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J Clin Microbiol. Dec 2004; 42(12): 5895–5898.
PMCID: PMC535263

Vancomycin Resistance, esp, and Strain Relatedness: a 1-Year Study of Enterococcal Bacteremia

Abstract

The prevalence of esp, a gene associated with infection-derived and outbreak strains, in enterococcal blood isolates from 2002 was determined. Fifty-five of 137 (40.1%) Enterococcus faecalis isolates, 30 of 58 (51.7%) E. faecium isolates, 1 of 1 E. raffinosus isolate, 0 of 4 E. gallinarum isolates, and 0 of 1 E. casseliflavus isolate were positive. esp wasn't associated with vancomycin resistance (VR) or clinical service. VR E. faecium isolates were less genetically diverse than vancomycin-susceptible strains. A large cluster of VR isolates, belonging to esp-positive E. faecium, was revealed. These data support the hypothesis that esp and VR may contribute to dissemination of particular clones.

Enterococci are gastrointestinal commensals that survive well outside the host. Identification of adhesion molecules might distinguish strains that can persist in a hospital environment and create the setting for nosocomially acquired infections. Shankar et al. identified esp, a gene encoding a surface protein of Enterococcus faecalis (13). esp has been associated with biofilm formation, which probably contributes to the ability of enterococci to cause infections (14). Both E. faecium and E. faecalis may harbor homologues of this protein that are heterogeneous in amino acid sequence and number of repeats (2). Recently, esp has been found to reside on an unique pathogenicity island in both species (6, 12). Frequency of esp among bloodstream and other infection-derived isolates ranges from 23 to 68%. Lower prevalence rates in isolates from the feces of healthy volunteers were determined (1, 2, 4, 13, 15). Importantly, some studies support associations of esp with outbreak strains and vancomycin resistance (VR) (11, 17).

The goals of this study were to determine the prevalence of esp in enterococci recovered from blood specimens between 1 January and 31 December 2002 at a large, urban teaching hospital and to assess any associations of esp with clustering of cases, VR, or clinical service. Two hundred one nonduplicate isolates of enterococci collected at The Johns Hopkins Hospital were tested. Multiple isolates from the same patient were tested if they were of different species, genotype, or VR phenotype. Eighteen isolates (8.2%) were lost, including 3 E. faecium isolates, 16 E. faecalis isolates, and 1 E. raffinosus isolate; 7 were from an intensive care unit (ICU), and 11 were from other services.

(This work was presented in part at the 103rd General Meeting of the American Society for Microbiology, Washington D.C., 2003.)

esp was determined by a PCR assay using esp-11 and esp-12 primers (13). Cycling conditions were as follows: 95°C for 10 min; 40 cycles of 95°C for 30 s, 60°C for 1 min, and 72°C for 2 min; and a final 72°C extension for 10 min. As an internal amplification control each suspension was simultaneously tested with primers L1 and G1, which amplify the 16S and 23S RNA gene spacer regions, under the same cycling conditions, except for a 55°C annealing temperature (5). Thirty of 58 (51.7%) E. faecium isolates, 55 of 137 (40.1%) E. faecalis isolates, 0 of 4 E. gallinarum isolates, 0 of 1 E. casseliflavus isolate, and 1 of 1 E. raffinosus isolate were positive (Table (Table1).1). The difference between E. faecium and E. faecalis was not statistically significant. In an evaluation of virulence determinants for enterococcal bacteremia Vergis found that 32% of 219 E. faecalis isolates carry esp but did not assess E. faecium (15). Similar esp-positive rates from blood culture isolates were found in other studies conducted in Europe (1, 4, 13, 16). Since the detection of esp in an E. raffinosus isolate is to our knowledge the first report of esp in an enterococcus other than E. faecium and E. faecalis, species identification was confirmed by 16S sequencing and arabinose fermentation (3). It is difficult to speculate on the importance of this finding. However, given that IS256- and IS905-like elements flank esp in E. faecalis MMH594 and that a phage-like open reading frame is located downstream of esp in E. faecium E300, this gene may be able to mobilize among species given appropriate conditions (6, 12).

TABLE 1.
esp rate by species

Any association of esp and VR was assessed. Vancomycin susceptibility (VS) was determined by an agar dilution assay according to NCCLS guidelines (10). For E. faecalis, 2 of 137 (1.5%) isolates were VR; 0 of the 2 VR isolates were esp positive, and 41% of the 135 VS isolates were esp positive. For E. faecium 42 of 58 isolates (72.4%) were VR; 21 of 42 (50%) were esp positive, and 56% of the 16 VS isolates were esp positive. Most importantly, 70% of the esp-positive E. faecium isolates and 75% of the esp-negative isolates were VR. In spite of this high rate of VR in E. faecium, esp was not associated with VR. Although Rice et al. found esp to be more prevalent among VR E. faecium in the United States, our findings agree with those of both Leavis et al. and Woodford et al., who found no association of VR and esp in E. faecium (7, 11, 18).

E. faecium and E. faecalis were genotyped by pulsed-field gel electrophoresis (PFGE) to assess clonal dissemination, and charts were reviewed to ascertain clinical service. Genomic DNA was extracted by an established protocol (9), digested with 2 U of SmaI (New England Biolabs), and separated in 1% agarose with a CHEF DRII (Bio-Rad). Electrophoresis was performed with 5- to 30-s ramped pulse times for 24 h at 14°C. Bands were visualized by ethidium bromide staining, and analysis was performed with the Molecular Analyst Fingerprinting Plus software (version 1.61; Bio-Rad). Dice coefficients were calculated, and the unweighted pair group method with arithmetic mean (UPGMA) was used to generate dendrograms. A similarity coefficient of 90% typically groups strains with three or less band differences. With this definition for grouping closely related isolates, 43 of the 82 (52.4%) esp-negative E. faecalis isolates grouped into 17 clusters with 2 to 7 members. Thirty-two of the 55 (58.2%) esp-positive E. faecalis isolates grouped into 10 clusters with 2 to 8 members (Fig. (Fig.1).1). This species difference was not statistically significant; therefore, these data suggest that esp does not impact genetic diversity for E. faecalis.

FIG. 1.
Dendrogram of PFGE results for E. faecalis (n = 137). The percentages of similarity are based on Dice coefficients and UPGMA. To the right are numbers of isolates in a group matching at 90% similarity. + and −, esp PCR results. ...

For esp-positive E. faecium 24 of 30 (80%) isolates were highly related, grouping into seven clusters with between 2 and 9 members (Fig. (Fig.2).2). The esp-negative isolates did not cluster like the positive isolates; 15 of 28 (53.5%) isolates grouped into five clusters with 2 to 4 members. The difference between esp-positive and esp-negative E. faecium was statistically significant (P = 0.0324; Z test). Lund and Edlund also report less genetic diversity for esp-positive E. faecium (8). esp was present in the isolates grouping in one large cluster of E. faecium wherein seven of nine isolates had the same PFGE pattern (Fig. (Fig.2).2). The association of esp with clustering is influenced by this group. If multiple isolates with indistinguishable PFGE patterns are removed from the clustering analysis, esp is no longer associated with clustering. Resistance to vancomycin was associated with clustering for E. faecium: 33 of 42 (78.6%) VR isolates were highly related compared to 5 of 16 (31.2%) VS isolates (P = 0.0007; Z test). The association of VR with clustering remained when multiple isolates with indistinguishable PFGE patterns were removed from the analysis. For the 42 VR E. faecium isolates, 18 of 21 (86%) esp-positive isolates compared to 11 of 21 (52%) esp-negative isolates were highly related (P = 0.0195; Z test). However, the association of esp with clustering in VR strains is also lost if only single isolates with distinguishable PFGE patterns are evaluated.

FIG. 2.
Dendrogram of PFGE results for E. faecium (n = 58). Percentages of similarity are based on Dice coefficients and UPGMA. To the right are numbers of isolates in a group at 90% similarity. + and −, esp PCR results. S, VS; R, VR. ...

The seven bacteremias in the large group of 9 E. faecium isolates took place over 6 months and occurred on five clinical services; the isolates had the vanA genotype. This strain differs by only one or two bands from a strain expressing both esp and vanA that caused an outbreak in the neonatal ICU and pediatric service. A database search (excluding the outbreak) of strains previously typed by PFGE demonstrated that this strain type was prevalent in 20 of 244 (8.2%) isolates tested between 1999 and 2001. These isolates were mainly from ICU, oncology, and solid-organ transplant patients. Our findings agree with previous reports showing esp to be highly prevalent in outbreak isolates of VR E. faecium (7, 17) and support the concept that some clones harboring both esp and VR genes may be maintained, leading to further dissemination.

Enterococcal bacteremias occurred in small numbers on many different medical and surgical services. Almost one-half of all bacteremias due to E. faecium occurred in internal medicine and pulmonary medicine (Table (Table2).2). Although the E. faecium esp-positive rates from these services (61 and 71%, respectively) are higher than the overall rate (52%), this difference was not significant. Furthermore, while several isolates were found in predominating PFGE clusters, strains from internal medicine and pulmonary medicine did not cluster together significantly (Fig. (Fig.22).

TABLE 2.
esp-positive enterococci by species and clinical service

The ratio of E. faecalis to E. faecium bacteremia was 2.4 to 1. For pediatric patients (pediatrics and pediatric oncology) the ratio was 6.5 to 1 and the presence of esp in E. faecalis was 53.8% compared to the overall rate of 40.1% (Table (Table2).2). This high rate of bacteremia due to E. faecalis was not significantly associated with esp. However, four different strains (representing 11 of 26 isolates) found in the pediatric population matched other pediatric isolates at >90% similarity. The pediatric bacteremia rate may correlate with dissemination of a few different strain types in pediatric patients independent of esp (Fig. (Fig.11).

In summary, esp was present in 52% of E. faecium isolates and 40% of E. faecalis isolates causing bacteremia, suggesting that the associated surface protein may contribute to development of invasive infections, depending on host factors and the contribution of other bacterial virulence elements. Although VR was not associated with esp for E. faecium, VR isolates were common (70%) and were more highly related than VS isolates. In a clinical setting in which VR E. faecium is endemic, the combination of a highly prevalent VR phenotype and esp could lead to dissemination of particular clones. Although this was a prevalence study, a VR, esp-positive strain related to those from a previous outbreak was detected, demonstrating the potential of strains with these virulence traits to be maintained within the hospital. esp has been associated with biofilm formation in E. faecalis, and thus it may be a determinant of environmental persistence (14). A strain with a surface adhesin such as that encoded by esp could also be more competitive in vivo, causing infection upon a breach of the epithelial barrier. Further study of esp and other genes, such as those on the pathogenicity islands, is needed to fully characterize the roles of the virulence elements of enterococci in persistence and strain dissemination (6, 12).

Acknowledgments

We thank Mary Farkosh for excellent technical assistance and Karen Carroll for review of the manuscript.

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