-150.[CrossRef][Medline]
4
4. Distel, J. W., J. F. Hatton, and M. J. Gillespie. 2002. Biofilm formation in medicated root canals. J. Endod. 28:689-693.[CrossRef][Medline]
5
5. Donelli, G., and E. Guaglianone. 2004. Emerging role of Enterococcus spp in catheter-related infections: biofilm formation and novel mechanisms of antibiotic resistance. J. Vasc. Access. 5:3-9.[Medline]
6
6. Donlan, R. M. 2002. Biofilms: microbial life on surfaces. Emerg. Infect. Dis. 8:881-890.[Medline]
7
7. Donlan, R. M., and J. W. Costerton. 2002. Biofilms: survival mechanisms of clinically relevant microorganisms. Clin. Microbiol. Rev. 15:167-193.[Abstract/Free Full Text]
8
8. Dworniczek, E., L. Wojciech, B. Sobieszczanska, and A. Seniuk. 2005. Virulence of Enterococcus isolates collected in Lower Silesia (Poland). Scand. J. Infect. Dis. 37:630-636.[CrossRef][Medline]
9
9. Fujimoto, S., and Y. Ike. 2001. pAM401-based shuttle vectors that enable overexpression of promoterless genes and one-step purification of tag fusion proteins directly from Enterococcus faecalis. Appl. Environ. Microbiol. 67:1262-1267.[Abstract/Free Full Text]
10
10. Hancock, L. E., and M. Perego. 2004. The Enterococcus faecalis fsr two-component system controls biofilm development through production of gelatinase. J. Bacteriol. 186:5629-5639.[Abstract/Free Full Text]
11
11. Hufnagel, M., S. Koch, R. Creti, L. Baldassarri, and J. Huebner. 2004. 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. 189:420-430.[CrossRef][Medline]
12
12. Iwen, P. C., D. M. Kelly, J. Linder, S. H. Hinrichs, E. A. Dominguez, M. E. Rupp, and K. D. Patil. 1997. Change in prevalence and antibiotic resistance of Enterococcus species isolated from blood cultures over an 8-year period. Antimicrob. Agents Chemother. 41:494-495.[Medline]
13
13. Klare, I., C. Konstabel, S. Mueller-Bertling, G. Werner, B. Strommenger, C. Kettlitz, S. Borgmann, B. Schulte, D. Jonas, A. Serr, A. M. Fahr, U. Eigner, and W. Witte. 2005. Spread of ampicillin/vancomycin-resistant Enterococcus faecium of the epidemic-virulent clonal complex-17 carrying the genes esp and hyl in German hospitals. Eur. J. Clin. Microbiol. Infect. Dis. 24:815-825.[CrossRef][Medline]
14
14. Kristich, C. J., Y. H. Li, D. G. Cvitkovitch, and G. M. Dunny. 2004. Esp-independent biofilm formation by Enterococcus faecalis. J. Bacteriol. 186:154-163.[Abstract/Free Full Text]
15
15. Leavis, H., J. Top, N. Shankar, K. Borgen, M. Bonten, E. J. Van, and R. J. Willems. 2004. A novel putative enterococcal pathogenicity island linked to the esp virulence gene of Enterococcus faecium and associated with epidemicity. J. Bacteriol. 186:672-682.[Abstract/Free Full Text]
16
16. Leavis, H. L., R. J. Willems, J. Top, and M. J. Bonten. 2006. High-level ciprofloxacin resistance from point mutations in gyrA and parC confined to global hospital-adapted clonal lineage CC17 of Enterococcus faecium. J. Clin. Microbiol. 44:1059-1064.[Abstract/Free Full Text]
17
17. Leavis, H. L., R. J. Willems, W. J. Van Wamel, F. H. Schuren, M. P. Caspers, and M. J. Bonten. 2007. Insertion sequence-driven diversification creates a globally dispersed emerging multiresistant subspecies of Enterococcus faecium. PLoS. Pathog. 3:e7.[CrossRef][Medline]
18
18. Leung, J. W., Y. L. Liu, T. D. Desta, E. D. Libby, J. F. Inciardi, and K. Lam. 2000. In vitro evaluation of antibiotic prophylaxis in the prevention of biliary stent blockage. Gastrointest. Endosc. 51:296-303.[CrossRef][Medline]
19
19. Lund, B., H. Billstrom, and C. Edlund. 2006. Increased conjugation frequencies in clinical Enterococcus faecium strains harbouring the enterococcal surface protein gene esp. Clin. Microbiol. Infect. 12:588-591.[CrossRef][Medline]
20
20. Lund, B., and C. Edlund. 2003. Bloodstream isolates of Enterococcus faecium enriched with the enterococcal surface protein gene, esp, show increased adhesion to eukaryotic cells. J. Clin. Microbiol. 41:5183-5185.[Abstract/Free Full Text]
21
21. Mohamed, J. A., W. Huang, S. R. Nallapareddy, F. Teng, and B. E. Murray. 2004. Influence of origin of isolates, especially endocarditis isolates, and various genes on biofilm formation by Enterococcus faecalis. Infect. Immun. 72:3658-3663.[Abstract/Free Full Text]
22
22. Murray, B. E. 2000. Vancomycin-resistant enterococcal infections. N. Engl. J. Med. 342:710-721.[Free Full Text]
23
23. Muscholl-Silberhorn, A., and R. Wirth. 2000. Electrotransformation of Enterococci, p. 134-140. In N. Eynard and J. Teissie (ed.), Electrotransformation of bacteria. Springer, Berlin, Germany.
24
24. Nallapareddy, S. R., K. V. Singh, and B. E. Murray. 2006. Construction of improved temperature-sensitive and mobilizable vectors and their use for constructing mutations in the adhesin-encoding acm gene of poorly transformable clinical Enterococcus faecium strains. Appl. Environ. Microbiol. 72:334-345.[Abstract/Free Full Text]
25
25. National Nosocomial Infections Surveillance System. 2004. National Nosocomial Infections Surveillance (NNIS) System report, data summary from January 1992 through June 2004. Am. J. Infect. Control 32:470-485.[CrossRef][Medline]
26
26. Oancea, C., I. Klare, W. Witte, and G. Werner. 2004. Conjugative transfer of the virulence gene, esp, among isolates of Enterococcus faecium and Enterococcus faecalis. J. Antimicrob. Chemother. 54:232-235.[Abstract/Free Full Text]
27
27. Pillai, S. K., G. Sakoulas, G. M. Eliopoulos, R. C. Moellering, Jr., B. E. Murray, and R. T. Inouye. 2004. Effects of glucose on fsr-mediated biofilm formation in Enterococcus faecalis. J. Infect. Dis. 190:967-970.[CrossRef][Medline]
28
28. Raad, I. I., H. A. Hanna, M. Boktour, G. Chaiban, R. Y. Hachem, T. Dvorak, R. Lewis, and B. E. Murray. 2005. Vancomycin-resistant Enterococcus faecium: catheter colonization, esp gene, and decreased susceptibility to antibiotics in biofilm. Antimicrob. Agents Chemother. 49:5046-5050.[Abstract/Free Full Text]
29
29. Sabbuba, N., G. Hughes, and D. J. Stickler. 2002. The migration of Proteus mirabilis and other urinary tract pathogens over Foley catheters. BJU Int. 89:55-60.[Medline]
30
30. Shankar, N., A. S. Baghdayan, and M. S. Gilmore. 2002. Modulation of virulence within a pathogenicity island in vancomycin-resistant Enterococcus faecalis. Nature 417:746-750.[CrossRef][Medline]
31
31. Shankar, N., C. V. Lockatell, A. S. Baghdayan, C. Drachenberg, M. S. Gilmore, and D. E. Johnson. 2001. Role of Enterococcus faecalis surface protein Esp in the pathogenesis of ascending urinary tract infection. Infect. Immun. 69:4366-4372.[Abstract/Free Full Text]
32
32. Tendolkar, P. M., A. S. Baghdayan, M. S. Gilmore, and N. Shankar. 2004. Enterococcal surface protein, Esp, enhances biofilm formation by Enterococcus faecalis. Infect. Immun. 72:6032-6039.[Abstract/Free Full Text]
33
33. Tendolkar, P. M., A. S. Baghdayan, and N. Shankar. 2005. The N-terminal domain of enterococcal surface protein, Esp, is sufficient for Esp-mediated biofilm enhancement in Enterococcus faecalis. J. Bacteriol. 187:6213-6222.[Abstract/Free Full Text]
34
34. Tendolkar, P. M., A. S. Baghdayan, and N. Shankar. 2006. Putative surface proteins encoded within a novel transferable locus confer a high-biofilm phenotype to Enterococcus faecalis. J. Bacteriol. 188:2063-2072.[Abstract/Free Full Text]
35
35. Toledo-Arana, A., J. Valle, C. Solano, M. J. Arrizubieta, C. Cucarella, M. Lamata, B. Amorena, J. Leiva, J. R. Penades, and I. Lasa. 2001. The enterococcal surface protein, Esp, is involved in Enterococcus faecalis biofilm formation. Appl. Environ. Microbiol. 67:4538-4545.[Abstract/Free Full Text]
36
36. Tomita, H., S. Fujimoto, K. Tanimoto, and Y. Ike. 1997. Cloning and genetic and sequence analyses of the bacteriocin 21 determinant encoded on the Enterococcus faecalis pheromone-responsive conjugative plasmid pPD1. J. Bacteriol. 179:7843-7855.[Abstract/Free Full Text]
37
37. Top, J., R. Willems, H. Blok, M. de Regt, K. Jalink, A. Troelstra, B. Goorhuis, and M. Bonten. 2007. Ecological replacement of Enterococcus faecalis by multiresistant clonal complex 17 Enterococcus faecium. Clin. Microbiol. Infect. 13:316-319.[CrossRef][Medline]
38
38. Treitman, A. N., P. R. Yarnold, J. Warren, and G. A. Noskin. 2005. Emerging incidence of Enterococcus faecium among hospital isolates (1993 to 2002). J. Clin. Microbiol. 43:462-463.[Abstract/Free Full Text]
39
39. Trieu-Cuot, P., C. Carlier, C. Poyart-Salmeron, and P. Courvalin. 1991. Shuttle vectors containing a multiple cloning site and a lacZ alpha gene for conjugal transfer of DNA from Escherichia coli to gram-positive bacteria. Gene 102:99-104.[CrossRef][Medline]
40
40. van den Bogaard, A. E., P. Mertens, N. H. London, and E. E. Stobberingh. 1997. High prevalence of colonization with vancomycin- and pristinamycin-resistant enterococci in healthy humans and pigs in The Netherlands: is the addition of antibiotics to animal feeds to blame? J. Antimicrob. Chemother. 40:454-456.[Free Full Text]
41
41. Van Wamel, W. J., A. P. Hendrickx, M. J. Bonten, J. Top, G. Posthuma, and R. J. Willems. 2007. Growth condition-dependent Esp expression by Enterococcus faecium affects initial adherence and biofilm formation. Infect. Immun. 75:924-931.[Abstract/Free Full Text]
42
42. Willems, R. J., W. Homan, J. Top, M. van Santen-Verheuvel, D. Tribe, X. Manzioros, C. Gaillard, C. M. Vandenbroucke-Grauls, E. M. Mascini, E. van Kregten, J. D. Van Embden, and M. J. Bonten. 2001. Variant esp gene as a marker of a distinct genetic lineage of vancomycin-resistant Enterococcus faecium spreading in hospitals. Lancet 357:853-855.[CrossRef][Medline]
43
43. Willems, R. J., J. Top, N. van den Braak, A. van Belkum, H. Endtz, D. Mevius, E. Stobberingh, A. van den Bogaard, and J. D. Van Embden. 2000. Host specificity of vancomycin-resistant Enterococcus faecium. J. Infect. Dis. 182:816-823.[CrossRef][Medline]
44
44. Willems, R. J., J. Top, N. van den Braak, A. van Belkum, D. J. Mevius, G. Hendriks, M. van Santen-Verheuvel, and J. D. Van Embden. 1999. Molecular diversity and evolutionary relationships of Tn1546-like elements in enterococci from humans and animals. Antimicrob. Agents Chemother. 43:483-491.[Abstract/Free Full Text]
45
45. Willems, R. J., J. Top, M. van Santen, D. A. Robinson, T. M. Coque, F. Baquero, H. Grundmann, and M. J. Bonten. 2005. Global spread of vancomycin-resistant Enterococcus faecium from distinct nosocomial genetic complex. Emerg. Infect. Dis. 11:821-828.[Medline]
46
46. Zegans, M. E., H. I. Becker, J. Budzik, and G. O'Toole. 2002. The role of bacterial biofilms in ocular infections. DNA Cell Biol. 21:415-420.[CrossRef][Medline]

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Journal of Bacteriology, November 2007, p. 8233-8240, Vol. 189, No. 22
0021-9193/07/$08.00+0     doi:10.1128/JB.01205-07
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

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