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Appl Environ Microbiol. Jul 2009; 75(14): 4904–4908.
Published online May 15, 2009. doi:  10.1128/AEM.02945-08
PMCID: PMC2708421

Dispersion of Multidrug-Resistant Enterococcus faecium Isolates Belonging to Major Clonal Complexes in Different Portuguese Settings[down-pointing small open triangle]

Abstract

The population structure of 56 Enterococcus faecium isolates selected from a collection of enterococci from humans, animals, and the environment in Portugal (1997 to 2007) was analyzed by multilocus sequence typing. We identified 41 sequence types clustering into CC17, CC5, CC9, CC22 and CC94, all clonal lineages comprising isolates from different hosts. Our findings highlight the role of community-associated hosts as reservoirs of enterococci able to cause human infections.

Enterococci are organisms widely distributed in nature that are recognized as one of the leading causes of nosocomial infections (1, 20). Despite their ubiquity, the population structure of Enterococcus faecium comprises a diversity of sequence types, with overrepresentation of particular clonal complexes (CCs) associated with swine (CC5), poultry (CC9), veal calf (CC1), or humans (CC17, CC22, and CC94) (1, 5, 15, 17, 30, 37). Particular host-specific human clonal lineages are considered high-risk CCs (HiRCCs) since they are recovered mostly from hospitalized patients (17). CC17 is the only recognized E. faecium HiRCC, nowadays globally disseminated, which has been sporadically isolated from nonhospitalized humans (3, 8-10). Different studies have analyzed the population structure of local enterococci, but they are focused mainly on clinical strains with a specific phenotype, such as vancomycin resistance, generally isolated in a short temporal frame (2, 7, 16, 18, 27, 29, 36).

We analyzed 56 representative E. faecium isolates from a Portuguese collection comprising 1,700 enterococci from different geographical locations (1997 to 2007), some included in previous studies (22-26). They were recovered from hospitalized patients in five hospitals of different regions (n = 20), swine excrement (n = 6) and environmental piggery samples (n = 16), retail poultry (n = 6) of four national commercial brands, feces from healthy humans (n = 5), hospital wastewater (n = 2), and the estuary of the river Douro (n = 1). Susceptibility testing with 15 antibiotics was performed following CLSI guidelines (6). Species identification and detection of genes encoding vancomycin resistance were performed by using multiplex PCR (11). Clonal relatedness was established by pulsed-field gel electrophoresis (PFGE) and multilocus sequence typing (MLST) as described previously (15, 22, 28). Sequence types (STs) differing in one or two of the seven housekeeping genes were considered single-locus variants (SLVs) and double-locus variants, respectively. Clusters of related STs differing in ≤2 loci that were thought to be descendants from a common ancestor were grouped into CCs by using the eBURST software program (12, 13) (http://www.mlst.net). Genes coding for virulence factors such as enterococcal surface protein (encoded by esp), hyaluronidase (encoded by hyl), cytolysin (encoded by cyl), gelatinase (encoded by gel), and aggregation substance (encoded by agg) and the backbone structure of Tn1546 harbored by vancomycin-resistant E. faecium (VRE) were investigated by using PCR (21, 34, 38).

The isolates studied (35 VRE isolates and 21 vancomycin-susceptible E. faecium [VSE] isolates) corresponded to 49 PFGE types and 41 different STs, including 24 newly identified STs, which clustered into CC5, CC9, CC17, CC22, and CC94 (Fig. (Fig.11 and Table Table11).

FIG. 1.
Clustering of 41 E. faecium STs by use of eBURST. STs are indicated by colored circles, representing 56 isolates from the present study, with 439 MLST profiles representing 1,358 E. faecium isolates from the database (www.mlst.net). Each ST is represented ...
TABLE 1.
Epidemiological features of Enterococcus faecium isolates from human, swine, poultry, and environmental samples in Portugal

CC17 was identified in 24 isolates (18 VRE isolates and 6 VSE isolates) from hospitals, healthy volunteers, swine, piggeries, and the environment in different regions from 1997 to 2007. In agreement with other studies, the CC17 meroclone consisted of a high diversity of STs (ST16, ST18, ST78, ST80, ST125, ST132, ST280, ST368, ST369, ST390, ST393, ST430, and ST431), particularly enriched by ST18 (7, 18, 29, 36, 37) (Table (Table1).1). All CC17 isolates were resistant to ampicillin and erythromycin, and most of them were associated with resistance to glycopeptides (75%) and ciprofloxacin (71%) and a high level of resistance to kanamycin (58%) and streptomycin (50%). esp was detected among isolates from most sources (38%), while hyl (17%) was mostly associated with the clinical setting. The detection of CC17 among nonclinical sources might indicate a hospital input of community strains with different genetic contents besides contamination from the hospital setting and could explain its high prevalence and global spread (3, 8-10).

Clonal dissemination and maintenance of particular resistant E. faecium strains among food animals have previously been documented, but MLST data are absent in almost all of these works (14, 19, 26, 32, 33). Although swine and poultry are considered potential reservoirs of pathogenic E. faecium, transmission to humans has been demonstrated in a few cases and is always linked to persons in close contact with farms (4, 19, 32, 33). CC5 comprised isolates of ST5 (n = 1), ST6 (n = 1), ST150 (n = 1), and ST185 (n = 2). Isolates classified as ST5 and ST185, which is an SLV of ST5, were recovered from two hospitals over 2 years (22) and from soil of an extensive piggery, respectively. They were clonally related to the ST6 isolate which represents the VRE strain “A” widespread among swine of four European countries since 1997 (26). CC5 isolates did not contain the tested putative virulence factors and exhibited various resistance phenotypes (Table (Table11).

CC9 included eight clonally unrelated isolates from community (ST148), swine (ST433 and ST437), and poultry (ST236, ST394, ST395, and ST405), generally resistant to ciprofloxacin (88%) and vancomycin (62%). Most of the CC9 poultry strains were persistently recovered from commercial brands of retail poultry products over the years and corresponded to different ampicillin-resistant VRE or VSE strains (23). All isolates lacked the virulence genes tested. CC9 strains associated with purK6 (ST236 and their SLVs, ST395 and ST405) or purK3 (ST148) have also been detected in chickens from Korea and Spain, respectively, which might mirror particular globally disseminated poultry lineages (http://efaecium.mlst.net/).

CC22 (ST32, ST392, ST435, and ST436) comprised isolates mostly resistant to tetracycline and ciprofloxacin and highly resistant to kanamycin from different piggeries and from human feces. This genogroup was recently associated with isolates from human origin (5, 17), with this study representing the first description of isolates among swine.

The remaining STs were recovered from humans and animals in nine different locations and corresponded to ST30, ST89, ST190, ST366, ST367, ST391, ST404, ST406, ST428 (CC94), ST429, ST432, ST434, and ST443, mostly identified as singletons. It is of note that ST366/ST367 and ST391 from hospitals are double-locus variants of STs belonging to CC17 and were resistant to ampicillin and contained esp. Also, ST443 from swine harbored purK1, which is associated with CC17, and contained the esp and hyl genes.

Tn1546 among CC17 isolates was highly diverse, with a number of variants containing ISEf1. Tn1546 types A, D, and S, lacking insertion sequences and associated with animals, were detected in the non-CC17 VRE isolates CC5 and CC9 (21, 38; this study).

In summary, this is the first study describing the population structure of E. faecium from different origins and locations in Portugal, one of the European nations with the highest VRE rates in the nosocomial setting (see the EARSS Annual Report 2006 [http://www.rivm.nl/earss]) (35). Our results pose new insights into E. faecium host specificity, since all the identified clonal complexes comprised isolates from different host origins. The frequent recovery from the community of HiRCC17 and the emergence of CC5 in the hospital setting highlight the role of nonhospital hosts as possible reservoirs of pathogenic enterococci. This community reservoir of multidrug-resistant strains able to cause human infections might contribute to their spread in hospitals and counteract any containment measure at the hospital level.

Acknowledgments

We are indebted to Rob J. Willems (University Utrecht, The Netherlands) for excellent scientific support, assignment of new MLST types, and allocation of new types to clonal complexes (http://www.mlst.net). We are grateful to Jorge M. A. Oliveira (University Porto, Portugal) for his assistance with the eBURST figure.

Ana Freitas was funded by a fellowship from Fundação para a Ciência e Tecnologia (SFRH/BD/24604/2005). This work was funded by grants from Fundação para a Ciência e Tecnologia (POCI/SAL/61385/2004 and POCI/AMB/61814/2004) of Portugal, from Ministerio de Educación y Ciencia of Spain, Programa Acciones Integradas Hispano-Portuguesas (H2004-0092), from the Fondo de Investigaciones Sanitarias, Ministerio de Sanidad (PI 06/1141), and from the European Union Sixth Framework Programme (LSHE-CT-2007-037410).

Footnotes

[down-pointing small open triangle]Published ahead of print on 15 May 2009.

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