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Antimicrobial Agents and Chemotherapy, July 2005, p. 2693-2700, Vol. 49, No. 7
0066-4804/05/$08.00+0     doi:10.1128/AAC.49.7.2693-2700.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

Population Structure of Enterococcus faecium Causing Bacteremia in a Spanish University Hospital: Setting the Scene for a Future Increase in Vancomycin Resistance?

Teresa M. Coque,1* Rob J. L. Willems,2,4 Jesús Fortún,3 Janetta Top,2,4 Sergio Diz,3 Elena Loza,1 Rafael Cantón,1 and Fernando Baquero1

Departamento de Microbiología,1 Departamento de Enfermedades Infecciosas, Hospital Universitario Ramón y Cajal, Madrid, Spain,3 Eijkman-Winkler Institute for Microbiology, Infectious Diseases and Inflammation,2 Division of Acute Internal Medicine and Infectious Diseases, University Medical Center Utrecht, Utrecht, The Netherlands4

Received 29 November 2004/ Returned for modification 16 January 2005/ Accepted 4 March 2005


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ABSTRACT
 
Over an 8-year period (1995 to 2002), 86 Enterococcus faecium blood isolates from 84 patients, of which 54 were ampicillin resistant (AREF) and 32 were ampicillin susceptible (ASEF), were studied in a university hospital (1,200 beds; serving a population of 600,000) in Spain, a country characterized by a near-absence of resistance to vancomycin and very high rates of ampicillin resistance among enterococci. Clonal relatedness by pulsed-field gel electrophoresis (PFGE), antibiotic susceptibility, presence of the virulence/epidemicity genes espEfm and hylEfm, and identification of purK alleles were studied. A group of isolates was also analyzed by amplified fragment length polymorphism (AFLP) and multilocus sequence typing. Medical charts (30 variables collected) were reviewed for 60/84 patients. ASEF showed high clonal diversity (32 PFGE types, 11 purK alleles, 4 AFLP genogroups), did not harbor putative virulence genes, and had no specific association with hospital acquisition. AREF isolates belonged to a clonal complex (CC) of genetically related strains (purK-1, AFLP genogroup C), occasionally harboring putative virulence traits, and were from patients with particular risk factors. Within this CC, previously associated with vancomycin-resistant E. faecium isolates causing outbreaks worldwide (W. L. Homan et al., J. Clin. Microbiol. 40:1963-1971, 2002), a great genetic diversity of antibiotic resistance and virulence/epidemicity profiles was found. Associations between esp and a >7-day hospital stay and between purK-1, hospital location, and nosocomial acquisition were noted (P < 0.001). These findings reflect the importance of local environmental differences in the evolution of this CC, suggesting that the emergence of vancomycin resistance among AREF strains in Spain may be a question of time.


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INTRODUCTION
 
The emergence and spread of antibiotic-resistant enterococci are not fully understood, since striking differences in different geographical areas have been observed. In the United States, the emergence of Enterococcus faecium as a nosocomial pathogen started in the 1980s with an increase in resistance to ampicillin (7, 33). Acquisition of vancomycin resistance by a few of these ampicillin-resistant E. faecium (AREF) clones, and further dissemination of specific vancomycin resistance transposons to multiple genetic backgrounds, led to an increase in vancomycin-resistant enterococci (VRE) in the following decade (12, 16, 23, 24, 31, 39). The occurrence of VRE in European hospitals remained low throughout the '90s, with a polyclonal enterococcal population structure containing a great variety of Tn1546 types in the community setting (7, 45, 48). However, the high prevalence of AREF in all European Union (EU) countries, with rates of VRE above 10% in six countries in recent years, suggests a change in the epidemiology of enterococci in Europe (Annual Report of the European Antibiotic Resistance Surveillance System [EARSS], 2002 [http//:www.earss.rivm.nl]). It has been demonstrated that endemic vancomycin-susceptible E. faecium (VSEF) clones have provided a key biological substrate for vancomycin resistance in European and American hospitals (20, 24, 31, 36). Widely disseminated VSEF clones have been described recently in Norway, Sweden, and Spain (11, 19, 40), and they might constitute the base for the emergence and successful spread of multiresistant enterococcal organisms in the EU.

E. faecium is a highly host adapted organism that has been divided into different ecovars or host-specific groups on the basis of phenotypic and/or genotypic characteristics (1, 47). Moreover, genetic differences between epidemic and nonepidemic strains among isolates colonizing humans have been reported (8, 17, 22, 47). Epidemic E. faecium strains belong to a human-specific ecovar classified as genogroup C on the basis of amplified fragment length polymorphism (AFLP) profiles (47), frequently harbor a specific allele of the housekeeping gene purK (coding for the phosphoribosylaminoimidazole carboxylase ATPase subunit), and eventually harbor a pathogenicity island (PAI) encoding an enterococcal surface protein (Esp) (5, 17, 21, 22, 46). Other traits potentially involved in the virulence processes, including the gene coding for HylEf, a possible hyaluronidase, have been found in a number of epidemic VRE strains (27, 32, 37).

In this work, the population structure of all invasive E. faecium isolates recovered over an 8-year period (1995 to 2002) in a single hospital in Spain was studied. Differences between ampicillin-susceptible E. faecium (ASEF) and AREF populations were found; most of the AREF isolates constituted a highly clonal population with features of the previous identified clonal lineage 1 (C1), renamed clonal complex 17 (CC-17) (purK-1, AFLP genogroup C) (4, 17, 44). The reasons for the different evolutionary outcomes of this CC in different countries remain to be clarified, but local availability of resistance determinants and gene capture elements might contribute to explaining these differences.


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MATERIALS AND METHODS
 
Bacterial strains. All E. faecium blood isolates recovered from June 1995 to December 2002 at the Hospital Ramón y Cajal, a 1,200-bed teaching institution, were included in our study. One isolate per patient (or more than one when different susceptibility patterns were observed) was selected for further studies. Preliminary bacterial identification and antibiotic susceptibility testing were performed by the semiautomatic PASCO (Difco, Detroit, MI) or WIDER (Francisco Soria Melguizo, Spain) system. Identification was confirmed by testing for the presence of aac(6')-Ii, which is specific for E. faecium, by PCR (10). Antimicrobial susceptibility to 13 antibiotics was determined by the standard agar dilution method (28).

Virulence/epidemicity (VIEP) markers. The presence of genes presumptively involved in virulence or epidemicity, esp, hyl, cyl, gel, and agg, coding for an enterococcal surface protein (Esp), hyaluronidase (Hyl), cytolysin/hemolysin (Cyl), gelatinase (Gel), and aggregation substance (Agg), respectively, was tested by multiplex PCR (14, 21, 32, 42). Tests with negative results were repeated in experiments using primers specific for individual genes. Three different oligonucleotide sets covering different regions of the pathogenicity island in which espEfm is located were used to test isolates with a initial negative result for this gene (21): esp4F (5'-GGA ACG CCT TGG TAT G-3') and esp6R (5'-CCG CTT TTG GTG ATT C-3'), esp14F (5'-AGATTT CAT CTT TGA TTC TTG G-3'), and esp-12R (5'-AAT TGA TTC TTT AGC ATC TGG-3'). Production of hemolysin and gelatinase was searched for as previously reported (9). The following strains were used as controls: E. faecium E-774 (espEfm+ hylEfm+ gel– cyl– agg–), Enterococcus faecalis OG1X containing pAD1 (espEfm– hylEfm– gel– cyl+ agg+), and E. faecalis OG1RF (espEfm– hylEfm– gel+ cyl– agg–). The identities of the amplified espEfm and hylEfm DNA fragments were verified by sequencing different PCR products. E. faecalis control strains were kindly provided by Barbara Murray (University of Texas at Houston).

Pulsed-field gel electrophoresis (PFGE). Genomic DNA was prepared as previously described (10) and digested with SmaI or ApaI (Amersham Life Science, Uppsala, Sweden). Migration of DNA fragments was carried out in a 1.4% agarose (SeaKemGold; Iberlabo, Spain) gel with 0.5% Tris-borate-EDTA at 13°C in a CHEF DR-II device (Bio-Rad, La Jolla, CA). Pulse times were ramped from 1 to 20 s for 37 h. Strains with more than six distinct restriction fragments were considered different as previously described (26, 38).

Computer-monitored fingerprinting analysis. Acquisition of the gel images was performed by using a charge-coupled device EDAS 2000 camera (Kodak, PACISA, Spain) with a 587- by 768-pixel resolution. Computer analysis of the banding patterns obtained by PFGE was performed with the Phoretix software package (Nonlinear Dynamics, Newcastle, United Kingdom). The PFGE patterns were normalized by using SmaI-digested genomic DNA from E. faecalis strain OG1RF as an external size marker. Images analyzed included three reference lanes of DNA size markers, and all images were compatible after normalization. Bands were assigned by computer and corrected manually after visual checking when necessary. The similarity of the banding patterns was analyzed by the Dice coefficient, and cluster analysis was performed by the unweighted-pair group method using average linkages (UPGMA). We used a slightly different position tolerance to allow 100% matching of the banding patterns obtained by duplicate samples on different images. A cutoff equivalent to 80% fragment matching was used, in additional statistical analysis, to group possibly genetically related isolates classified as different PFGE types by standard criteria (see above) (35).

MLST. A subset of E. faecium isolates was selected for characterization by multilocus sequence typing (MLST) using seven housekeeping loci as previously described (17). Different sequences were assigned allele numbers, and different allelic profiles were assigned sequence types (STs) by interrogating the E. faecium MLST database (http://www.mlst.net).

AFLP. A subset of E. faecium isolates was selected for analysis by AFLP. The technique was performed as previously described with the addition of a final ethanol precipitation step to further purify the DNA (47). The amplification products were run on a capillary DNA sequencer (ABI PRISM 3700 DNA analyzer; Applied Biosystems, Foster City, CA). GeneScan collection software (Applied Biosystems) was used to collect data and to export the data to BioNumerics (version 3.5; Applied Maths, Sint-Martens-Lattem, Belgium) for further analysis. The Pearson product-moment correlation coefficient was used to calculate similarities of the AFLP-based densitometric curves, and UPGMA was used for cluster analysis (BioNumerics, version 3.5). AFLP banding patterns were matched with a library of 404 vancomycin-resistant E. faecium (VREF) strains representing the four different AFLP genogroups (A to D) discerned previously (47).

Sequence analysis. The purK gene was amplified using primers purK-1n and purK-2n (http://www.mlst.net), purified (Qiaquick PCR purification kit; QIAGEN, Hilden, Germany), and sequenced by using an ABI Prism 377 automated sequencer (Perkin-ElmerApplied Biosystems, Foster City, CA). purK alleles were assigned by interrogating the E. faecium MLST database (see above).

Patients and chart review. Patients' medical charts were reviewed, and the following demographic and clinical data were included: age, sex, nosocomial acquisition, hospital location, days of hospitalization, APACHE II score, baseline illness, recent surgery, assisted ventilation, central venous catheterization, hyperalimentation, urinary catheterization, renal insufficiency (serum creatinine level of >2 mg/dl), crude mortality, and related mortality. Exposure to antibiotics in the month prior to the date of the initial enterococcal blood culture was evaluated and included vancomycin (parenteral and oral), teicoplanin, metronidazole, clindamycin, beta-lactams, sulfonamides, quinolones, aminoglycosides, and tetracyclines. Patient outcomes were analyzed until hospital discharge or death while in hospital.

Statistical analysis. Characteristics of patients with bacteremia caused by AREF versus ASEF were compared using Student's t test for continuous data and chi-square analysis for categorical data. Yates' correction and a two-tailed Fisher exact test were performed when necessary. Epidemiological and clinical variables were related to molecular typing data provided by PFGE and AFLP and to the presence of the esp, hyl, and purK-1 genes. For PFGE, isolates showing more than 80% similarity were included in the same group and compared with the rest of the isolates; for AFLP, isolates of genogroup C were analyzed as one class and isolates of genogroups A, B, and R as another. Putative virulence factors were categorized as follows: for esp or hyl, isolates containing these traits were compared with those lacking them; for purK, comparison was made between isolates harboring the purK-1 allele and isolates containing other purK alleles. All variables with a P value of <0.1 were included in logistic regression modeling. Multivariate analysis was performed by using logistic regression, with significant variables selected by a backward stepwise procedure. Statistical significance for comparison proportions was calculated by the chi-square test (a P value of <0.05 was considered to be statistically significant). Epi-Info, version 6 (Centers for Disease Control and Prevention, Atlanta, GA), and SPSS 7.5 for Windows were used for analysis of the data.


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RESULTS
 
Epidemiological background and bacterial strains. We collected 626 enteroccocal blood isolates from 374 patients. E. faecalis was the most common species isolated (n = 481; 77%), followed by E. faecium (n = 96; 15%) and Enterococcus spp. (n = 49; 8%). Enterococci represented the fourth most common pathogen isolated from bloodstream infections after Escherichia coli, Staphylococcus aureus, and coagulase-negative staphylococci. The yearly incidence of bloodstream infections caused by E. faecium ranged from 0.1% to 1.7% during the study period. The rate of ampicillin resistance among clinical isolates of this species increased steeply, from 17% in 1991 to 53% in 1995 and 75% in 2002 (data obtained from the microbiology laboratory) (13).

Ninety-six E. faecium isolates corresponding to 90 patients were initially collected. Eighty-six E. faecium blood isolates corresponding to 84 patients were selected for further studies after the identification was confirmed by molecular methods (2 patients each had 2 isolates with different susceptibility profiles). The distribution of the E. faecium clinical isolates over hospital units is shown in Table 1. Of the 84 patients, 39% were in medical wards, 26% were in surgical wards, 15% were in intensive-care facilities, and 19% were outpatients. This distribution differs for patients with bacteremia caused by ASEF versus AREF: 31% versus 44% in medical wards, 19% versus 31% in surgical wards, 12% versus 17% in intensive-care facilities, and 37% versus 8% outpatients.


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  		TABLE 1. Features of Enterococcus faecium blood isolates recovered from 1995 to 2002 in the Ramón y Cajal Hospital: clinical data, PFGE, antibiotic resistance, and virulence profiles

Antimicrobial susceptibility. MICs of 13 antibiotics are shown in Table 2. AREF isolates were more resistant than ASEF isolates to the following antibiotics: ciprofloxacin (71% versus 22%), erythromycin (86% versus 40%), quinupristin-dalfopristin (50% versus 25%), streptomycin (high-level resistance [HLR]) (65% versus 12%), and kanamycin (HLR) (85% versus 12%). HLR to gentamicin was not found among ASEF isolates (11% versus 0%). All isolates were susceptible to linezolid. Isolates within a particular PFGE type (A, W, T, D, or Z) showed different susceptibility patterns (Table 1).


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  		TABLE 2. Antimicrobial susceptibilities of Enterococcus faecium blood isolates collected from the Ramón y Cajal Hospital (1995 to 2002)

PFGE. The 86 isolates studied were classified into 57 PFGE types, 25 AREF and 32 ASEF. Four PFGE types were found in more than one patient (A, T, D, W). Identical banding patterns were found among all the isolates within type A and within type T. However, a diversity of banding profiles (0 to 6 bands) was observed for isolates within PFGE types D (16 isolates, 15 subtypes) and W (5 isolates, 5 subtypes) The genetic diversity observed in PFGE types D and W was also reflected by differences in antimicrobial resistance and epidemicity/virulence profiles among isolates of these types. Interestingly, PFGE types representing isolates that persisted in the hospital for more that one year (types A, D, T, W) clustered together (80% similarity) and were obtained from patients located on the same hospital floor.

AFLP. Fifty-eight E. faecium isolates (34 of 54 AREF and 24 of 32 ASEF isolates) were selected for typing by AFLP; they included isolates belonging to different PFGE types and isolates representing different subtypes within a PFGE cluster showing different VIEP profiles. All AREF isolates studied belonged to AFLP group C, except for two that belonged to group A. ASEF isolates were distributed among AFLP genogroups A (n = 4), B (n = 7), C (n = 6), and R (n = 7).

Diversity of purK alleles. Most of the AREF isolates, 49 strains from hospitalized patients, contained purK-1, while 2 harbored purK-21, 1 harbored purK-6, and 1 contained purK-9. The fact that purK-21 differs from purK-1 by only 1 nucleotide suggests that isolates carrying purK-21 are evolutionarily closely related to purK-1-carrying isolates and thus probably belong to the same clonal complex. This was supported by a maximum-parsimony tree based on the purK gene sequences of the 12 alleles described in this study (Fig. 1).



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  		FIG. 1. Rooted maximum-parsimony tree using BioNumerics (version 4.0) based on the DNA sequences of 12 different purK alleles. Numbers on the branches refer to bootstrap percentages of 1,000 resamplings.

Conversely, ASEF isolates had a diversity of purK alleles: purK-1 (n = 3), purK-2 (n = 8), purK-3 (n = 2), purK-4 (n = 1), purK-6 (n = 3), purK-8 (n = 4), purK-9 (n = 5), purK-11 (n = 1), purK-16 (n = 1), purK-17 (n = 1), and purK-22 (n = 2). Certain purK alleles, such as purK-2, frequently encountered in isolates belonging to AFLP group B, purK-3, purK-16, and pur K-11, were found only in ASEF isolates from nonhospitalized patients. ASEF isolates containing purK-6, purK-8, or purK-9 were collected from inpatients located in different hospital wards (Table 1).

Virulence/epidemicity markers. The distribution of different VIEP markers among the E. faecium isolates studied is shown in Table 1. We found espEfm in 24/86 isolates (28%) corresponding to five PFGE types. All E. faecium isolates harboring the esp determinant were AREF and were assigned to AFLP group C. The hylEfm determinant was detected in 8/86 isolates (9%) corresponding to four PFGE types of AREF isolates that belong to AFLP genogroup C. The first isolates harboring espEfm or hylEfm were recovered in 1995 and 1997, respectively. Within specific PFGE types (types D and W), Esp or Hyl positive as well as negative isolates were found. In these cases Esp- and Hyl-negative isolates in time gave way to positive ones. Only 6/86 isolates (7%) corresponding to three PFGE-types (types D, W, and T) contained both espEfm and hylEfm. These isolates were recovered in 2001 and 2002 on the same floor of our hospital. The presence of gelatinase or hemolysin was not demonstrated for any isolate.

MLST. Fourteen AREF isolates representing PFGE types with different VIEP marker profiles and 6 ASEF isolates containing different purK alleles were studied by MLST (Table 3). STs obtained for AREF isolates with purK-1 were ST-16, ST-17, and ST-18, and those obtained for AREF isolates with purK-21 were ST-63 and ST-103; all clustered in a single clonal complex (CC-17). The only purK-1 ASEF isolate studied had ST-18 and also belonged to this CC. This clonal complex encompasses most of the outbreak isolates collected from hospitalized patients on different continents (17, 44).


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  		TABLE 3. Diversity of STs among E. faecium blood isolates with different antibiotic and virulence profiles

Clinical data. Data for analysis were available for 60 of the 84 patients included in the study. Data included in the clinical records of the other 24 patients were too scarce to be analyzed in detail, but they did not differ from those of the patients analyzed with respect to clinical data and outcome (data not shown). Of the 60 patients analyzed, bacteremia was caused by AREF in 39 (65%) and by ASEF in 21 (35%).

The following variables were significantly associated in univariate analysis with AREF: place of hospitalization (abdominal or digestive surgery departments versus other), urinary catheterization, previous exposure to beta-lactams or quinolones, length of hospitalization, and nosocomial acquisition. No significant association was found between the presence of central venous catheters, parenteral nutrition, abdominal surgery, or underlying diseases (diabetes, neoplasia, immunodeficencies) and bacteremia caused by AREF or ASEF. No association was found between previous therapy with vancomycin, metronidazole, clindamycin, or sulfonamides and isolation of AREF or ASEF.

Univariate analysis demonstrated a strong correlation between purK-1 determinants and AFLP type C (P < 0.001), so both factors were considered together for subsequent analysis. Strains with purK-1 also have a significantly higher proportion of espEfm (P = 0.001) but not of hylEfm. The presence of esp was significantly associated with nosocomial acquisition, prolonged hospitalization, and urinary catheterization. purK-1 was also significantly associated with nosocomial acquisition, particularly with a stay in the general surgery or gastroenterology ward, and with previous therapy with beta-lactam agents. In multivariate analysis, Esp was again significantly associated with prolonged hospitalization and purK-1 with nosocomial acquisition, particularly in the above-mentioned wards (Table 4).


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  		TABLE 4. Association between epidemiological variables of patients with bacteremia caused by Enterococcus faecium and the virulence/epidemicity traits esp, purK-1, and hyl


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DISCUSSION
 
AREF strains have been suggested to serve as substrates for the emergence of VREF outbreaks in European and American institutions (20, 24, 31, 36). MLST studies including epidemiologically unrelated isolates from different sources, hosts, and geographical areas demonstrated that recent E. faecium population biology has at least two main components: (i) a great number of genotypes at linkage equilibrium and (ii) a recently emerged clonal complex encompassing most of the VRE outbreak isolates collected from hospitalized patients on different continents (originally designated C1 and renamed CC-17) which are predominantly resistant to ampicillin (90%) and contain the esp trait (70%) (5, 17, 44). In our 8-year-follow-up study, we have also detected similar clear-cut differences between ASEF and AREF populations causing bacteremia in our institution. AREF isolates constitute a highly clonal population with features of CC-17 (purK-1 and -21; AFLP genogroup C). The low diversity of AREF clinical isolates has also been demonstrated in Norwegian and Swedish hospitals, suggesting the emergence and spread of an E. faecium subpopulation in the clinical setting both in geographical areas where VRE are epidemic and in those where they are not (17, 19, 40; this study).

Our results showed clear differences in the host-ecovar specificity of the VSEF population causing bacteremia in our institution. Most AREF isolates from our study clustered in genogroup C (isolates from hospitalized humans), while ASEF isolates were equally distributed among genogroups A (isolates from pigs and nonhospitalized humans), B (isolates from poultry), C, and R (human strains that do not fit in any of the other groups mentioned) (47), indicating different acquisition routes for different invasive E. faecium strains. Previous studies in different countries have shown that most VREF and VSEF clinical isolates clustered in genogroup C, while VREF and VSEF from community sources or colonized inpatients clustered in different genogroups (8, 17, 22, 47). Differences between our study and previous work might be due to the bias created by the selection of isolates, since other series included smaller numbers of ASEF or blood isolates (8, 17, 19, 22, 47), thereby underestimating/overlooking infections caused by endogenous ASEF isolates (19).

It has been demonstrated previously that specific clones can acquire different antibiotic resistance and virulence traits, highlighting the relevance of the local gene pool for the dissemination and persistence of antibiotic-resistant enterococci (4, 29, 34, 36, 48). In this study, epidemic and nonepidemic AREF isolates with different antibiotic and virulence profiles belonging to AFLP genogroup C and containing purK-1 or purK-21 correspond to STs within CC-17 (ST-16, -17, -18, -63, and -103). This was also true for one ASEF isolate. Since isolates of this clonal complex were able to persist for long periods in our hospital, the lack of epidemic VREF in our country and other European locations might be due to differences in the availability, ability of transfer, or stability of specific genetic elements coding for vancomycin resistance (7, 12, 16, 39-41, 45, 48).

The esp gene has been defined as an epidemicity marker of VREF isolates (17, 21, 46); however, it has been also detected in nonepidemic VSEF isolates, and it is absent in some epidemic AREF strains (3, 11, 19, 21, 49). That suggests that other factors might play a role in the dissemination of the main AREF clonal complex. The variable presence of esp in isolates of PFGE type D could be explained by horizontal transfer of either DNA fragments containing esp or the whole E. faecium PAI to only some isolates within the clone or by loss of this DNA region by some of the isolates. Transfer and deletion of PAIs is frequent (15), and interenterococcal transfer of large chromosomal fragments has been reported on different occasions (24). Furthermore, conjugative transfer of esp among isolates of E. faecium has been described recently (30). In addition to Esp, Hyl has also been associated with epidemic isolates (32). We detected only eight hyl+ isolates corresponding to five PFGE types (9%), a frequency lower than that previously reported for American isolates (34%) (32). Interestingly, for the clones representing both esp- and/or hyl-positive and -negative isolates, the negative variant gave way in time to a positive variant, possibly suggesting horizontal transfer of these traits in the nosocomial setting. The fact that the presence of esp was associated with prolonged hospitalization and urinary catheterization suggest that these traits may have been acquired to enhance fitness at least in some of the clones. Differences in the prevalence of esp and hyl between this and other studies could be due to the bias imposed by the selection of the strains or, again, by differences in the local availability of esp- or hyl-containing elements.

PFGE-types D and W, repeatedly recovered in bacteremic cases from inpatients in high-risk areas over 5 years and 1 year, respectively, were highly variable (for type D, 16 isolates and 15 subtypes; for type W, 5 isolates and 5 subtypes). In contrast, two PFGE types recovered from inpatients in medical wards were highly stable over time (types A and T, persisting 1 and 3 years, respectively). A remarkable polymorphism among isolates corresponding to the same PFGE type (0 to 7 bands) has been shown previously in different long-term studies (2, 6, 26, 36, 43). Differences in clone plasticity might be explained by a variable presence of "hot spots" for rearrangements of genetic fragments in the genomes, the local availability (ward, hospital, host) of adaptive genetic elements and the further evolution imposed by the environment (interplay with a patient's own flora or antibiotic selective pressure).

The analysis of patients' clinical records confirms our findings, as it strongly supports the nosocomial acquisition, from specific wards in our institution, of strains harboring the purK-1 allele and belonging to genogroup C, as well as the association of strains harboring esp with prolonged hospitalization. In agreement with previous studies (13, 25), the place of hospitalization (general surgery or gastroenterology ward versus other), urinary catheterization, previous exposure to ß-lactams or quinolones, and lengthy hospitalization were associated with the isolation of AREF.

This paper documents the population structure of isolates belonging to the "epidemic" clonal complex CC-17 of E. faecium in a setting with a near-absence of VREF. The reasons for the different evolutionary outcomes of this CC in different countries remain to be clarified, although local availability of antibiotic resistance determinants and/or specific gene capture elements, previously demonstrated to be important for successful dissemination and persistence of VRE (18, 29, 39), might contribute to explaining these differences.


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ACKNOWLEDGMENTS
 
We thank A. Rollán and M. van Santen-Verheuvel for technical assistance in the PFGE and AFLP typing, respectively. We are also grateful to D. Aanensen for developing the web-based E. faecium MLST database (available at http://www.mlst.net), which is hosted at Imperial College London.

T. M. Coque wishes to express her endless gratitude to B. E. Murray for introducing her to the field of molecular genetics and for the support and opportunities given during her years of training in the B.E.M. laboratory.


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FOOTNOTES
 
* Corresponding author. Mailing address: Servicio de Microbiología, Hospital Universitario Ramón y Cajal, Carretera de Colmenar, km. 9.1, Madrid 28034, Spain. Phone: 34-91-336 83 30. Fax: 34-91-336 88 09. E-mail: mcoque.hrc{at}salud.madrid.org. Back


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REFERENCES
 
    1
  1. Aarestrup, F. M., P. Butaye, and W. Witte. 2002. Nonhuman reservoirs of enterococci, p. 55-99. In M. S. Gilmore (ed.), The enterococci: pathogenesis, molecular biology, and antibiotic resistance. American Society for Microbiology, Washington, D.C.
    2. 2
  2. Baden, L. R., W. Thiemke., A Skolnik, R. Chambers, J. Strymish, H. S. Gold, R. C. Moellering, and G. Eliopoulos. 2001. Prolonged colonization with vancomycin resistant Enterococcus faecium in long-term care patients and the significance of clearance. Clin. Infect. Dis. 33:1654-1660.[CrossRef][Medline]
    3. 3
  3. Baldassarri, L., L. Bertuccini, M. G. Ammendolia, G. Gherardi, and R. Creti. 2001. Variant esp gene in vancomycin-sensitive Enterococcus faecium. Lancet 357:1802.
    4. 4
  4. Baquero, F. 2004. From pieces to patterns: evolutionary engineering in bacterial pathogens. Nat. Rev. Microbiol. 2:510-518.[CrossRef][Medline]
    5. 5
  5. Bonora, M. G., M. Ligozzi, M. De Fatima, L. Bragagnolo, A. Goglio, G. C. Guazzotti, and R. Fontana. 2004. Vancomycin-resistant Enterococcus faecium isolates causing hospital outbreaks in northern Italy belong to the multilocus sequence typing C1 lineage. Microb. Drug Resist. 10:114-123.[CrossRef][Medline]
    6. 6
  6. Bonten, M. J., M. K. Hayden, C. Nathan, T. Rice, and R. A. Weinstein. 1998. Stability of vancomycin resistant enterococcal genotypes isolated from long-term colonized patients. J. Infect. Dis. 177:378-382.[Medline]
    7. 7
  7. Bonten, M. J. M., R. J. Willems, and R. A. Weinstein. 2001. Vancomycin resistant enterococci: why are they here, and where do they come from? Lancet Infect. Dis. 1:314-325.[CrossRef][Medline]
    8. 8
  8. Bruinsma, N., R. J. L. Willems, A. E. van der Bogaard, M. van Santen-Verheuvel, N. London, C. Driessen, and E. E. Stobberingh. 2002. Different levels of genetic homogeneity in vancomycin-resistant and -susceptible Enterococcus faecium isolates from different human and animal sources analyzed by amplified-fragment polymorphisms. Antimicrob. Agents Chemother. 46:2779-2783.[Abstract/Free Full Text]
    9. 9
  9. Coque, T. M., J. M. Steckelberg, J. E. Patterson, and B. E. Murray. 1995. Incidence of hemolysin, gelatinase and aggregation substance among enterococci isolated from patients with endocarditis, other infections and from feces of hospitalized and community based persons. J. Infect. Dis. 171:1223-1229.[Medline]
    10. 10
  10. Coque, T. M., P. Seetulsingh, K. V. Singh, and B. E. Murray. 1998. Application of molecular techniques to the study of nosocomial infections caused by enterococci. Methods Mol. Med. 15:469-493.
    11. 11
  11. Coque, T. M., R. J. Willems, R. Cantón, R. Del Campo, and F. Baquero. 2002. High occurrence of esp among ampicillin resistant and vancomycin susceptible Enterococcus faecium clones from hospitalized patients. J. Antimicrob. Chemother. 50:1035-1038.[Abstract/Free Full Text]
    12. 12
  12. De Lencastre, H., A. E. Brown, M. Chung, D. Armstrong, and A. Tomasz. 1999. Role of the transposon Tn5482 in the epidemiology of vancomycin resistant Enterococcus faecium in the pediatric oncology unit of a New York City hospital. Microb. Drug Resist. 5:113-129.[Medline]
    13. 13
  13. Fortún, J., T. M. Coque, P. Dávila, L. Moreno, E. Loza, R. Cantón, F. Baquero, and S. Moreno. 2002. Risk factors associated with ampicillin-resistance in patients with bacteremia due to Enterococcus faecium. J. Antimicrob. Chemother. 50:1003-1009.[Abstract/Free Full Text]
    14. 14
  14. Gilmore, M. S., P. S. Coburn, S. R. Nallapareddy, and B. E. Murray. 2002. Enterococcal virulence, p. 301-354. In M. S. Gilmore (ed.), The enterococci: pathogenesis, molecular biology, and antibiotic resistance. American Society for Microbiology, Washington, D.C.
    15. 15
  15. Hacker, J., and J. B. Kaper. 2000. Pathogenicity islands and the evolution of microbes. Annu. Rev. Microbiol. 54:641-679.[CrossRef][Medline]
    16. 16
  16. Hanrahan, J., C. Hoyen, and L. B. Rice. 2000. Geographic distribution of a large mobile element that transfers ampicillin and vancomycin resístance between Enterococcus faecium strains. Antimicrob. Agents Chemother. 44:1349-1351.[Abstract/Free Full Text]
    17. 17
  17. Homan, W. L., D. Tribe, S. Poznanski, M. Li, G. Hogg, E. Spalburg, J. D. A. van Embden, and R. J. L. Willems. 2002. Multilocus sequence typing scheme for Enterococcus faecium. J. Clin. Microbiol. 40:1963-1971.[Abstract/Free Full Text]
    18. 18
  18. Johnsen, P. J., J. I. Østerhus, H. Sletvold, M. Sørum, H. Kruse, K. Nielsen, G. S. Simonsen, and A. Sundsfjord. 2005. Persistence of animal and human glycopeptide-resistant enterococci on two Norwegian poultry farms formerly exposed to avoparcin is associated with a widespread plasmid-mediated vanA element within a polyclonal Enterococcus faecium population. Appl. Environ. Microbiol. 71:159-168.[Abstract/Free Full Text]
    19. 19
  19. Jureen, R., J. Top, J. C. Mohn, S. Harthug, L. Langeland, and R. J. L. Willems. 2003. Molecular characterization of ampicillin-resistant Enterococcus faecium from hospitalized patients in Norway. J. Clin. Microbiol. 41:2330-2336.[Abstract/Free Full Text]
    20. 20
  20. Kawalec, M., M. Gniadkowski, M. Zaleska, T. Ozorowski, L. Konopka, and W. Hryniewicz. 2001. Outbreak of vancomycin-resistant Enterococcus faecium of the phenotype VanB in a hospital in Warsaw, Poland: probable transmission of the resistance determinants into an endemic vancomycin-susceptible strain. J. Clin. Microbiol. 39:1781-1787.[Abstract/Free Full Text]
    21. 21
  21. Leavis, H. L., J. Top, N. Shankar, K. Borgen, M. Bonten, J. van Embden, and R. J. L. 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]
    22. 22
  22. Leavis, H. L., R. J. L. Willems, J. Top, E. Spalburg, E. M. Mascini, A. C. Fluit, A. Hoepelman, A. J. de Neeling, and M. J. Bonten. 2003. Epidemic and nonepidemic multidrug-resistant Enterococcus faecium. Emerg. Infect. Dis. 9:1108-1115.[Medline]
    23. 23
  23. Mato, R., H. De Lencastre, R. B. Roberts, and A. Tomasz. 1996. Multiplicity of genetic backgrounds among vancomycin resistant Enterococcus faecium isolates recovered from an outbreak in a New York City hospital. Microb. Drug Resist. 2:309-317.[Medline]
    24. 24
  24. McAsahan, S. K., K. L. Vergin, S. J. Giovannoni, and D. S. Thaler. 1999. Interspecies recombination between enterococci: genetic and phenotypic diversity of vancomycin resistant transconjugants. Microb. Drug Resist. 5:101-112.[Medline]
    25. 25
  25. Mohn, S. C., S. Harthug, and N. Langeland. 2000. Outbreak of ampicillin-resistant Enterococcus faecium isolates: risk factors for faecal colonization. APMIS 108:296-302.[CrossRef][Medline]
    26. 26
  26. Morrison, D., N. Woodford, S. P. Barrett, P. Sisson, and B. D. Cookson. 1999. A banding pattern polymorphism in vancomycin-resistant Enterococcus faecium and criteria for defining strains. J. Clin. Microbiol. 37:1084-1091.[Abstract/Free Full Text]
    27. 27
  27. Nallapareddy, S. R., G. M. Weinstock, and B. E. Murray. 2003. Clinical isolates of Enterococcus faecium exhibit strain-specific collagen binding mediated by Acm, a new member of the MSCRAMM family. Mol. Microbiol. 47:1733-1747.[CrossRef][Medline]
    28. 28
  28. National Committee for Clinical Laboratory Standards. 2000. Methods for dilution antimicrobial susceptibility tests for bacteria that grow aerobically. Approved standard M7-A5. National Committee for Clinical Laboratory Standards, Wayne, Pa.
    29. 29
  29. Novais, C., T. M. Coque, J. C. Sousa, F. Baquero, and L. Peixe. 2004. Local genetic patterns within a vancomycin-resistant Enterococcus faecalis clone isolated in three hospitals in Portugal. Antimicrob. Agents Chemother. 48:3613-3617.[Abstract/Free Full Text]
    30. 30
  30. 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]
    31. 31
  31. Perlada, D. E., A. G. Smulian, and M. T. Cushion. 1997. Molecular epidemiology and antibiotic suscptibility of enterococci in Cincinnati, Ohio: a prospective citywide survey. J. Clin. Microbiol. 35:2342-2347.[Abstract]
    32. 32
  32. Rice, L. B., L. Carias, S. Rudin, C. Vael, H. Goossens, C. Konstabel, I. Klare, S. R. Nallapareddy, W. Huang, and B. E. Murray. 2003. A potential virulence gene, hylEfm, predominates in Enterococcus faecium of clinical origin. J. Infect. Dis. 187:508-512.[CrossRef][Medline]
    33. 33
  33. Shepard, B. D., and M. S. Gilmore. 2002. Antibiotic-resistant enterococci: the mechanism and dynamics of drug introduction and resistance. Microbes Infect. 4:215-224.[CrossRef][Medline]
    34. 34
  34. Smith, D. L., J. Dushoff, E. N. Perencevich, A. D. Harris, and S. A. Levin. 2004. Persistent colonization and the spread of antibiotic resistance in nosocomial pathogens: resistance is a regional problem. Proc. Natl. Acad. Sci. USA 101:3709-3714.[Abstract/Free Full Text]
    35. 35
  35. Struelens, M. J., R. de Ryck, and A. Deplano. 2001. Analysis of microbial genomic macrorestriction patterns by pulsed-field gel electrophoresis (PFGE) typing, p. 159-176. In K. Ldijkshoorn, J. Towner, and M. Struelens (ed.), New approaches for the generation and analysis of microbial typing data. Elsevier Science, Amsterdam, The Netherlands.
    36. 36
  36. Suppola, J. P., E. Kolho, S. Salmenlinna, E. Tarkka, J. Vuopio-Varkila, and M. Vaara. 1999. vanA and vanB incorporate into an endemic ampicillin-resistant vancomycin-sensitive Enterococcus faecium strain: effect on interpretation of clonality. J. Clin. Microbiol. 37:3934-3939.[Abstract/Free Full Text]
    37. 37
  37. Teng, F., M. Kawalec, G. W. Weinstock, W. Hryniewicz, and B. E. Murray. 2003. An Enterococcus faecium secreted antigen, SagA, exhibits broad-spectrum binding to extracellular matrix proteins and appears essential for E. faecium growth. Infect. Immun. 71:5033-5041.[Abstract/Free Full Text]
    38. 38
  38. Tenover, F. C., R. D. Arbeit, R. V. Goering, P. A. Mickelsen, B. E. Murray, D. H. Persing, and B. Swaminathan. 1995. Interpreting chromosomal DNA restriction patterns produced by pulsed-field gel electrophoresis: criteria for bacterial strain typing. J. Clin. Microbiol. 33:2233-2239.[Medline]
    39. 39
  39. Tomita, H., C. Pierson, S. K. Lim, D. B. Clewell, and Y. Ike. 2002. Possible connection between a widely disseminated conjugative gentamicin resistance (pMG1-like) plasmid and the emergence of vancomycin resistance in Enterococcus faecium. J. Clin. Microbiol. 40:3326-3333.[Abstract/Free Full Text]
    40. 40
  40. Torell, E., I. Kuhn, B. Olsson-Liljequist, S. Haeggman, B. M. Hoffman, C. Lindahl, and L. G. Burman. 2003. Clonality among ampicillin-resistant Enterococcus faecium isolates in Sweden and relationship with ciprofloxacin resistance. Clin. Microbiol. Infect. 9:1011-1019.[CrossRef][Medline]
    41. 41
  41. Torell, E., O. Cars, B. Olsson-Liljequist, B. M. Hoffman, J. Lindback, and L. G. Burman. 1999. Near absence of vancomycin-resistant enterococci but high carriage rates of quinolone-resistant ampicillin-resistant enterococci among hospitalized patients and nonhospitalized individuals in Sweden. J. Clin. Microbiol. 37:3509-3513.[Abstract/Free Full Text]
    42. 42
  42. Vankerckhoven, V., T. Van Autgaerden, C. Vael, C. Lammens, S. Chapelle, R. Rossi, D. Jabes, and H. Goossens. 2004. Development of a multiplex PCR for the detection of asa1, gelE, cylA, esp, and hyl genes in enterococci and survey for virulence determinants among European hospital isolates of Enterococcus faecium. J. Clin. Microbiol. 42:4473-4479.[Abstract/Free Full Text]
    43. 43
  43. Werner, G., R. J. L. Willems, B. Hildebrandt, I. Klare, and W. Witte. 2003. Influence of transferable genetic determinants on the outcome of typing methods commonly used for Enterococcus faecium. J. Clin. Microbiol. 41:1499-1506.[Abstract/Free Full Text]
    44. 44
  44. Willems, R. J. L., J. Top, M. van Santen, A. Robinson, T. M. Coque, F. Baquero, H. Grundmann, and M. J. M. Bonten. 2005. Global emergence of vancomycin-resistant Enterococcus faecium is caused by the clonal spread of a distinct nosocomially-adapted genetic complex. Emerg. Infect. Dis. 11:821-828.[Medline]
    45. 45
  45. Willems, R. J. L., J. Top, N. van der Braak, A. van Belkum, D. J. Mevius, G. Hendriks, M. van Santen-Verheuvel, and J. D. van Embden. 1999. Molecular diversity of Tn1546-like elements in enterococci from humans and animals. Antimicrob. Agents Chemother. 43:483-491.[Abstract/Free Full Text]
    46. 46
  46. Willems, R. J. L., 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. 200l. Variant esp gene as a marker of a distinct genetic lineage of vancomycin resistant Enterococcus faecium spreading in hospitals. Lancet 357:853-855.
    47. 47
  47. Willems, R. J. L., W. Homan, J. Top, N. van Den Braak, A. van Belkum, H. Endtz, D. Mevius, E. Stobberingh, A. van Den Bogaard, and J. van Embden. 2000. Host specificity of vancomycin resistant Enterococcus faecium. J. Infect. Dis. 182:816-823.[CrossRef][Medline]
    48. 48
  48. Woodford, N., A. M. A. Adebiyi, M. F. I. Palepou, and B. Cookson. 1998. Diversity of VanA glycopeptide resistance elements in enterococci from humans and animals. Antimicrob. Agents Chemother. 42:502-508.[Abstract/Free Full Text]
    49. 49
  49. Woodford, N., M. Soltani, and K. J. Hardy. 2001. Frequency of esp in Enterococcus faecium isolates. Lancet 358:584.

Antimicrobial Agents and Chemotherapy, July 2005, p. 2693-2700, Vol. 49, No. 7
0066-4804/05/$08.00+0     doi:10.1128/AAC.49.7.2693-2700.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.



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