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.
DISCUSSION
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.