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Journal of Clinical Microbiology, January 2007, p. 147-153, Vol. 45, No. 1
0095-1137/07/$08.00+0     doi:10.1128/JCM.01704-06
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

Clonal Structure of Enterococcus faecalis Isolated from Polish Hospitals: Characterization of Epidemic Clones{triangledown}

Magdalena Kawalec,1* Zbigniew Pietras,2 Emilia Danilowicz,3 Aleksandra Jakubczak,2 Marek Gniadkowski,1 Waleria Hryniewicz,1 and Rob J. L. Willems4

National Institute of Public Health, 00-725 Warsaw, Poland,1 Institute of Microbiology, Faculty of Biology, Warsaw University, 02-096 Warsaw, Poland,2 Warsaw Agriculture University, Faculty of Biotechnology, 02-798 Warsaw, Poland,3 University Medical Center Utrecht Eijkman-Winkler Laboratory for Microbiology, Infectious Diseases and Inflammation, 3584 CX Utrecht, The Netherlands4

Received 17 August 2006/ Returned for modification 16 October 2006/ Accepted 29 October 2006


   ABSTRACT
Top
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
To study the population structure of Enterococcus faecalis from Polish hospitals, 291 isolates were typed by pulsed-field gel electrophoresis and a novel multilocus sequence typing scheme (P. Ruiz-Garbajosa et al., J. Clin. Microbiol. 44:2220-2228, 2006). The isolates originated from geographically widespread medical institutions and were recovered during a 10-year period (1996 to 2005) from different clinical sources. The analysis grouped the isolates into five epidemic and 71 sporadic clones. The importance of the previously identified global clonal complexes CC2 and CC9 was corroborated by our findings that two of the Polish epidemic clones, A and J, were classified into these clonal complexes (CCs). However, the two most predominant clones, C (ST40) and F (CC87), did not cluster in the aforementioned CCs and may represent novel epidemic CCs. These clones may have emerged in Central Europe. Clone F, carrying glycopeptide resistance determinants of VanA or VanB phenotypes, caused several outbreaks in hematology units and appeared to be the most prevalent clone in recent years in Poland. Antimicrobial susceptibility testing and additional tests for pathogenicity-related phenotypes (hemolysin and gelatinase production) and genes (asa1 and esp) were performed to further characterize these epidemic clones. Multidrug resistance, glycopeptide resistance, presence of asa1, and production of hemolysin appeared to be statistically significant features related to epidemicity. Production of gelatinase was significant for two of the epidemic clones, whereas presence of the esp gene was not specific for the epidemic clones.


   INTRODUCTION
Top
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Enterococci are known to be opportunistic nosocomial pathogens capable of causing life-threatening infections, such as endocarditis and bacteremia, mostly in immunocompromised patients (17, 18, 20). Since Enterococcus spp. are resistant to multiple antibacterial drugs, there are only limited options for effective therapy and prophylaxis of serious infections (16, 21). The two enterococcal species most often isolated from clinical infections are Enterococcus faecalis and Enterococcus faecium, the first of which is responsible for the majority of infections, whereas in the latter, multidrug resistance has predominantly accumulated (18).

Molecular epidemiological studies of E. faecium using multilocus sequence typing (MLST) revealed the existence of host-specific lineages and a distinct genetic subpopulation named clonal complex 17 (CC17) (12, 34, 35) that is responsible for the majority of hospital-related infections and outbreaks and that has spread globally. Until now, less has been known about the population structure of E. faecalis. Analyzing 110 isolates by MLST, Ruiz-Garbajosa et al. identified 55 sequence types (ST) and four major CCs, two of which, CC2 and CC9, were significantly enriched among nosocomial isolates and were considered to represent hospital-adapted complexes, equivalent to E. faecium CC17. Furthermore, CC2 and CC9 corresponded to the previously identified E. faecalis BVE complex (ß-lactamase production, vancomycin resistance, and causing endocarditis) hosting the E. faecalis putative pathogenicity island and the ACB clone (Argentina-Connecticut-Bla+), respectively (22, 23). Moreover, MLST confirmed recombination as the important driving force for generating genetic variability in E. faecalis (29), as it was previously proposed after sequencing the V583 strain genome (27).

The aim of this study was to assess the clonal structure of E. faecalis isolates from Poland recovered nationwide and representing clinical and carrier isolates by pulsed-field gel electrophoresis (PFGE) and MLST and to link the molecular typing data with the presence of putative virulence determinants and glycopeptide resistance genes. This information may provide further insight into the population structure and genetic evolution of E. faecalis and the dynamics that may limit attempts of controlling E. faecalis infections in the future.

(Part of this work has been presented at the Second International American Society for Microbiology-Federation of European Microbiological Societies Conference on Enterococci, Helsingør, Denmark, 2005.)


   MATERIALS AND METHODS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Bacterial isolates. A total of 291 E. faecalis isolates were collected by the National Institute of Public Health in Warsaw from 64 Polish hospitals over a 10-year period (1996 to 2005). In detail, 120 isolates originated from 15 hospitals in Warsaw, and groups of more than 10 isolates were obtained from Bydgoszcz (19 isolates, 3 hospitals), Poznan (18 isolates, 5 hospitals), Kraków (16 isolates, 2 hospitals), Sosnowiec (11 isolates), and Szczecin (11 isolates, 2 hospitals). Smaller groups or sporadic isolates represented centers in 31 other cities that were distributed all over the country. Twenty-three percent of the isolates were collected in 1996, 3% in 1997, 9% in 1998, 5% in 1999, 7% in 2000, 6% in 2001, 6% in 2002, 15% in 2003, 18% in 2004, and 8% in 2005. For each isolate, the clinical source and limited demographic data were documented. Based on this information, three categories of isolates could be identified. Thirty-seven percent were isolates from invasive infections (n = 104), including 70 blood isolates, 19 isolates from peritoneum and peritoneal fluid, 10 isolates from cerebrospinal fluid and isolates from pleura and bile. Thirty-four percent (n = 101) were isolates from noninvasive infections (61 wound isolates and isolates from urine), and 29% (n = 86) represented carrier isolates (28 vagina isolates, 17 rectal swab and stool isolates, skin, sputum, bronchoalveolar lavage fluid, sperm isolates, and 6 isolates from the hospital environment).

Growth of isolates. Isolates were stored at –70°C in brain heart infusion broth (Becton Dickinson, Detroit, MI) with 15% glycerol and cultured twice on Columbia agar supplemented with 5% sheep blood (Becton Dickinson) for 16 h at 35°C in oxygen atmosphere before experimental testing.

Bacterial reidentification. Genus identification of the isolates was performed according to the scheme of Facklam and Collins (9). All species were identified using the API 20 STREP test (bioMérieux, Charbonnieres-les-Bains, France), supplemented by potassium tellurite reduction, motility, and pigment production tests (9).

Antimicrobial susceptibility testing. For susceptibility testing, the agar dilution method was used, performed, and interpreted according to the Clinical Laboratory Standards Institute guidelines (3). The following antimicrobials were tested: penicillin, ampicillin, gentamicin, rifampin (Polfa Tarchomin, Warsaw, Poland); tetracycline, chloramphenicol (Sigma-Aldrich Chemie, St. Louis, MO); streptomycin (ICN Biomedicals, Irvine, CA); ciprofloxacin (Bayer, Wuppertal, Germany); vancomycin (Eli Lilly, Indianapolis, IN); teicoplanin (Marion Merrell, Denham, United Kingdom); and linezolid (Pfizer, New York, NY). E. faecalis ATCC 29212 and Staphylococcus aureus ATCC 29213 were used as reference strains. Isolates which were nonsusceptible to three or more antimicrobials of different chemical groups were assessed as multidrug-resistant (MDR). ß-Lactamase production was tested directly with nitrocefin discs for all of the non-penicillin-susceptible isolates (3).

Detection of gelatinase and hemolysin activities. Screening for gelatinase (GelE) and hemolysin (Cyl) production was performed as described previously (4) with the incubation step at 37°C for 24 h.

Detection of genes coding for pathogenicity factors and glycopeptide resistance. Total DNA of the isolates was purified using a Genomic DNA Prep Plus kit (A&A Biotechnology, Gdansk, Poland). The presence of asa1 and esp genes was examined by dot blot hybridization of genomic DNA (at least 200 ng DNA in a dot), immobilized on the Hybond N+ membrane (Amersham Bioscience, Bucks, United Kingdom), with molecular probes labeled with the ECL Random-Prime labeling and detection system (Amersham Bioscience). All of the steps were performed according to the manufacturer's protocol. The probes were site-specific PCR products amplified as described before for asa1 (36) and esp (primers esp11-F and esp12-R) (31) on the genomic DNA templates of E. faecalis FA2-2/PAM714 (1) and MMH594 (31), respectively. In case of indecisive hybridization signals, confirmatory site-specific PCRs were performed using the same conditions as for obtaining the DNA probes. The vanA and vanB genes were detected by PCR as described previously (2, 5).

PFGE typing. Total genomic DNA for PFGE was purified according to the procedure of Clark et al. (2) in agarose plugs made as described by de Lencastre et al. (7) and digested with the SmaI restriction enzyme (Fermentas, Vilnius, Lithuania). PFGE was performed using a CHEF DRIII system (Bio-Rad, Hercules, CA) under the following conditions: temperature, 14°C; voltage, 6 V/cm; initial pulse time, 1 s; final pulse time, 30 s; electrophoresis time, 24 h. The results were interpreted according to the criteria set by Tenover et al. (32) and additionally digitalized and analyzed in silico by the Fingerprint Informatix II software (Bio-Rad, Salt Lake City, UT). For calculations, the Dice algorithm (24), with 3.11% optimization and 0.87% tolerance parameters was used. The cutoff level of similarity for all of the dendrogram branches was 35%.

MLST. The MLST analysis was performed as described previously (29), using an ABI PRISM Big Dye Cycle Sequencing Ready Reaction kit (Applied Biosystems, Boston, MA) in a 3100 AVANT Genetic Analyzer (Applied Biosystems, Foster City, CA). Allelic sequences and allele profiles were compared with sequences of known alleles and ST submitted to the Internet-accessible database (http://efaecalis.mlst.net/). The eBURST V3 software (10), available at the MLST website, was used to cluster allelic profiles.

Statistical analysis. A two-by-two contingency table with {chi}2 test (P < 0.05) was used to calculate significance of association of virulence and resistance determinants with clonal identity of isolates. Concordance of typing was determined by cross-classifying all isolates on the basis of matched and mismatched PFGE types and STs according to the method described by Robinson et al. (28) using EpiCompare software (version 0.99; RidomGmbH, Wurzburg, Germany). The index of diversity was calculated to assess the discriminatory power of PFGE and MLST as described previously (11, 13) using the EpiCompare software (version 0.99).


   RESULTS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Antimicrobial susceptibility testing and detection of vancomycin resistance genes. One-hundred seventy-two E. faecalis isolates (59%) exhibited the MDR phenotype. Of all antimicrobials tested, nonsusceptibility to tetracycline (82.5%), rifampin (72%), and ciprofloxacin (72%) was found most frequently. Approximately half of the tested isolates were resistant to chloramphenicol (42.6%), streptomycin (50.2%), and gentamicin (49.1%). Antibiotics that were effective in vitro against the majority of isolates were ampicillin and penicillin (resistant isolates, 0.69% and 8%, respectively), with none of the resistant isolates being a ß-lactamase producer. Similarly, almost 94% of isolates were susceptible in vitro to linezolid, 89.7% to vancomycin, and 94.5% to teicoplanin. The presence of the vanA gene was confirmed in 16 E. faecalis isolates (5.5%) and that of vanB in 14 isolates (4.8%), making a total of 30 vancomycin-resistant enterococcus (VRE) isolates.

Detection of gelatinolytic and hemolytic phenotypes and of the genes coding for pathogenicity factors. Forty-seven percent (n = 137 isolates) of the studied E. faecalis isolates produced gelatinase, while 31% (n = 90) showed ß-hemolysis on agar plates supplemented with horse blood. These phenotypes coexisted only in 24 isolates (8.2%), whereas 88 isolates (30.25%) lacked both of them. The presence of the aggregation substance gene asa1 was detected in 157 isolates (54%), while the esp gene encoding enterococcal surface protein Esp was found in 211 isolates (72.5%).

PFGE and MLST. The visual analysis of PFGE patterns revealed 76 different PFGE types (clones) among all of the study isolates. Of these, five PFGE types grouped at least 14 of the isolates each, 11 types represented 2 to 5 isolates each, and 60 types were encountered only once. The five most prevalent clones, clones A (15% of the isolates), C (20%), F (21%), I (9%), and J (5%), accounted for 70% of all of the isolates. An important characteristic of these five clones was that they were represented by isolates recovered from numerous hospitals in different cities during the entire study period, whereas the other clones were observed in single centers. Therefore, these five prevalent clones were considered epidemic for the entire country.

Clones A, F, and C were clearly distinguished also by the numerical analysis of the PFGE data with the Fingerprint Informatix II software that generated a three-dimensional snapshot instead of a classical dendrogram (Fig. 1, panel A). For clone A there was an exact match of the results obtained in both analyses. Similar accuracy of visual and in silico approaches was found for 42 and 37 isolates classified into PFGE types F and C, respectively. However, the numerical analysis eroded two clones, PFGE types J and I, which were discerned by the visual interpretation, by clustering only 6 of the 13 isolates of PFGE type J and 5 of the 25 isolates of PFGE type I. Moreover, the in silico analysis classified 99 single isolates into separate PFGE types, only 38 of which were assigned to sporadic PFGE types by the visual analysis. On the other hand, the Dice algorithm clustered three separate groups of isolates, with 16, 15, and 4 isolates, respectively, while these isolates were not grouped using visual analysis of the PFGE data.


Figure 1
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  		FIG. 1. The clonal structure of the collection of 291 E. faecalis isolates collected from 1996 to 2005 in hospitals in Poland. (A) The three-dimensional snapshot depicting the similarity of PFGE patterns obtained with the in silico analysis. Each figure represents a single isolate; shapes correspond to PFGE types identified by visual interpretation of PFGE. (B) eBURST analysis of the MLST data. The epidemic clones are called as in panel A; the sizes of points are in direct proportion to numbers of isolates of each ST in the online database (http://efaecalis.mlst.net).

 
MLST analysis was performed for 150 selected E. faecalis isolates. The isolates were selected from different geographic origins and different years of isolation. Furthermore, at least one representative of each PFGE type was selected. Therefore, the epidemic clones were underrepresented in this subgroup of isolates. MLST revealed 53 STs, 34 of which were novel types according to the MLST database (ST81 to ST89, and ST119 to ST143). Seven of these new STs (ST88, ST133 to ST137, and ST142) arose from new combinations of known alleles; the others corresponded to new alleles in the E. faecalis population from Polish hospitals. Allele sequences, allelic profiles, and information about the E. faecalis strains can be found at http://efaecalis.mlst.net.

Five STs were found to be highly prevalent and comprised almost half of the isolates; 16.7% of the isolates were ST40, 10.7% were ST21, 10% were ST87, 6.7% were ST6, and 5.3% were ST28. Of these, ST87 was a new ST, observed only in Poland so far. The five STs were classified into three different CCs (Fig. 1, panel B). In particular, CC2, represented in Poland mostly by ST6, and ST21, the predicted founder of CC21, were both previously identified as major E. faecalis CCs (29). On the other hand, ST28 and ST87 constituted CC87, a novel major CC. ST40, the most prevalent ST in our collection, did not belong to any of the major complexes but clustered together with an ST114 isolate from the United States (http://efaecalis.mlst.net).

Comparison of the PFGE (visual interpretation) and MLST results. As shown in Table 1, the concordance of PFGE and MLST, calculated by cross-classifying isolates based on matched and mismatched PFGE types and STs, was high (96%). All isolates identified as clone A by PFGE (n = 9) were ST6. Similarly, all PFGE type I isolates that were analyzed by MLST (n = 7) were ST21. Twenty-three PFGE type F isolates represented three highly related STs, ST87 (n = 15), ST28 (n = 7), and ST83 (n = 1), all belonging to CC87. Also, isolates recognized as PFGE type J (n = 7) represented three different but highly related STs, ST9 (n = 4), ST88 (n = 2), and ST142 (n = 1), all classified into CC9. The only exception was the PFGE clone C (n = 26). Twenty-three type C isolates were ST40, while three were ST16. Both STs differ in all seven loci, demonstrating that isolates belonging to these STs do not share a common evolutionary history and thus do not represent a single clone.


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  		TABLE 1. Concordance between PFGE and MLSTa

 
Several isolates that belonged to different PFGE clones constituted a single ST, e.g., PFGE types called CZ, AI, AO, BA, BE, and CO were all ST30. However, PFGE patterns classified into PFGE types did not provide a more discriminatory data set than MLST (Table 2). When PFGE patterns were classified into subtypes, PFGE appeared to be more discriminatory than MLST (Table 2).


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  		TABLE 2. Genetic diversity of the strain set based on PFGE types, PFGE subtypes, and MLSTa

 
Distribution of resistance and virulence determinants among the epidemic and sporadic clones. Seventy-four percent of isolates with the MDR phenotype belonged to one of the five epidemic clones, while only 24% of the sporadic clones exhibited this phenotype. This statistically significant enrichment of MDR among the epidemic clones suggests that MDR contributes to the epidemic spread of E. faecalis in hospitals. Also, vancomycin resistance alone was found significantly more frequently among the epidemic isolates (13.3%) than among the sporadic isolates (3.4%).

Of the different virulence determinants, hemolysin production and the presence of asa1 were specifically linked with the epidemic clones, while the presence of esp was not (Table 3). The production of gelatinase was not significantly different between epidemic and sporadic isolates. All of these data are summarized in Fig. 2.


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  		TABLE 3. Characteristics of E. faecalis clones as distinguished by PFGE (visual analysis), with results of MLST (STs), phenotypes and genotypes of antimicrobial resistance, and some pathogenicity-related factors

 

Figure 2
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  		FIG. 2. Characteristics of the epidemic and sporadic PFGE clones. MDR, multidrug resistance; Cyl, hemolysin production; GelE, gelatinase production; asa1, presence of the asa1 gene; esp, presence of the esp gene; VRE, presence of the vanA or vanB gene.

 
Characteristics of the five major epidemic clones. The characteristics of the five major epidemic clones are presented in more detail below and in Table 3.

(i) Clone F. Clone F (CC87: ST28, ST83, and ST87) was represented by 62 isolates (21% of the studied isolates) and recovered from 32 different hospitals located in 23 cities all over the country. The time of isolation covered almost the entire study period, with the exception of 1997. Almost half of the isolates (n = 26) originated from invasive infections, while 16 were from noninvasive ones and 20 represented carrier isolates. Fifty-one isolates were of the MDR phenotype, while 21 isolates were VRE (13 VanA and 8 VanB). Forty-two isolates produced hemolysin (phenotype Cyl+), while one isolate was gelatinase positive (GelE+). Forty isolates harbored asa1, and esp was found in 46 isolates. MDR, Cyl+, VRE and the lack of GelE were statistically significant features of clone F.

(ii) Clone C. Clone C (ST40; ST16) was represented by 60 isolates (20% of the studied isolates) originating from 41 hospitals in 19 cities all over the country. Similarly to clone F, it was isolated during the whole study period, with the exception of 1997. Isolates of the PFGE type C were carrier isolates (n = 26), "invasive" isolates (n = 21, almost exclusively from blood), and "noninvasive" isolates (n = 13, including 9 from urine). All of these isolates were MDR, and two were VRE (1 VanA and 1 VanB). Forty-one isolates produced gelatinase, 21 isolates produced hemolysin, and both traits together were found in 13 isolates. The asa1 gene was detected in 32 isolates, and esp was detected in 48 isolates. MDR and GelE+ were statistically significant characteristics of clone C.

(iii) Clone A. Clone A (CC2: ST16) was represented by 43 isolates (15% of the studied isolates) from 16 different hospitals located in 11 cities in almost all geographic regions of Poland, and it was identified during the entire study period. Isolates of PFGE type A were collected mostly from noninvasive infections (n = 26, 19 isolates from wounds), 11 were from invasive infections, and 6 were carrier isolates. Forty-two isolates were MDR, but none was resistant to vancomycin. Forty isolates were GelE+, 6 isolates were Cyl+, and both phenotypes were found in 4 isolates. Thirty-four isolates harbored asa1, and 21 isolates contained esp. The combination of MDR, GelE+, Cyl+, asa1, and low incidence of esp were statistically significant for clone A.

(iv) Clone I. Clone I (CC21: ST21) was represented by 25 isolates (9% of the studied isolates) from 13 hospitals located in 12 cities of several different regions. Isolates of PFGE type I were collected from noninvasive infections (n = 11, almost uniformly from wounds), invasive infections (n = 8), and from carriage (n = 6). Thirteen PFGE subtypes were distinguished. Eleven isolates were MDR, including four VRE (1 VanA and 3 VanB). Gelatinase was produced by 7 isolates, 6 were beta-hemolytic, and none showed both activities together. The asa1 gene was detected in 7 isolates, and esp was detected in 14 isolates. The low incidence of GelE and asa1 were statistically significant for this clone.

(v) Clone J. Clone J (CC9: ST9 and ST88; ST142) was represented by 13 isolates (5% of the studied isolates) from seven different hospitals in two cities separated by almost 300 km (Bydgoszcz and Warsaw). Isolates of the PFGE type J were collected from noninvasive infections (n = 9), invasive infections (n = 3), and carriage (n = 1). Eleven isolates were MDR, but none was VRE. Gelatinase was produced by 9 isolates, 3 isolates were beta-hemolytic, and 2 of them were of both phenotypes. The asa1 gene was detected in 5 isolates, and esp was detected in 12 isolates. MDR was a statistically significant characteristic of clone J.


   DISCUSSION
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
This research was aimed at analyzing the population structure and molecular epidemiology of E. faecalis from hospital-acquired infections and from noninfective sites. The overrepresentation of isolates from Warsaw and the nonequal temporal distribution of isolates in the analyzed collection implicate the necessity of careful interpretation of the data obtained. However, the length of the period the isolates were collected (1996 to 2005) and the geographic allocation of the samples allowed us to draw some general conclusions on the existence of epidemic nosocomial clones and their characteristics that may have contributed to their ecological success in this particularly competitive environment. It should be underlined that wider studies on the clonality of E. faecalis have just been commenced (23, 29) and this has been one of the very first countrywide analyses of this species ever performed.

The genetic relatedness of E. faecalis isolates was studied using two typing schemes, PFGE, which is considered the standard typing method for the analysis of hospital-related isolates (7), and MLST (29), which is a relatively new DNA-based approach that allows worldwide comparisons of easily storable and exchangeable data. Furthermore, MLST provides not only information on the global and long-term epidemiology of bacterial species but also insights in their population structure and patterns of evolutionary descent (10). PFGE and MLST revealed the existence of five epidemic clones spread in the country and isolated during the whole study period, namely A (ST6; 15%), C (ST40, ST16; 22%), F (CC87; 21%), I (ST21; 9%), and J (CC9; 5%). Clone names and percentages of the isolates belonging to each of them were derived from the visual analysis of the results of PFGE typing (32). Grouping of isolates based on visual analysis of PFGE differed slightly from classifications based on computer-based interpretation of PFGE patterns and MLST. However, in general, the concordance between the three methods was high (>90%). The nonperfect parity between PFGE and MLST was already shown previously for E. faecalis (29) as well as for other microorganisms (8, 25).

With exception of the PFGE type I, all of the epidemic clones were significantly associated with MDR. This was not the case for sporadic isolates. Of all resistance phenotypes, high-level resistance to aminoglycosides was most prominently associated with clones A and F. In addition, vancomycin resistance, not described for E. faecalis in Poland prior to 1998 (14), was particularly frequent in clone F (CC87). The association between antibiotic resistance and increased fitness in the hospital environment is not unexpected and was reported previously (19).

In general, no correlation was found between the clinical source of isolation and the clonal structure of the examined collection of E. faecalis, although 50% of isolates belonging to clone F (CC87) were from invasive sites, representing primarily bacteremias and illustrating the domination of this clone in hematology units in Poland. Clone F (CC87) was also, in general, the most frequently encountered clone, causing four VRE outbreaks, one VanB and three VanA, in hematology wards in three cities in Poland, Kraków, Poznan, and Warsaw, in 2002 to 2004 (15, 15a). This clone, which included ST28, ST83, and ST87, did not belong to the two important hospital-adapted complexes CC2 and CC9 described previously (29). The finding that only one-third of the isolates belonging to this clone were vancomycin resistant may suggest that this clone has spread as vancomycin susceptible and that later subsets of this clone acquired vancomycin resistance genes through horizontal gene transfer. Such an event, vancomycin-susceptible spread followed by acquisition of vancomycin resistance, has been reported previously in Spain and Portugal (6, 26). Both of these Iberian clones were typed with MLST but had no relation to CC87 (P. Ruiz-Garbajosa, personal communication). Other characteristics of clone F included frequent hemolysin activity and the lack of gelatinase production. The absence of gelatinase was especially remarkable because this was one of the significant features of the other epidemic clones. Since gelatinase production can decrease rates of horizontal gene transfer (33), the absence of this enzyme may, at least partially, explain frequent acquisition of the different resistance determinants, especially of vanA and vanB gene clusters, by this clone.

Similarly to clone F, PFGE clone C (ST40), the second most prominent clone, did not belong to any of the previously identified hospital-adapted clones or clonal complexes. Previously, this ST was only found in three isolates recovered from a Dutch seal, from a hospitalized patient, and from a healthy volunteer from Spain (29). In this study, clone C (ST40) was found widespread in the country, representing isolates from invasive infections as well as carrier isolates, and was recovered during the whole study period. This may suggest that this clone originated in Poland or its neighboring countries and may, in the near future, emerge also in other countries.

PFGE clone I (ST21) is part of the previously identified CC21, encompassing animal and community surveillance isolates, including VRE, and only a few clinical isolates. This CC was not thought to be of specific relevance for the hospital environment (29). In our study, clone I (ST21) lacked the MDR phenotype, which was generally found among the main epidemic clones, but encompassed three VRE isolates, including the first glycopeptide-resistant E. faecalis isolated in Poland (14).

Two other prevalent clones, PFGE type A (ST6) and PFGE type J (ST9, ST88, and ST142) belonged to the two previously identified hospital-adapted lineages of E. faecalis, CC2 (or the BVE clone) and CC9, respectively (23, 29), with the exception of the clone J isolate with ST142. In contrast to what was previously reported for these clonal complexes, clones A and J described in this study did not represent vancomycin-resistant or ß-lactamase-producing isolates. Clone J represented the highest proportion of isolates carrying the esp gene (92%), while clone A significantly differed from the other epidemic clones by a low percentage of isolates (48%) carrying the esp gene. As the presence of other genes of the putative pathogenicity island was not checked (30), it is possible that esp-negative isolates may still contain parts of the pathogenicity island. This has been reported previously for isolates belonging to the BVE clone (23).

In conclusion, we described in this study the population structure of E. faecalis isolated in Polish hospitals. The high prevalence of five distinct clones, of which only two (CC2 and CC9) represent globally dispersed hospital-related lineages, suggested that at least three clonal complexes (CC87, CC21, and ST40) might be endemic to a restricted geographic region. The lack of information on the population structure of E. faecalis in neighboring countries makes it difficult to speculate about the regional spread of these "local" clones. Local availability of resistance and virulence genes determine to what extent these factors are acquired by the epidemic clones through horizontal gene transfer, which may direct and fine-tune their evolutionary development toward genetic subpopulations fully adapted to the hospital environment. This may explain why MDR characteristics, including vancomycin resistance, hemolytic activity, and presence of the asa1 gene, were found to be statistically significant features for epidemic clones in Poland, while ß-lactamase-producing isolates were not found.


   ACKNOWLEDGMENTS
 
We thank Iza Wasko and Ewa Sadowy for help with the MLST technique and Patricia Ruiz-Garbajosa for fruitful discussions on the manuscript.

This work was supported in part by a grant from the Polish Ministry of Education and Science (no. 2P05D01528).


   FOOTNOTES
 
* Corresponding author. Mailing address: Department of Molecular Microbiology, National Institute of Public Health, Chelmska 30/34, 00-725 Warsaw, Poland. Phone: 48 22 8514388. Fax: 48 22 8412949. E-mail: madziak{at}cls.edu.pl. Back

{triangledown} Published ahead of print on 8 November 2006. Back


   REFERENCES
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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Journal of Clinical Microbiology, January 2007, p. 147-153, Vol. 45, No. 1
0095-1137/07/$08.00+0     doi:10.1128/JCM.01704-06
Copyright © 2007, American Society for Microbiology. All Rights Reserved.



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