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Previous Article | Next Article 
Applied and Environmental Microbiology, October 2001, p. 4538-4545, Vol. 67, No. 10
Instituto de Agrobiotecnología y
Recursos Naturales and Departamento de Producción Agraria,
Universidad Pública de Navarra-Consejo Superior de
Investigaciones Científicas, Campus de Arrosadia, 31006 Pamplona,1 Unit of Biochemistry,
Department of Basic Biomedical Sciences, Cardenal Herrera-CEU
University, 46113 Moncada,2 and
Department of Microbiology, University Clinics, 31008 Pamplona,3 Spain
Received 24 April 2001/Accepted 11 July 2001
The enterococcal surface protein, Esp, is a high-molecular-weight
surface protein of unknown function whose frequency is significantly increased among infection-derived Enterococcus faecalis
isolates. In this work, a global structural similarity was found
between Bap, a biofilm-associated protein of Staphylococcus
aureus, and Esp. Analysis of the relationship between the
presence of the Esp-encoding gene (esp) and the biofilm
formation capacity in E. faecalis demonstrated that the
presence of the esp gene is highly associated
(P < 0.0001) with the capacity of E.
faecalis to form a biofilm on a polystyrene surface, since
93.5% of the E. faecalis esp-positive isolates were
capable of forming a biofilm. Moreover, none of the E. faecalis
esp-deficient isolates were biofilm producers. Depending on the
E. faecalis isolate, insertional mutagenesis of
esp caused either a complete loss of the biofilm formation phenotype or no apparent phenotypic defect. Complementation studies revealed that Esp expression in an E. faecalis
esp-deficient strain promoted primary attachment and biofilm
formation on polystyrene and polyvinyl chloride plastic from urine
collection bags. Together, these results demonstrate that (i) biofilm
formation capacity is widespread among clinical E.
faecalis isolates, (ii) the biofilm formation capacity is
restricted to the E. faecalis strains harboring esp, and (iii) Esp promotes primary attachment and
biofilm formation of E. faecalis on abiotic surfaces.
Enterococcus faecalis is
a saprophytic commensal that inhabits the oral cavity and
gastrointestinal flora of humans and animals, although it can behave as
an opportunistic pathogen causing severe urinary tract infections,
surgical wound infections, bacteremia, and bacterial endocarditis
(21, 32, 47). Over the past 2 decades, E. faecalis has become responsible for up to 12% of nosocomial infections, with mortality rates for bloodstream infections ranging from 20 to 68% depending on the patient population (11).
The increased incidence of E. faecalis infection has been
related to the innate resistance of this microorganism to many commonly used antimicrobial agents and to its ability to become resistant to
most, and in some cases to all, of the presently available antibiotics,
either by mutation or by incorporation of foreign genetic material
(6, 24). However, antibiotic resistance alone does not
explain the prevalence of E. faecalis in enterococcal nosocomial infections, since Enterococcus faecium, a species
less susceptible to commonly used antimicrobial agents, is responsible for only 20% of hospital-acquired enterococcal infections whereas E. faecalis is responsible for most of the remaining
infections (33). This observation strongly supports the
existence of additional virulence properties that may facilitate or
enhance virulence of the E. faecalis isolates associated
with infections.
Although knowledge on the pathogenic factors of E. faecalis
is still limited, several virulence molecules associated preferentially with infection-derived E. faecalis strains have been
described; these include cytolysin (16, 25, 26),
aggregation substance (7, 28, 35), extracellular
superoxide (22, 23), surface carbohydrates (17,
24), and surface proteins, such as Ace (34), EfaA
(30), and Esp (43). Among these molecular
species, Esp is the only one whose role in virulence has not been
defined. Esp is a large surface protein of 1,873 amino acids with an
N-terminal domain (amino acids 50 to 743) without significant
similarity to other proteins in the database. The central core region
(amino acids 744 to 1665) consists of a series of two distinct tandem repeat units encoded by nearly identical DNA sequence repeats and shows
global structural similarity to C alpha and Rib proteins of group B
streptococci. The C-terminal domain (amino acids 1666 to 1873) contains
a membrane-spanning hydrophobic region and includes a slight variation
of the LPXTGX motif found in most wall-associated surface proteins of
gram-positive bacteria. It is presently hypothesized that the
N-terminal region of Esp might participate in interactions with the
host and that the central repeat region might serve to retract the
protein from the surface, hiding the protein from the immune system.
In previous work aimed at identifying new factors involved in
Staphylococcus aureus biofilm formation, we reported a
high-molecular-weight cell wall-associated protein of 2,276 amino acids
named Bap (9). The corresponding bap gene was
found among staphylococcal species isolated from bovine mastitis but
not among the human S. aureus isolates tested.
Interestingly, all of the S. aureus isolates harboring Bap
were strong biofilm producers, and transposon-insertional mutagenesis
of bap caused the loss of the biofilm formation phenotype. Bap showed global organizational similarities to an outer membrane protein-like protein of Pseudomonas putida involved
in adhesion to seeds (12), to a proline/threonine-rich
protein of unknown function of Salmonella enterica serovar
Typhi, and to the Esp protein of E. faecalis.
Taking into account the global structural similarity between Bap and
Esp, in this work we analyzed the association between the ability of
E. faecalis isolates to produce a biofilm and the presence
of esp. Furthermore, we performed insertional mutagenesis of
esp in different E. faecalis isolates and
analyzed the effects produced in different steps of the biofilm
formation process at the macroscopic and microscopic levels. A role as
a putative virulence factor involved in bacterial biofilm formation was
assigned to Esp.
Bacterial strains, plasmids, and culture conditions.
A total
of 200 enterococcal isolates were used: 152 E. faecalis
isolates, 39 E. faecium isolates, 7 E. avium
isolates, 1 E. gallinarum isolate, and 1 E. durans isolate. They were isolated from patients during 1999 and
2000 at the Microbiology Department of the University Clinics of
Navarra, Pamplona, Spain. Bacterial species were identified with
API STREP (bioMérieux, Marcy l'Etoile, France) or GPI-Vitek
(bioMérieux).
0099-2240/01/$04.00+0 DOI: 10.1128/AEM.67.10.4538-4545.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
The Enterococcal Surface Protein, Esp, Is Involved
in Enterococcus faecalis Biofilm Formation
ABSTRACT
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
INTRODUCTION
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
MATERIALS AND METHODS
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
DNA manipulations.
Routine DNA manipulations were performed
using standard procedures (4, 41) unless otherwise stated.
Plasmid DNA from E. coli was purified with a Quantum Prep
Plasmid Miniprep (Bio-Rad) or Midiprep (QIAGEN) kit. Plasmids were
transformed into E. faecalis by electroporation using a
previously described method (48). Enterococcal
transformations were enhanced by inducing chloramphenicol acetyltransferase translation with a subinhibitory concentration of
chloramphenicol (0.2 µg/ml) for 2 h after electroporation. Restriction enzymes were purchased from Boehringer Mannheim and used
according to the manufacturer's instructions. Oligonucleotides were
obtained from Life Technologies (Table
1).
|
Epidemiology study on the presence of the esp gene
and repeat number variation.
Primers esp11 and
esp12 (Table 1) were used to amplify a 950-bp fragment
within the N-terminal region of esp and to detect its
presence in DNAs from the different enterococcal isolates. To assess
the repeat number variation among the esp-positive
enterococcal isolates, primers esp46 and esp47
(Table 1) were used for amplification across the A repeat region,
whereas primers esp2 and esp5 (Table 1) were used
for amplification across the C repeat region. According to the
sequences of the esp repeat regions described by Shankar et
al. (43), the number of A repeats was calculated as
follows: nA = (faPCR 
182)/252, where nA is the number of A repeats and faPCR is the size of the DNA fragment
after PCR amplification using primers esp46 and
esp47 (Table 1). Similarly, the number of C repeats was
calculated as follows: nC = (fcPCR 384)/246, where
nC is the number of C repeats and
fcPCR is the size of the DNA fragment
after PCR amplification using primers esp2 and
esp5 (Table 1).
Disruption of esp For disruption of esp in three esp-positive biofilm-forming isolates (Table 1), a 950-bp PCR fragment within the N-terminal region of the esp gene amplified with primers esp11 and esp12 was cloned into the pGEM-T Easy vector (Promega, Madison, Wis.). The esp fragment was then cloned into the EcoRI site of the shuttle vector pBT2, and the resulting plasmid (pTA1) was transformed into E. faecalis by electroporation. After electroporation, bacterial strains were incubated for 24 to 48 h at 30°C on BHI agar with chloramphenicol. Ten milliliters of BHI broth-chloramphenicol was subsequently inoculated with a single colony which had been previously resuspended in 100 µl of BHI broth, and the culture was incubated for 24 h at 43.5°C without shaking. Tenfold serial dilutions of this culture in sterile BHI broth were plated on BHI agar-chloramphenicol and incubated for 24 h at 43.5°C. After overnight incubation, colonies were analyzed for disruption of the esp gene by colony PCR with primers esp3S and esp4S (Table 1), and the results were confirmed by Southern blot analysis.
Biofilm assay on polystyrene plates and adherence to polyvinyl chloride (PVC) plastic. The ability of the enterococcal strains to form a biofilm on an abiotic surface was quantified essentially as described elsewhere (37). Briefly, E. faecalis strains were grown overnight in TSB with 0.25% glucose at 37°C. The culture was diluted 1:40 in TSB-0.25% glucose, and 200 µl of this cell suspension was used to inoculate sterile 96-well polystyrene microtiter plates (Iwaki, Tokyo, Japan). After 24 h at 37°C, wells were gently washed three times with 200 µl of phosphate-buffered saline (PBS), dried in an inverted position, and stained with 1% crystal violet for 15 min. The wells were rinsed again, and the crystal violet was solubilized in 200 µl of ethanol-acetone (80:20, vol/vol). The optical density at 595 nm (OD595) was determined using a microplate reader (Multiskan EX; Labsystems). Each assay was performed in triplicate and repeated three times.
Bacterial adherence to PVC plastic was studied using a phase-contrast microscope (magnification, ×1,000; Nikon Optiphot) as described elsewhere (39), with the following modifications. E. faecalis strains were grown overnight in TSB-0.25% glucose at 37°C. Subsequently, the culture was diluted 1:40 in TSB-0.25% glucose, and 200 µl of this cell suspension was inoculated into the wells of 96-well microtiter dishes containing sterile PVC disks from urine collection bags and incubated for 24 h at 37°C. To compare biofilm formation ability and adherence to PVC of wild-type, recombinant, and complemented strains, bacteria were cultured overnight in TSB-0.25% glucose, supplemented with chloramphenicol (20 µg/ml) when appropriate, and subcultured in TSB-0.25% glucose using microtiter dishes.Primary adherence assay. Early adherence of E. faecalis to a polystyrene surface was determined as previously described (14), with the following modifications. E. faecalis strains were grown in TSB-0.5% glucose, supplemented with chloramphenicol (20 µg/ml) when appropriate, overnight at 37°C. Cultures were then adjusted with TSB-0.5% glucose to an OD578 of 0.1. Ten milliliters of each suspension was added to two polystyrene petri dishes. After incubation for 2 h at 37°C, petri dishes were washed three times with PBS. Cells were fixed with Bouin solution (Sigma) and Gram stained. Adherent bacterial cells were observed by oil immersion microscopy, and the mean count was determined in five microscopic fields. Each experiment was repeated three times.
Analysis of cell surface expression of Esp. E. faecalis strains were grown overnight in TSB-0.25% glucose, supplemented with chloramphenicol (20 µg/ml) when appropriate, at 37°C. The culture was diluted 1:40 in TSB-0.25% glucose, and 200 µl of this cell suspension was used to inoculate sterile 96-well polystyrene microtiter plates (Iwaki). After 24 h at 37°C, wells were gently washed three times with 200 µl of PBS containing 0.1% Tween 20. Wells were blocked with 5% bovine serum albumin at 37°C for 1 h prior to a 2-h incubation with anti-Esp serum diluted 1:5,000 in PBS containing 0.1% Tween 20. Bound antibodies were detected with a peroxidase-conjugated goat anti-rabbit immunoglobulin G antibody (Jackson ImmunoResearch Laboratories, Inc., Bar Harbor, Maine) diluted 1:2,500.
Cell surface hydrophobicity.
The cell surface
hydrophobicities of E. faecalis strains were verified as
previously described (40), with the following modifications. Cells were grown overnight in TSB-0.25% glucose, supplemented with chloramphenicol (20 µg/ml) when appropriate, at
37°C. Three hundred microliters of the test hydrocarbon
(n-hexadecane; Merck, Darmstadt, Germany) was added to
round-bottom test tubes containing 3 ml of washed cells which had been
suspended in PUM buffer (22.2 g of
K2HPO4 · 3H2O, 7.26 g of
KH2PO4, 1.8 g of urea, 0.2 g of MgSO4 · 7H2O, and distilled water to 1,000 ml [pH 7.1]) (39) to an OD470 of 1.0. Following a
10-min preincubation at 37°C, tubes were shaken for 30 s. The
aqueous phase was carefully removed with a Pasteur pipette, and light
absorbance was determined at 470 nm, using a Milton Roy 20D model
spectrophotometer. The percentage of bacterial adhesion to hydrocarbon
was calculated as follows: [1 (ODF/ODI)] × 100, where
ODI and ODF are the ODs of
cells resuspended in PUM buffer determined at the beginning and the end
of the experiment, respectively.
Complementation studies.
The esp gene was
amplified with high-fidelity thermophilic DNA polymerase (Expand Long
Template PCR System; Roche) from E. faecalis 14377 because
the esp gene of this strain contains only one A repeat and
four C repeats with primers pbacS and esp2S
(Table 1). The pbacS primer includes the 35, 10, and
ribosome binding sites of the constitutive promoter of the
bacA gene of E. faecalis (15, 45).
The chimeric pbac-esp gene was cloned into the plasmid pCU1,
and the resulting plasmid, pTA2, was transformed by electroporation into E. faecalis strain 23. Stable expression of Esp was
analyzed by enzyme-linked immunosorbent assay (ELISA) as described above.
Statistical analysis. A nonparametric Kendall rank correlation analysis was used to assess the association between the presence of esp and biofilm formation. For analysis of primary adherence and biofilm formation, a two-tailed Student's t test was applied.
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RESULTS |
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|
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
|
|---|
Relationship between biofilm formation and presence of
esp.
We searched eubacterial genome databases for
sequence homologies to the S. aureus Bap protein using the
gapped BLASTP program (1) and found that Bap and Esp share
33% sequence identity and 50% sequence similarity in an overall
alignment. Individual pairwise alignments between the two proteins
revealed that the N-terminal domain of Bap (region B, amino acids 361 to 819) shows 33% identity with the 694-residue N-terminal domain of
Esp. Furthermore, the C repeat region of Bap, which accounts for 52%
of the protein, also shows 33% identity with the C repeat region of
Esp (Fig. 1).
|
1), weak biofilm forming (1 < OD595 2), medium biofilm forming (2 < OD595 3), and strong biofilm forming
(OD595, >3) (Table
2). Simultaneously, the presence of the
esp gene in all of the isolates was analyzed by PCR. The results indicate that biofilm formation ability is highly and significantly associated with the presence of esp (Kendall
rank correlation, 0.845; P < 0.0001). The biofilm
formation ability was restricted to E. faecalis isolates
harboring esp, and esp was detected neither in
any of the E. faecium, E. avium, or E. gallinarum isolates tested, as previously described
(43), nor in any of the E. faecalis isolates
unable to produce a biofilm. The majority (93.5%) of the E. faecalis esp-positive isolates were able to form biofilm, being
classified as strong, medium, or weak biofilm producers (Table 2).
|
Disruption of the esp gene in E.
faecalis strains with different abilities to form a
biofilm.
To further analyze the possible role of the Esp protein
in E. faecalis biofilm formation, disruption of the
esp gene was performed in strong (strain 54), medium (strain
11279), and weak (strain 11262) biofilm-forming strains. For this
purpose, a 950-bp PCR fragment corresponding to an internal N-terminal
region of esp was cloned into the shuttle vector pBT2,
producing the pTA1 plasmid. The natural biofilm-forming strains were
transformed with pTA1, and transformants were grown at a nonpermissive
replicative temperature. Recombinants (Fig.
2A) were confirmed by PCR using primers
esp3S and esp4S (data not shown) and Southern
blotting of PstI- and EcoRI-digested chromosomal
DNA (Fig. 2B).
|
Cell surface hydrophobicities of parental and mutant strains.
Figure 3 illustrates the hydrophobic
nature of the cell surfaces of parental strains and isogenic
esp mutants. Parental esp-positive strains always
showed similar (strain 54) or higher affinity for n-hexadecane compared with mutant strains. Remarkably, a
similar decrease in hydrophobicity was observed when the hydrophobicity of a bap-positive S. aureus strain (V329) was
compared with that of its bap-negative mutant (M556) (data
not shown).
|
Variation in number of A and C repeats of esp in relation to biofilm formation capacity. In order to determine whether the number of A or C repeats present in Esp was related to the intensity of the biofilm formation ability, we calculated the number of repeats in each E. faecalis strain. In agreement with previously reported results (43), the number of A repeat units ranged from 1 to 3 whereas the number of C repeat units ranged from 4 to 10, with a majority of the isolates displaying 7 C repeat units. No association was found between the number of A or C repeat units within the esp gene and the biofilm formation capacity in the E. faecalis esp-positive isolates (data not shown).
Involvement of Esp in primary attachment and biofilm formation on
PVC plastic.
Early adherence of the wild-type E. faecalis 11279 and its isogenic esp mutant 11279M is
illustrated in Fig. 4A. Strain 11279 adhered to polystyrene much more efficiently than mutant 11279M. In
addition, direct observation of PVC plastic tabs from a urine collection bag using phase-contrast microscopy showed that the parental
strain produced a layer of cells covering the PVC surface (Fig. 4B). In
contrast, very few cells of the esp mutant were attached to
PVC plastic. These results on early adherence are consistent with those
described above on biofilm formation in polystyrene microtiter plates.
|
Esp allows primary attachment to an abiotic surface.
Complementation tests were performed to determine whether
overexpression of Esp was able to confer biofilm formation ability to
an E. faecalis esp-negative strain. Since the nucleotide
sequence upstream of the ATG codon of the esp gene
(accession number AF034779) is very short, we were uncertain whether
the promoter of esp was included in this sequence. To solve
this problem, esp was overexpressed under the control of a
constitutive promoter in E. faecalis, as described in
Materials and Methods. The expression of the Esp protein in the
complemented strain 23C measured by ELISA is shown in Fig.
5A. The complemented strain showed an
enhanced capacity to form a biofilm on a polystyrene surface (Fig. 5B).
However, the complemented strain was always classified into the group
of weak biofilm-forming strains (1 < OD595 2). The number of cells bound to polystyrene was quantified in order
to determine whether the enhanced capacity for biofilm formation in the
complemented strain was due to a modification of primary adherence to
polystyrene. It was observed that the number of attached cells in the
complemented strain was at least 20-fold higher than that in the
wild-type strain (P < 0.0001), demonstrating that Esp
highly enhances primary binding to polystyrene (Fig. 5C). In addition,
we assessed the ability of 23C to attach to PVC plastic from urinary
bags using phase-contrast microscopy. The complemented strain 23C
showed multiple groups of cells adhered to the PVC plastic. In
contrast, very few individual cells were attached to the plastic in the case of the wild-type strain (data not shown).
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DISCUSSION |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Like other gram-positive microorganisms, enterococci are able to produce biofilms on abiotic surfaces (27), increasing their high innate resistance to antibiotics (13), yet the factors controlling enterococcal biofilm formation and maintenance remain unknown (36). The initial step in the colonization of catheters and biofilm formation is bacterial adherence to the biomaterial (20). To date, mutagenesis studies on the initial attachment of gram-positive bacteria to abiotic surfaces have been performed mainly with Staphylococcus epidermidis (19, 42, 46) and have shown that a variety of bacterial surface proteins are involved in the process. Among these, the best characterized protein is AtlE, a cell surface-localized autolysin which has also vitronectin-binding activity and therefore is involved in binding to both biotic and abiotic surfaces (19). Since not all S. epidermidis strains harboring the atlE gene produce a biofilm in vitro, the presence of atlE appears to be necessary but insufficient to induce primary attachment to abiotic surfaces.
The esp gene has been identified in a random sequencing project on the genome of a clinical strain of E. faecalis (43). It has been preferentially detected in infection-derived E. faecalis strains but not in other less pathogenic enterococcal species, strongly suggesting a role of Esp in the pathogenesis of E. faecalis (43). Further supporting this hypothesis are the results obtained in this study on the highly significant association (P < 0.0001) between the presence of the esp gene and the ability to produce a biofilm in vitro. Eighty-seven out of 93 E. faecalis strains harboring esp were able to produce a biofilm in vitro, whereas none of the 59 esp-deficient strains tested were able to produce a biofilm. Furthermore, Esp expression determined by ELISA revealed that five out of the six esp-positive, biofilm-negative E. faecalis isolates analyzed did not produce Esp, strongly suggesting that the absence of biofilm formation in these strains is associated with the lack of Esp expression. These results strongly suggest that esp is involved in the biofilm formation process of E. faecalis.
Insertional mutagenesis of esp has shown no significant effect on biofilm formation in the strongest biofilm-producing E. faecalis strain analyzed (strain 54). This could be attributed to additional surface adhesins that might mediate the initial attachment to the abiotic surface in the absence of esp. On the other hand, the finding that none of the esp-defective E. faecalis strains was able to produce a biofilm suggests the existence of a genetic association between the presence of esp and the presence of these adhesins. A striking common feature between esp and bap genes, both of which are associated with abiotic surface attachment, is that they are flanked by a sequence similar to that of the transposase IS905 in the case of esp (24) and IS431 in the case of bap (our unpublished results). Based on this observation and in agreement with other authors (10, 44) it is tempting to speculate that esp could be part of a pathogenicity island (PAI). It is known that adhesins, which mediate the capacity of bacteria to attach to specific eukaryotic receptor molecules, are major virulence factors encoded by PAIs (18). The possibility that additional adhesins might flank esp and bap and constitute a PAI is under study.
The high degree of conservation of the nucleotide sequences of A and C repeats present in both esp (43) and bap (9) strongly suggests that these repeats could have an important role in the function of this protein. Although our data reveal that the number of repeats is not related to the amount of biofilm produced, we found, in agreement with a previous report (43), that none of the esp-positive strains exhibited a complete loss of either A or C repeats, strongly suggesting that both regions are important for the functionality of the protein. These A and C repeats are not the only structural feature shared by Esp and Bap proteins. Analysis of their amino acid sequence reveals the presence of dimerization domains and calcium binding motifs in both of them. We are presently evaluating the contribution of these domains to the function of Esp and Bap.
Esp exhibits characteristics of surface protein receptors designated microbial surface components recognizing adhesive matrix molecules (38) that adhere to components of the host to initiate colonization. Many of these proteins have a modular design and contain a number of tandem repeat domains that probably arise from a series of recombination and/or duplication events. Although we have no evidence for the presence of domains for binding to host factors in Esp, we cannot exclude for Esp under in vivo conditions a direct ligand-binding activity to the extracellular matrix or an indirect role modulating ligand-binding activity of other molecules. If this is the case, Esp-mediated adherence of E. faecalis to plastic biomaterials would not be the primary role of this protein in the host-bacterium interaction. It has been proposed that the presence of Esp could increase cell surface hydrophobicity and facilitate hydrophobic interactions (43). In contrast with previous results (27) where it was observed that adherence of E. faecalis to urinary catheters was not related to bacterial hydrophobicity, our results demonstrate that the presence of Esp in the cell surface increases hydrophobicity, adherence to abiotic surfaces, and biofilm formation. This apparent discrepancy could be at least partially explained if the E. faecalis clinical strains analyzed were esp deficient, which would likely result in a low hydrophobicity.
Routinely, antibiotic susceptibility is determined in clinical laboratories using the broth microdilution susceptibility test. However, it is well established that antimicrobials directed to planktonic cells may not be efficient against biofilm cells (8, 29). In E. faecalis, improvement of the selection of effective antimicrobial agents against recalcitrant infections is urgently needed. Taking into account the strong correlation between the presence of esp and the ability to produce a biofilm, it may be possible to screen for putative biofilm-forming E. faecalis strains by testing for the presence of the esp gene by PCR or for its product by ELISA. The genetic (presence of esp) rather than phenotypic (adherence or biofilm formation) nature of this screening is advantageous in that it allows preliminary identification of strains which are highly adherent and are thus good candidates for antibiotic susceptibility testing in biofilms (2).
It has been estimated that over 65% of nosocomial infections in the developed world are derived from biofilm-related infections. This represents a warning signal, since in the near future the use of medical implants is likely to increase. In this context, bacterial molecules involved in attachment mediated by host proteins and others, such as Esp, involved in adherence to abiotic surfaces and biofilm formation could become promising therapeutic targets in control programs for eradicating persistent enterococcal infections associated with the presence of biofilms.
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ACKNOWLEDGMENTS |
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Alejandro Toledo-Arana and Jaione Valle contributed equally to this work.
We express our gratitude to V. Shankar for providing the antibodies against Esp, to F. Götz for plasmid pCU1, and to R. Brückner for plasmid pBT2.
This work was supported by grant BIO99-0285 from the Comisión Interministerial de Ciencia y Tecnología and grants from the Cardenal Herrera-CEU University and from the Departamento de Educación y Cultura del Gobierno de Navarra. Alejandro Toledo-Arana is a predoctoral Mutis program fellow from Agencia Española de Cooperación Internacional (AECI), Spain. Jaione Valle is a predoctoral fellow from the Ministerio de Ciencia y Tecnologia (FPI), Spain. C. Cucarella is a predoctoral fellow from the Cardenal Herrera-CEU University.
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FOOTNOTES |
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* Corresponding author. Mailing address: Instituto de Agrobiotecnología y Recursos Naturales, Universidad Pública de Navarra, Campus de Arrosadia, Pamplona-31006, Spain. Phone: 34 948 24 28 34. Fax: 34 948 23 21 91. E-mail: ilasa{at}unavarra.es.
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REFERENCES |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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