ORIGINAL ARTICLE 10.1111/j.1469-0691.2006.01496.x Multilocus sequence typing and analysis of putative virulence factors in vancomycin-resistant and vancomycin-sensitive Enterococcus faecium isolates from Brazil I. L. B. C. Camargo 1, M. S. Gilmore 2 and A. L. C. Darini 1 1 Faculdade de Ciencias Farmaceuticas de Ribeirao Preto, Universidade de São Paulo, São Paulo, Brazil and 2 Schepens Eye Research Institute, Harvard Medical School, Boston, MA, USA ABSTRACT Enterococci are leading causes of hospital-acquired infections that are often difficult to treat because of high-level aminoglycoside and glycopeptide resistance. Vancomycin-resistant enterococci are a global problem, and have been isolated with increasing frequency in hospitals in Brazil. The objective of this study was to determine the genetic relatedness of vancomycin-resistant Enterococcus faecium (VREFM) and vancomycin-sensitive E. faecium (VSEFM) isolated from human infections and faecal sources in Brazil, and to compare these isolates with those from domesticated animals. Isolates (n = 56) were classified by multilocus sequence typing (MLST) and assessed for putative virulence traits. The acm gene was detected in 98% of all isolates. The 56 isolates studied comprised 26 different MLST types. VSEFM isolates from the faeces of pigs were found to be distinct from all human isolates characterised previously by MLST, and were assigned new sequence type (ST) numbers. VREFM isolates were represented by four different STs (ST-114, ST-17, ST-281, ST-50). Among the 26 STs identified in this study, eburst detected three groups of STs with related allelic profiles, and 19 unrelated STs. Among E. faecium isolates from Brazil, the esp gene was restricted to vancomycin-resistant isolates. Furthermore, isolates classified as ST-17 by MLST, an epidemic strain type isolated internationally with the purk-1 gene, were found among VREFM isolates from Brazil that also harboured the esp and hyl genes. Keywords resistance Enterococcus faecium, epidemiology, esp gene, hyl gene, multilocus sequence typing, vancomycin Original Submission: 10 March 2005; Revised Submission: 6 February 2006; Accepted: 27 February 2006 Clin Microbiol Infect 2006; 12: 1123 1130 INTRODUCTION Enterococcus faecium causes a large proportion of human enterococcal infections, which can be extremely difficult to treat because of resistance to glycopeptides and ampicillin, and high-level resistance to aminoglycosides [1]. Six different phenotypes for vancomycin-resistant enterococci (VRE) have been described: VanA, VanB, VanC, VanD, VanE and VanG [2 4]. In Brazil, VRE have recently become endemic in hospitals. The first vancomycin-resistant enterococcal strain identified was a divergent VanD strain of E. faecium isolated in 1996 from a patient with aplastic Corresponding author and reprint requests: A. L. C. Darini, Avenue do Cafe s n, Monte Alegre, Ribeirao Preto, SP 14040-903, Brazil E-mail: aldarini@fcfrp.usp.br anaemia [3,5]. The first documented case of VanA E. faecium infection occurred in Brazil during 1997. The strain was isolated from a case of meningitis in São Paulo [6]. One year later, several vancomycin-resistant E. faecium and Enterococcus faecalis strains were isolated from the same hospital in São Paulo, providing evidence of an outbreak caused by VanA enterococci [7]. A study of these isolates identified several different clones of E. faecium. VRE have also been isolated from different hospitals in São Paulo, indicating interhospital dissemination [8 10]. In addition to antibiotic resistance, putative virulence factors described in vancomycin-resistant E. faecium (VREFM) include a collagen-binding adhesin (acm) and hyaluronidase (hyl) [11,12]. Rice et al. [12] described an open reading frame, designated hyl Efm, which had a deduced aminoacid sequence with significant identity to known Ó 2006 Copyright by the European Society of Clinical Microbiology and Infectious Diseases
1124 Clinical Microbiology and Infection, Volume 12 Number 11, November 2006 hyaluronidase genes. hyl Efm has been detected mostly in multiresistant E. faecium isolates from non-stool cultures from patients in hospitals in the USA [12], but has also been found in an endemic VREFM strain in a survey in Taiwan [13]. A collagen-binding adhesin of E. faecium (Acm), expressed on the surface of the bacterium, was characterised as a new member of the MSCRAMMs (Microbial Surface Components Recognising Adhesin Matrix Molecules) family. Despite the detection of the acm gene in all 32 isolates studied, the Acm phenotype is identifiable only in certain clinical isolates [11]. The attachment of Acm-positive E. faecium to collagen type I has been studied, but a role for Acm in E. faecium pathogenesis has not yet been demonstrated [11]. Gelatinase, aggregation substance and cytolysin are virulence factors found in E. faecalis, although they have also been sought among E. faecium isolates [14 16]. Gelatinase (GelE) is an extra-cellular zinc metallo-endopeptidase that hydrolyses collagen, gelatin and small peptides [17] and plays a role in biofilm development [18 20]. Aggregation substance, encoded by asc10, asa1 and other related genes, represents a family of pheromone-inducible surface proteins that promote aggregation during bacterial conjugation [21]. Cytolysin has bacteriocin activity and is also a haemolytic toxin for human, horse and rabbit erythrocytes [22]. Cytolysin and aggregation substance appears to act synergically to enhance the severity of endocarditis [23]. A virulence factor found in both E. faecalis and E. faecium is enterococcal surface protein (Esp), encoded by the esp gene [15,24,25]. Esp is a surface protein that enhances biofilm formation by E. faecalis in the presence of glucose [26], and is one of the virulence factors encoded by the pathogenicity island that is present in both E. faecalis [27] and E. faecium [28]. Some studies have observed an association between the occurrence of esp and specific variants of the housekeeping gene purk (phosphoribosylaminoimidazole carboxylase ATPase subunit), which is a gene that has been used for typing VREFM [24,25,29,30]. Willems et al. [25] suggested that the purk allele may be useful as a marker for epidemic VREFM genogroups in infection control. Homan et al. [30] have proposed a scheme for characterisation of E. faecium by multilocus sequence typing (MLST). An advantage of MLST for epidemiological investigations and surveillance is that it allows easy comparison of local isolates with all strains contained in an electronic database (http://efaecium.mlst.net/). The eburst program permits analysis of the MLST dataset into non-overlapping groups of related sequence types (STs) or clonal complexes, and then indicates the predicted progenitor and the evolutionary relationship of isolates [31]. Recent studies have shown that VanA E. faecium isolates in Brazil have a number of different pulsed-field gel electrophoresis profiles, indicating that several strains are involved in infection and colonisation of patients [7,8]. However, the carriage of virulence factors and the MLST profiles of E. faecium isolates from Brazil have not been investigated previously. The present study aimed to determine the sequence-based relatedness of isolates from Brazil by MLST, and then to relate these findings to the presence of known virulence traits. MATERIALS AND METHODS Bacteria Fifty-six isolates of E. faecium were investigated in this study: ten vancomycin-sensitive E. faecium isolates that colonised hospitalised patients (VSERM chp) and one infection-derived vancomycin-sensitive isolate from a hospitalised patient (VSEFM ihp) were chosen randomly from the collection of Maschieto et al. [32]; 12 VSEFM isolates that colonised healthy humans (chh), unrelated to the hospital environment, were collected from undergraduate students of the University of São Paulo; ten VSEFM isolates from the faeces of pigs (fp) were collected randomly from two different farms, 300 km apart (Braganca Paulista and Ribeirao Preto); ten vancomycinresistant infection-derived isolates from hospitalised patients (VREFM ihp), and 13 vancomycin-resistant isolates that colonised hospitalised patients (VREFM chp) were obtained from the southern part of Brazil [5,7,8,10]. More details concerning the source of isolates are shown in Table 1. All isolates were tested for high-level resistance to aminoglycosides on plates of brain heart infusion agar (Difco, Detroit, MI, USA) containing gentamicin (500 mg L) or streptomycin (2000 mg L). MICs of vancomycin, quinupristin dalfopristin and penicillin were determined using the CLSI (NCCLS) agar dilution method [33]. PCR was used to determine the genotype (vana, vanb, vand or vanc-1 vanc- 2), as described previously [34]. MLST scheme Internal fragments of seven housekeeping genes were amplified with specific primers, using reaction conditions described previously [30]. The genes amplified were adk, atpa, ddl, gyd, gdh, purk and psts. All PCR-amplified fragments were sequenced, using both forward and reverse primers and the
Camargo et al. MLST and virulence factors in E. faecium 1125 Table 1. Phylogenetic characteristics, virulence factors, multilocus sequence typing (MLST) and pulsed-field gel electrophoresis (PFGE) type for isolates of Enterococcus faecium Strains Alleles E. faecium Source Date Susceptibility profile P Q-D HG HS + Virulence factors atpa ddl gdh purk gyd psts adk ST PFGE VSfp 4 RP 2003 S R S S acm 11 8 10 8 20 9 6 229* ND VSfp 10 RP 2003 S R S S acm 36 8 10 8 20* 9 6 222* ND VSfp 6 BP 2003 S R S S acm 36 8 10 8 20* 9 6 222* ND VSfp 5 RP 2003 S R S S acm 36 8 10 8 20* 9 6 222* ND VSchp 21 HCFMRP 2001 S S R R acm 13 21* 8 17 10 27 6 225* ND VSihp JF9 Lemos-MG 2003 R R R S acm 13 8 8 8 6 10 6 94 ND VSchh 04A RP 2004 S R S S acm 13 8 8 8 6 10 6 94 ND VSchh 18A RP 2004 S R S S acm 13 8 8 8 6 27 6 178 ND VSchh 13C RP 2004 S R S S acm 13 8 8 8 6 27 11 213* ND VSchh 05A RP 2004 S R S S acm 13 8 8 8 6 27 11 213* ND VSfp 7 BP 2003 S I S S acm 13 8 8 23 6 10 11 218* ND VSfp 17 RP 2003 S I S S acm 5 13 26 7 10 6 6 221* ND VSfp 12 RP 2003 S R S S acm 5 13 26* 17 10 26 6 226* ND VSchp 23 HCFMRP 2001 S S S S acm 5 13 9 15 8 37 6 163 ND VSchp 22 HCFMRP 2001 S S S R acm 5 13 9 15 8 37 6 163 ND VSchp 20 HCFMRP 2001 S S S S acm 5 13 9 15 8 37 6 163 ND VSchp 3 HCFMRP 2001 S I S S acm 5 13 9 15 8 37 6 163 ND VSchp 5 HCFMRP 2001 S I S S acm 5 13 9 15 8 37 6 163 ND VSfp 11 RP 2003 S R S S acm 10 20* 9 17 17 19 6 223* ND VSchp 9 HCFMRP 2001 S S S S acm 5 13 18 17 17 19 6 211* ND VSchh 23 RP 2004 S R S S acm 36* 13 18 17 10 41* 14* 224* ND VSchp 18 HCFMRP 2001 S S S S acm 35* 3 25* 2 1 1 1 220* ND VSchp 14 HCFMRP 2001 S S S S acm 2 3 1 2 1 1 1 22 ND VSchp 8 HCFMRP 2001 S S S S acm 2 3 1 2 1 1 1 22 ND VSchh 15B RP 2004 S S S S acm 2 3 1 2 1 1 1 22 ND VSchh 08A RP 2004 S R S S acm 2 3 1 2 1 1 1 22 ND VSchh 06A RP 2004 S S S S acm 2 3 2 2 1 1 1 214* ND VSfp 15 RP 2003 S I S S acm 25 3 1 2 10 10 1 219* ND VRihp 16 CSV-RJ 2001 R R R R acm esp hyl 1 1 1 1 1 1 1 17 F VRihp 17 CSV-RJ 2002 R R R R acm esp hyl 1 1 1 1 1 1 1 17 F VRchp 15 CSSM-SP 1998 R R R S acm hyl esp 1 1 1 1 1 1 1 17 C VRihp 13 CSSM-SP 1998 R R R S acm esp hyl 1 1 1 1 1 1 1 17 C VRihp 22 HCSP 1999 R R R R acm 15 1 6 6 1 1 1 50 F VSchh 16B RP 2004 S R S S acm 3 7 3 6 1 1 1 215* ND VSchh 02A RP 2004 S S S S acm 15 7 5 7 1 1 1 212* ND VSfp 1 BP 2003 S R S R acm 21 2 12 9 1 1 5 217* ND VSchh 25C RP 2004 S R S S None 8 4 12 9 1 1 5 130 ND VSchh 12C RP 2004 S R S S acm 15 4 12 6 1 1 5 216* ND VRchp 24 CSSM-SP 1998 R R R S acm esp 5 3 21 18 6 32 3 114 ND VRchp 14 CSSM-SP 1998 R R R S acm esp 5 3 21 18 6 32 3 114 C VRchp 12 CSSM-SP 1998 R R R S acm 5 3 21 18 6 32 3 114 C VRchp 11 CSSM-SP 1998 R R R S acm 5 3 21 18 6 32 3 114 C VRchp 10 CSSM-SP 1998 R R R S acm esp 5 3 21 18 6 32 3 114 C VRchp 9 CSSM-SP 1998 R R R S acm 5 3 21 18 6 32 3 114 C VRchp 8 CSSM-SP 1998 R R R S acm 5 3 21 18 6 32 3 114 C VRchp 7 CSSM-SP 1998 R R R S acm 5 3 21 18 6 32 3 114 C VRchp 6 CSSM-SP 1998 R R R S acm esp 5 3 21 18 6 32 3 114 C VRchp 5 CSSM-SP 1998 R R R R acm 5 3 21 18 6 32 3 114 C VRchp 4 CSSM-SP 1998 R R R S acm 5 3 21 18 6 32 3 114 G VRchp 3 CSSM-SP 1998 R R R S acm 5 3 21 18 6 32 3 114 G VRihp 23 HSPSP 1999 R R R S acm esp 5 3 21 18 6 32 3 114 C VRihp 19 CSSM-SP 1999 R R R S acm esp 5 3 21 18 6 32 3 114 C VRihp 18 CSSM-SP 1999 R R S R acm esp 5 3 21 18 6 32 3 114 ND VRihp 2 CSSM-SP 1998 R R R S acm esp 5 3 21 18 6 32 3 114 C VRihp 1 CSSM-SP 1998 R R R S acm esp 5 3 21 18 6 32 3 114 C VRihp 25 HCC-PR 1996 R R R R acm 9 24* 1 18 1 1 3 281* ND P Q-D HG HS + represents the tested drugs: P, penicillin; Q-D, quinupristin dalfopristin; HG, high-level gentamicin resistance; HS, high-level streptomycin resistance. VS, vancomycin-susceptible; VR, vancomycin-resistant; RP, Ribeirao Preto; BP, Braganca Paulista; HCFMRP, Hospital de Clinicas da Faculdade de Medicina de Ribeirao Preto, Lemos (Minas Gerais), ; CVS-RJ, Clinica Sao Vicente (Rio de Janeiro); CSSM-SP, Casa de Saude Santa Marcelina (Sao Paulo); HCSP, Hospital Sâo Paulo; HSPSP, Hospital do Servidor Público de São Paulo; HCC-PR, Hospital das Clínicas de Curitiba, Parana; R, resistant; S, sensitive; I, intermediately-resistant; ND, not determined. The strains that possess the esp, acm and hyl genes are in bold type. *A new allele or sequence type (ST). ABI PRISM Big Dye Cycle Sequencing Ready Reaction (PE Applied Biosystems, Warrington, UK), and were analysed using an ABI 3100 sequencer (PE Applied Biosystems). In order to determine single-locus variants (SLVs) within groups of related isolates (clonal complexes), and to obtain a population snapshot, the eburst v.2 program (http://efaecium.mlst.net/) was used [31]. Genotypic test for virulence traits Genomic DNA was extracted by detergent lysis [35], and then 200 ng of each DNA sample was bound to a Zeta-Probe nylon membrane (Bio-Rad, Hercules, CA, USA), using a 96-well Bio-Dot microfiltration apparatus (Bio-Rad). DNA was fixed to the membrane using an ultraviolet multilinker (UVC 515;
1126 Clinical Microbiology and Infection, Volume 12 Number 11, November 2006 Ultra-Lum, Carson, CA, USA). Cytolysin (cyll L, cyll S, cyla), esp, gele, acm and hyl genes were detected by hybridisation with specific probes prepared by PCR using the primers listed in Table 2. Aliquots (50 ng) of each probe were denatured before labelling with [a- 32 P]dCTP using the RadPrime DNA Labeling System (Invitrogen, Carlsbad, CA, USA), and were then hybridised with the membranes overnight at 65 C. The membranes were then washed twice with 2 SSPE (1 SSPE is 180 mm NaCl, 10 mm NaH 2 PO 4, 1 mm EDTA, ph 7.4) plus SDS 0.1% w v for 10 min at room temperature, and then washed once with 1 SSPE plus SDS 0.1% w v for 15 min at 65 C. The membranes were exposed to BioMax MR-1 Film for 18 24 h. The following strains were used as positive controls for each gene: E. faecium TX0016 (hyl Efm gene) [12]; E. faecium TX2555 (acm) [12]; E. faecalis MMH594 (esp and cytolysin genes) [36]; and E. faecalis OG1RF (gele) [37]. Phenotypic tests for virulence traits Cytolysin production was detected by observing b-haemolysis surrounding colonies on brain heart infusion agar supplemented with human erythrocytes 5% v v [22]. Gelatinase production was detected on plates made with Todd Hewitt broth (Difco) containing gelatin 30 g L, and solidified with agar. A turbid zone around the colony after incubation for 24 h at 37 C and 4 h at 4 C was measured as an index of production of gelatinase [17]. RESULTS Susceptibility profiles and virulence factors Susceptibility profiles and virulence factors determined for each isolate are listed in Table 1. All VRE ST-17, all VREFM ihp ST-114, and four of 12 VREFM chp ST-114 isolates carried the esp gene (Table 1). Fifty-five (98%) isolates carried the acm gene, with the exception being VSEFM chh 25C (ST-130), which carried none of the virulence genes tested. The hyaluronidase gene (hyl) was detected in only four isolates (VREFM chp 15, VREFM ihp 17, VREFM ihp 16 and VREFM ihp 13). All hyl-positive isolates were VRE and belonged to ST-17. The cytolysin genes and the gele gene were not detected in any isolate in this study, although isolate VREFM ihp 17 showed apparent gelatinase activity. MLST Each housekeeping gene sequenced was assigned an allelic number corresponding to sequences already in the database, or was assigned a new number by the curator of the database (http://efaecium.mlst.net/). Based on the seven allelic numbers assigned, each isolate was allocated to a specific ST. The ten VSEFM fp isolates represented eight new STs, and eight of 12 VSEFM chh isolates were allocated to seven new STs. The new allelic numbers and new STs identified in this study are indicated by asterisks in Table 1. In total, 26 different STs were identified among the 56 isolates studied. All purk-1 alleles occurred in ST-17 isolates. Among the 26 STs, eburst identified three groups of related isolates and 19 ungrouped isolates. The first group comprised five isolates belonging to three STs, ST-178, ST-94 and ST-213. Only one isolate (VSEFM chh 18A) belonged to ST-178, while ST-94 (VSEFM chh 04 and VSEFM iph JF9) and ST-213 (VSEFM chh 05A Gene (GenBank accession number) Cytolysin cylll (L37110) cyll S (L37110) cyla (L37110) Enterococcal surface protein esp a (AY322150) Hyaluronidase hyl efm (AF544400) Adhesin acm (AY135217) Gelatinase gele (M37185) Primers F5 -AACTAAGTGTTGAGGAAATG R5 - AAAGACACAACTACAGTTAC F5 -AGAACTTGTTGGTCCTTC R5 -GCTGAAAATAATGCACCTAC F5 -ACAGGTTATGCATCAGATCT R5 -AATTCACTCTTGGAGCAATC 14F 5 -AGATTTCATCTTTGATTC TTGG 12R 5 -AATTGATTCTTTAGCATCTGG F5 -CGATGCGCAAGAATTAGACA R5 -CATGATTGGACAACCGAGTG F5 -GGCCAGAAACGTAACCGATA F5 -CGCTGGGGAAATCTTGTAAA F5 -AATTGCTTTACACGGAACGG R5 -GAGCCATGGTTTCTGGTTGT Amplicon size (bp) 159 52 134 52 507 52 500 50 308 52 353 52 548 52 Annealing temperature ( C) Table 2. Sequences of primers designed to amplify probes for virulence factor genes of Enterococcus faecium a The sequences of the E. faecium esp primers were provided by N. Shankar, College of Pharmacy, Oklahoma University Health Sciences Center, OK, USA.
Camargo et al. MLST and virulence factors in E. faecium 1127 Fig. 1. A, eburst diagram of the population analysis of Enterococcus faecium isolates from Brazil. B, eburst diagram of the analysis of the sequence types (STs) of the Brazilian isolates and the entire public E. faecium MLST database (all STs assigned). The size of the dots depends on the number of strains. Grey dots indicate the progenitor of each group. The numbers correspond to the STs. The dashedcircled STs are those found in this study, with the STs forming clonal complexes being linked by lines. The arrow indicates ST-17, one of the STs determined among the vancomycinresistant enterococci from Brazil. and VSEFM chh 13C) are double-locus variants of each other, and both are SLVs of ST-178 (Fig. 1A). When the STs found in this study were compared with all STs registered in the MLST database, it was observed that ST-94 was related to three additional SLV STs (ST-60, ST-61 and ST-40). Therefore, ST-94 is considered to be a founder of group number 1 (Fig. 1B). The second group shows that ST-22 and ST-214 are SLVs of each other. Three isolates belonged to ST-22 (VSEFM chh 08A, VSEFM chp 14 and VSEFM chp 8), and one isolate was identified as ST-214 (VSEFM chh 06A). When compared with the public MLST database, ST-22 appears to be the founder of a large group of STs, and ST-214 has ST-36 as an SLV (Fig. 1B). The third group consisted of ST-222 and ST- 229, and this group was not connected to any ST identified previously in the database. Only one isolate was typed as ST-229 (VSEFM fp 4), and three isolates belonged to ST-222 (VSEFM fp 5, VSEFM fp 6 and VSEFM fp 10) (Fig. 1). All remaining isolates were ungrouped, with no SLVs identified from this study. However, when compared with the database, eburst identified a number of SLV groups, and identified possible progenitors. Thus, ST-217 (VSEFM fp 1) formed a group with ST-44; ST-212 formed a group with ST-27 and ST-28; and ST-17 was part of a group with several related STs (ST-78, ST-209, ST-42 and ST-16) (Fig. 1). Among the 23 vancomycin-resistant enterococcal isolates from Brazil, four were ST-17, 17 were ST-114 (including VREFM ihp and VREFM chp), one was ST-281 (VREFM ihp 25, a VanD variant), and one was ST- 50. DISCUSSION Genes for the enterococcal virulence factors cytolysin and gelatinase were not detected among the 56 E. faecium isolates studied, consistent with the findings of other studies [38, 39]. Interestingly, one isolate was positive for gelatinase activity, but
1128 Clinical Microbiology and Infection, Volume 12 Number 11, November 2006 no gene was detected. This may indicate the rare occurrence of a divergent gelatinase gene in some strains of E. faecium, or alternatively, the presence of an additional protease that yields cleavage products which behave similarly to those generated by E. faecalis gelatinase in this test. The esp gene was restricted to vancomycinresistant isolates, with 13 (56%) of the VREFM isolates analysed being positive for esp. Five of 13 esp-positive strains were from VREFM colonising hospitalised patients (chp), and eight of 13 were infection isolates, representing 80% of the VREFM ihp group. The hyl gene was rare among VREFM isolates from Brazil, being found in only 17% of VREFM isolates in this study. All hyl-positive isolates were also esp-positive. Rice et al. [12] detected hyl in 38% of non-stool VREFM isolates and in 33% of stool VREFM isolates from the USA. Vankerckoven et al. [16] detected esp in 65% and hyl in 17% of 271 isolates from Europe, with hyl being detected in 27% of VREFM clinical isolates, compared with only 14% of the faecal isolates. Similarly, esp was detected in 92% of clinical and 73% of faecal VREFM isolates, and the prevalence of esp was higher in VREFM than in VSEFM (77% vs. 53%, respectively) [16]. In Brazil, none of the VSEFM isolates were esp-positive. The present study included isolates from 1998 (after the first outbreak of VRE in Brazil) and from 1999 (collected during a survey in the same hospital). All isolates from 1999 were esp-positive, as were some of the isolates from 1998. It is known that esp can be deleted from the pathogenicity island of E. faecalis at a high frequency [27], but the mechanism of its acquisition is unknown. Oancea et al. [40] demonstrated that the esp gene is transferable by conjugation among enterococcal isolates. Because of the temporal relationship between related isolates that were esp-positive in the present study, it seems that esp may have been acquired by E. faecium during the first outbreak. MLST analysis showed that VSEFM isolates from the faeces of pigs in Brazil are distinct from all human isolates studied previously. All VREFM isolates studied could be assigned to four different STs (ST-114, ST-17, ST-281 and ST-50). Most isolates from the first outbreak in Brazil were classified as ST-114, and only two were ST-17. However, the VREFM iph 23 isolate, from a different hospital in the city of São Paulo, was also ST-114, which indicates possible inter-hospital dissemination. Two isolates from 1999 (São Paulo city), and two from Rio de Janeiro (one in 2000 and the other in 2001), were allocated to ST-17. The housekeeping gene purk allele 1 (purk-1) is present in ST-17, and is linked to the presence of esp. All ST-17 isolates possessed the hyl gene, which was not detected in any of the other isolates. The E. faecium MLST database contains several VRE registered as ST-17 from different geographical areas, namely the UK, the USA, France and The Netherlands. Willems et al. [25] reported several isolates that harboured purk-1, and noted that all were also esp-positive. More recently, Bonora et al. [29] identified purk-1 in all epidemic VREFM isolates studied between 2000 and 2001 in northern Italy. All were esp-positive and clustered in ST-78, an SLV of ST-17. Werner et al. [41] described a profile related to ST-17 in a clinical isolate from Germany, and Willems et al. [42] considered that ST-17 was an example of cumulative evolutionary processes that improved the relative fitness of bacteria in hospital environments. These observations indicate that VREFM ST-17 and its SLVs are distributed unusually widely. The presence of four ST-17 isolates in the present study reinforces the importance of surveillance programmes to avoid the spread of this lineage in hospitals. ACKNOWLEDGEMENTS The authors thank I. C. V. Palazzo, J. Ferreira and A. S. Bagdahyan for technical assistance. H. S. Sadder, L. Dalla Costa, R. Cobo Zanella and P. Del Peloso are thanked for providing some of the isolates included in this study. The work was supported by Fundação de Amparo a Pesquisa do Estado de São Paulo (FAPESP, 02 11518-6) and Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES, BEX1684 03-5). REFERENCES 1. Cereda RF, Pignatari AC, Hashimoto A, Sader HS. In vitro antimicrobial activity against enterococci isolated in a university hospital in São Paulo, Brazil. Brazil J Infect Dis 1997; 1: 83 90. 2. Cetinkaya Y, Falk P, Mayhall CG. Vancomycin-resistant enterococci. Clin Microbiol Rev 2000; 13: 686 707. 3. Dalla Costa LM, Reynolds PE, Souza HAPHM, Souza DC, Palepou MFI, Woodford N. Characterization of a divergent vand-type resistance element from the first glycopeptide-resistant strain of Enterococcus faecium isolated in Brazil. Antimicrob Agents Chemother 2000; 44: 3444 3446.
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