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Genetic features and molecular epidemiology of Enterococcus faecium isolated in two university hospitals in Brazil
Diagnostic Microbiology and Infectious Disease 74 (2012) 267–271
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Diagnostic Microbiology and Infectious Disease j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / d i a g m i c r o b i o
Genetic features and molecular epidemiology of Enterococcus faecium isolated in two university hospitals in Brazil☆ Leila Priscilla Pinheiro da Silva a, André Pitondo-Silva a, Roberto Martinez b, Ana Lúcia da Costa Darini a,⁎ a b
Departamento de Análises Clínicas, Toxicológicas e Bromatológicas, Faculdade de Ciências Farmacêuticas de Ribeirão Preto, Universidade de São Paulo, São Paulo, Brazil Departamento de Clínica Médica, Faculdade de Medicina de Ribeirão Preto, Universidade de São Paulo, São Paulo, Brazil
a r t i c l e
i n f o
Article history: Received 28 May 2012 Accepted 30 July 2012 Available online 6 September 2012 Keywords: CC17 E. faecium MLST PFGE Tn1546 Virulence factors VREs
a b s t r a c t The global emergence of vancomycin-resistant Enterococcus faecium (VREfm) has been characterized by a clonal spread of strains belonging to clonal complex 17 (CC17). Genetic features and clonal relationships of 53 VREfm isolated from patients in 2 hospitals in Ribeirao Preto, São Paulo, Brazil, during 2005–2010 were determined as a contribution to the Brazilian evolutionary history of these nosocomial pathogens. All isolates were daptomycin susceptible, vancomycin-resistant, and had the vanA gene. The predominant virulence genes were acm and esp. Only 5 VREfm isolated in 2005–2006 had intact Tn1546, while 81% showed Tn1546 with deleted left extremity and insertion of IS1251 between the vanS and vanH genes. Multilocus sequence typing analysis permitted the identification of 9 different sequence types (STs), with 5 being new ones (656, 657, 658, 659, and 660). Predominant STs were ST412 and ST478, all belonging to CC17, except ST658. This is the first report of the ST78 in Brazil. © 2012 Elsevier Inc. All rights reserved.
1. Introduction Enterococci, formerly considered as part of the microbiota in the gastrointestinal tract of humans and animals, have emerged as important nosocomial pathogens in the last 2 decades. Enterococcus faecalis and E. faecium are the species most frequently isolated from human clinical specimens. A recent significant increase in the incidence of nosocomial infections caused by E. faecium has been reported (Top et al., 2008). The change in the epidemiology of enterococcal infections has been attributed to the presence of a genetic lineage of E. faecium, inserted in clonal complex 17 (CC17) as determined by multilocus sequence typing (MLST), that has spread globally (Willems et al., 2005). This lineage is characterized by resistance to ampicillin and quinolone and by the presence of putative virulence genes such as esp and hyl (Freitas et al., 2010; Willems et al., 2001). Although it is recognized that enterococci cause serious infections such as endocarditis and sepsis, they are generally considered lowvirulence microorganisms. Virulence factors in E. faecium are not well known, but some have been pointed out as possible contributors: aggregation substance (agg gene), cytolysin (cyl gene), collagen
☆ This study was supported by the Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP, grants 2010/01463-6 and 2011/18574-8). The funding source had no role in the study design; in the collection, analysis, and interpretation of data; in the writing of the manuscript; or in the decision to submit the manuscript for publication. ⁎ Corresponding author. Tel.: +55-1636024290; fax: +55-1636024725. E-mail address: [email protected] (A.L. da Costa Darini). 0732-8893/$ – see front matter © 2012 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.diagmicrobio.2012.07.012
binding protein (acm gene), enterococcal surface protein (espfm gene), gelatinase (gel gene), and hyaluronidase (hyl gene) (Billsstrom et al., 2008; Top et al., 2008). Currently, 7 phenotypes of acquired vancomycin resistance (types VanA/B/D/E/G/L/M) are known in enterococci, but VanA and VanB are reported most frequently (Werner et al., 2008). The VanA phenotype is characterized by the presence of intact Tn1546 or VanA element, which may result from mutations, deletions, and/or insertion sequences. Therefore, molecular characterization of the Tn1546 structure is important because it provides epidemiologic information about the spread of resistance determinants by horizontal transfer and also about transmission of vancomycin resistance between different ecologic niches (López et al., 2010). In Brazil, vancomycin-resistant Enterococcus faecium (VREfm) with vanD phenotype was first described in 1996 in the city of Curitiba, State of Parana, whereas VanA E. faecium was isolated in 1997 from a meningitis case in São Paulo, SP (Dalla Costa et al. 2000; Zanella et al., 1999). Afterwards, vancomycin-resistant enterococci (VRE) were detected at hospitals in various cities in the country, including São Paulo, Marilia, Campinas, Rio de Janeiro, Uberlândia, Porto Alegre, and others (Camargo et al., 2006; D'azevedo et al., 2000; Palazzo et al., 2011). The recent enterococci epidemiology in Brazil has shown great changes, but very limited data are available regarding clonal profiles characterized by MLST. This study was designed to determine the enterococcal genetic features of isolates from 2 hospitals in the city of Ribeirao Preto, State of São Paulo, as a contribution to the evolutionary history of these nosocomial pathogens in Brazil.
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2. Materials and methods Of the 53 VREfm isolates included in this study, 43 were from different patients hospitalized at the University Hospital of the Faculty of Medicine of Ribeirao Preto, University of São Paulo (HCFMRP-USP) between September 2008 and September 2010. Of these, 29 were isolated from surveillance cultures (rectal swabs), 13 from different infected patients (urine, 6; blood, 3; ear, 1; surgical wounds, 2; and peritoneal cavity, 1), and only 1 VREfm from an unknown origin. Five VREfm (HC-61, HC-62, HC-63, HC-64, and HC-65), which were isolated at the same hospital between 2005 and 2006, were also included. Of the remaining 5 VREfm isolated in another university hospital, from July 2009 to mid-2010, 4 were from infections (urine, 2; bile, 1; and abdominal secretion, 1) and 1 from an unknown origin. The identification and antimicrobial susceptibility of VREs isolated at HCFMRP-USP were determined by the Vitek2® system (bioMérieux Vitek Systems, Hazelwood, MO, USA). Conventional biochemical tests were used to identify the VREs from the other hospital. Minimum inhibitory concentrations (MICs) of vancomycin and daptomycin were determined by E-test® (AB Biodisk, Solna, Sweden), and MIC values of susceptibility to both antibiotics were based on the guidelines issued by the Clinical and Laboratory Standards Institute (CLSI, 2009). The total genomic DNA of the isolates was extracted as described by Bolano et al. (2001) with some modifications. Briefly, cells recovered by centrifugation from 5 mL of Brain Heart Infusion broth of an 18- to 20-h bacterial culture, washed once with saline, and centrifuged during 5 min at 2.655 × g were mixed with glass beads and lysis buffer and kept under stirring for 10 min. Addition of phenol chloroform was followed by centrifugation for 20 min at 15.294 × g. After the addition of absolute isopropanol, the tubes were maintained at −20 °C for 18–20 h. DNA was purified and recovered by ethanol precipitation. The genomic DNA obtained was used as template in polymerase chain reactions (PCR), which were performed to confirm species and to investigate the presence of vanA, vanB, and vanC genes in the VREs investigated (Dutka-Malen et al., 1995; Woodford et al., 1993). Amplification of VanA elements was by the long-PCR 1 reaction using primer (P1) complementary to the inverted repeats flanking the transposon. Long-PCR 2/3 reaction was used for amplification of vanRSHAX genes, using specific primers (P2 and P3). Long PCRs were performed using a commercial kit (Long PCR Enzyme Mix, Fermentas, Foster City, CA, USA) according to the manufacturer's recommendations. DdeI restriction endonuclease was used to digest the enterococci vanRSHAX gene clusters that produced long-PCR 2/3 products different from the prototype Tn1546. The long PCR and restriction enzyme cleavage products were analyzed in 1% agarose gel electrophoresis and visualized with ethidium bromide under ultraviolet light (Palepou et al., 1998). PCR overlapping was performed for all enterococci showing Tn1546 different from the prototype (Woodford and Stigter, 1998). The IS1251 insertion element was investigated by PCR using primers described in the literature, and the MegaBace™ 1000 DNA Sequencing System (GE Healthcare, Chalfont St Giles, Buckinghamshire, UK) was used to sequence the PCR products of some isolates to determine the exact location of the insertion sequence in the Tn1546 (Camargo et al., 2004). The following virulence genes were analyzed by PCR: genes codifying collagen-adhesin protein (acm), aggregation substance (agg), cytolysin (cylLL, cylLS, cylA), enterococcal surface protein (esp), gelatinase (gel), and hyaluronidase (hyl). Positive and negative controls from our collection were used in all PCR assays (Camargo et al., 2006). Pulsed-field gel electrophoresis (PFGE) was performed after SmaI digestion of genomic DNA of the 53 VREfm in a Gene Navigator apparatus (Amersham, Uppsala, Sweden), following the protocol described by Kaulfmann (1998). Among all 53 E. faecium in the study, 31 VREfm selected according to PFGE results were characterized by MLST. Those with different
pulsotypes were selected, but others with the same pulsotype were also chosen based on some characteristics as date of isolation, type of clinical specimens, isolation hospital, and hospital sector. E. faecium MLST was performed using the scheme proposed by Homan et al. (2002), and internal fragments of 7 housekeeping genes (adk, atpA, ddl, gyd, gdh, purK, and pstS) were amplified with specific primers, according to conditions described in the online MLST database (http://efaecium.mlst.net/). All amplified fragments were purified using GFX PCR (GE Healthcare) and were sequenced using both forward and reverse primers with the MegaBace™ 1000 DNA Sequencing System. An allelic number corresponding to a sequence already in the database was assigned to each housekeeping gene sequenced, or a new one was assigned by the curator of the database. Based on the 7 allelic numbers assigned, each isolate was allocated to a specific ST. Phylogenetic analyses were performed using the online eBURST v3 algorithm (http://eburst.mlst.net/) following the criteria described by Pitondo-Silva et al. (2009). 3. Results All 53 isolates included in the study were identified as E. faecium, harbored the vanA gene, and showed vancomycin MICs N256 μg/mL, whereas all VREfm were daptomycin susceptible with MICs varying from ≤0.5 to 2 μg/mL. Only the first 5 VREfm isolated showed an intact Tn1546, whereas in 81% of the remaining enterococci (n = 43), Tn1546 had the deleted left extremity and an insertion of IS1251 between the vanS and vanH genes. The sequencing of IS1251 showed that it was inserted downstream of the 5.820 nucleotide of the VanA element in an inverted position, beginning 17 nucleotides beyond the stop codon of the vanS gene. Only 1 isolate (HC-42) showed a Tn1546-like element without either extremity, although it had the IS1251 insertion (Fig. 1). Digestion of the vanRSHAX gene cluster produced 3 extra bands (964, 851, and 558 bp) indicating the enterococci, which had a base-pair variant (T nucleotide instead of G) in vanX at position 8234 and was interfering in the DdeI cleavage site (data not shown). Virulence genes codifying subunits of cytolysin and hyaluronidase were not detected in VREfm isolates. In contrast, among all E. faecium analyzed, 100% (n = 53) showed amplified fragments of the esp gene, 98.1% (n = 52) of the acm gene, 20.8% (n = 11) of the agg gene, and 18.9% (n = 10) of the gel gene. Most VREfm isolated in 2010 had acm and esp genes as well as agg and gel genes, in contrast to the 2008 and 2009 isolates with only esp and acm genes. According to the PFGE dendrogram, the strains belonged to 4 predominant pulsotypes (A, D, F, and H) among a total of 14, each containing 6 or more isolates, which were considered of the same clonal type by a criterion of above 90% genomic similarity (Fig. 2). The first 5 VREfm isolated in HCFMRP showed pulsotypes (M, M1, and M2), which, although predominant in a period when only a few isolates were obtained, were not detected in later isolates, suggesting the occurrence of clonal disseminations of some PFGE profiles at specific times. PFGE indicated high genomic similarity among strains from both hospitals (Fig. 2). MLST of 31 VREfm showed 9 different STs (Fig. 3), all belonging to CC17, except for ST658, which was a singleton. Among the 9 STs identified, 5 are first reported in this study (656, 657, 658, 659, and 660). ST659 and ST660 were detected owing to the presence of new alleles for the ddl gene, identified as 52 and 53, respectively. According to eBURST analysis, among the first 5 E. faecium isolated at the HCFMRP, 3 showed ST78, a single-locus variant (SLV) of ST17, and the other 2 showed ST659 and ST660, SLVs of ST78. ST78 is a worldwide-spreading clone first identified in Brazil by this study (strains HC-61, HC-64, and HC-65). Although ST78 was not detected in later years, most isolates from 2008 to 2010 showed genetic variations like double-locus (DLV) or triple-locus variants (TLV) of ST78, generating ST412, ST478, and ST656. The major STs identified,
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Fig. 1. Schematic representation showing intact Tn1546 (gray arrows) and different VanA elements with their deletions and the site of insertion of IS1251 of the VREfm studied. The deletions are represented and delimited by dotted and dashed lines, respectively. The position and orientation of IS1251 are indicated by black arrows.
Fig. 2. Pulsed-field gel electrophoresis (PFGE) of SmaI-digested DNA of VREfm in the study. PFGE profiles are represented by uppercase letters, followed by an identification number of the VREfm, hospital sector, date of isolation, and ST (MLST). HC-HCFMRP = University Hospital of the Faculty of Medicine of Ribeirao Preto; SCMRP = initials of the other university hospital in the study; HEM = hematology; GAM = gastroenterology; IMU = immunology; TMO = bone marrow transplantation; ORT = orthopaedics; CTI = intensive care unit; CTIP = pediatric intensive care unit; arrows indicate isolates from patients with infection (15, 16, 17, 22, 26, 27, 33, 34, 37, 38, 39, 43, 47, 54, 55, 56, and 57).
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Fig. 3. Application of eBURST algorithm to MLST data, which includes E. faecium collected in this study and existing isolates in the database. Each ST is represented by a dot in a size proportional to the number of E. faecium involved, considering all isolates in the database. ST related VREfm strains from Brazil are indicated by arrows. The broken line indicates STs found in this study.
ST412 and ST478, were found in 38.7% (n = 12) and 32.3% (n = 10) of the strains, respectively. ST17 is a worldwide-spreading epidemic clone, but the only VREfm showing ST17 (HC-07) did not spread in the hospitals involved in the study. Results obtained by MLST showed that VREfm strains isolated in both hospitals had a clonal relationship, showing the same STs (412 and 478). 4. Discussion The genotypic characterization of glycopeptide resistance for all VREfm isolates showed that only the first 5 had an intact Tn1546, whereas most of the other isolates showed the same alteration in the VanA element characterized by deletion of the Tn1546 left extremity and insertion of IS1251 between the vanS and vanH genes. This insertion sequence (IS1251) was first described in the United States and, more recently, in Taiwan (Handwerger et al., 1995; Hsieh et al., 2010; Jensen et al., 1998; Willems et al., 1999). In Brazil, IS1251 was first described in a VanA element of VRE in 2004 and was also detected during an outbreak, which occurred during 2007 in a university hospital in Campinas, São Paulo (Camargo et al., 2004; Palazzo et al., 2011). The enterococci in this study showed a base-pair variant (T nucleotide instead of G) in vanX which was first reported in Brazil in 2004 by Camargo et al. (2004). For many years, the prevalent characteristic among determinants of VREfm glycopeptide resistance in Brazil was the presence of the Tn1546 prototype (Palazzo et al., 2006), but the scenario is being modified owing to the alterations found in VanA elements in VREs isolated in the last few years (Camargo et al., 2004; Darini et al., 2000; Palazzo et al., 2011). It could be inferred that the enterococci in the present study initially showed intact transposon but, over time, acquired insertion sequences and deletions, thus showing polymorphisms among variants of Tn1546. The presence of polymorphisms in different Tn1546 subtypes of enterococci has been widely debated by several authors worldwide. Furthermore, some researchers suggested a presumptive association among specific VanA elements, geographic areas, and environmental sources (Novais et al., 2008; Woodford et al., 1998).
Enterococci are considered microorganisms of low virulence, but some genes that codify virulence factors can contribute to enhancing their capacity to cause infections. Predominant virulence genes among enterococci studied were the esp gene, which is involved in the initial adhesion and biofilm formation in E. faecium, and the acm gene, which has also been involved in the adhesion of E. faecium (Van Wamel et al., 2007; Werner et al., 2008). In contrast with previous findings, none of the isolates showed amplified fragments for the hyl gene (Billsstrom et al., 2008; Freitas et al., 2010; Palazzo et al., 2011). Since VREfm isolated in 2010 showed more virulence factors than those from 2008 and 2009, further investigations are needed to elucidate whether these differences influenced the pathogenesis of E. faecium infections. Both PFGE profiles and MLST data indicated that there was a clonal relationship between VREfm isolates at both hospitals, an evidence of horizontal transmission between them, possibly through patients hospitalized in both hospitals, or transferred from one to other, or even through employees who work in both institutions. PFGE could not determine a common ancestry, but MLST results indicated that VREfm in this study could be direct descendants of or could have evolved from a common ancestor, since most isolates from 2008 to 2010 showed ST412, ST478, and ST656, closely linked to ST78, which was identified in 3 isolates among the first 5 VREfm isolated in HCFMRP. ST78 in Brazil was identified for the first time in this study. According to the literature and to information deposited in the E. faecium MLST database, several countries such as France, Taiwan, Italy, Korea, Japan, Portugal, Paraguay, Germany, China, Hungary, Austria, Latvia, and the Netherlands have identified enterococci with ST78 in hospitalized patients (Fallico et al., 2011; Freitas et al., 2009; Hsieh et al., 2010; Khan et al., 2010; Ko et al., 2005; Libisch et al., 2008; Qu et al., 2007; Watanabe et al., 2009; Werner et al., 2008, 2011). These data should be taken as a warning, which indicates an increase in the worldwide distribution of enterococci pertaining to ST78 clone. A comparative study involving Brazilian VREfm included all isolates characterized by MLST. It was observed that most VREfm identified in outbreaks reported until recently showed ST114, which is not inserted in CC17 (Camargo et al., 2006). VREfm strains also
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belonging to CC17 were identified in some cases in Brazil (ST50, ST115, ST97, ST281), and ST17 in 2 strains isolated in 1998 (São Paulo, SP) and in 2 strains from Rio de Janeiro (1 in 2000 and another in 2001) (Camargo et al., 2006). According to MLST results concerning VREfm strains in this study, 9 different STs were found, with 5 being new ones (656, 657, 658, 659, and 660). All STs identified belonged to clonal complex 17 (CC17), except for ST658, a singleton, showing changes in the VREfm profiles causing outbreaks in Brazil and suggesting that VREfm strains belonging to CC17 are beginning to predominate in the country, a fact that was suggested in a previous study (Palazzo et al., 2011). ST412 and ST478, both belonging to CC17, predominated during the study period and were identified in the various wards of the 2 hospitals. Panesso et al. (2010) showed that ST412 was also the most frequent among VREs isolated in 4 countries in South America, indicating the presence of strains associated with the hospital environment. New STs, 659 and 660, were detected in this study owing to the presence of new alleles for the ddl gene. Mutations in the ddl gene can produce vancomycin dependence which has been recently reported in Brazil (Kerbauy et al., 2011). Although the 2 new alleles of the ddl gene of the VRE studied have shown 2 mutations previously identified (GGC → AGC in position 615 and TGT → CGT in position 739), those mutations were not enough to determine the vancomycin-dependent phenotype in the isolates studied. This study confirms that VREfm isolated in recent years in Brazil belong to CC17 and have genetic determinants, such as virulence genes and antimicrobial resistance, contributing as a selective advantage for these organisms to adapt to the hospital environment. Acknowledgments The authors are grateful to W.A. Silva, Jr., A.A., Marques, and C. Ayres for DNA sequencing; to J.C. Ferreira for technical assistance; and to I.L.B.C. Camargo for suggestions in some experiments. References Billsstrom H, Lund B, Sullivan A, Nord CE. Virulence and antimicrobial resistance in clinical Enterococcus faecium. Int J Antimicrob Agents 2008;32:38–49. Bolano A, Stinchi S, Preziosi R, Bistoni F, Allegrucci M, Baldelli F, et al. Rapid methods to extract DNA and RNA from Cryptococcus neoformans. FEMS Yeast Res 2001;1: 221–4. Camargo ILBC, Del Peloso PF, Da Costa CF, Goldman GH, Darini ALC. Identification of an unusual VanA element in glycopeptide-resistant Enterococcus faecium in Brazil following international transfer of a bone marrow transplant patient. Can J Microbiol 2004;50:767–70. Camargo ILBC, Gilmore MS, Darini ALC. Multilocus sequence typing and analysis of putative virulence factors in vancomycin-resistant and vancomycin-sensitive Enterococcus faecium isolates from Brazil. Clin Microbiol Infect 2006;12:1123–30. Clinical and Laboratory Standards Institute (CLSI). Zone diameter and MIC interpretive standards for Enterococcus spp. v. 29, n. 3; approved standard, M2 M7. Wayne, PA: CLSI; 2009. D'azevedo PA, Kacman SB, Schmalfuss T, Silva A, Rodrigues LF. Primeiro caso de Enterococcus resistente a vancomicina isolado em Porto Alegre, RS. J Bras Pat 2000;36:258. Dalla Costa LM, Reynolds PE, Souza HAPHM, Souza DC, Palepou MFI, Woodford N. Characterization of a divergent van D-type resistance element from the first glycopeptide-resistant strain of Enterococcus faecium isolated in Brazil. Antimicrob Agents Chemother 2000;44:3444–6. Darini ALC, Palepou MF, Woodford N. Effects of the movement of insertion sequences on the structure of VanA glycopeptide resistance elements in Enterococcus faecium. Antimicrob Agents Chemother 2000;44:1362–4. Dutka-Malen S, Evers S, Courvalin P. Detection of glycopeptides resistance genotype and identification to the species level of clinically relevant enterococci by PCR. J Clin Microbiol 1995;33:24–7. Fallico L, Boldrin C, Grossato A, Franchin E, De Canale E, Tommasini T, et al. Molecular epidemiology of Enterococcus faecium isolates from an Italian hospital. Infect 2011;39:127–33. Freitas AR, Novais C, Ruiz-Garbajosa P, Coque TM, Peixe L. Dispersion of multidrugresistant Enterococcus faecium isolates belonging to major clonal complexes in different Portuguese settings. Appl Environ Microbiol 2009;75:4904–8. Freitas AR, Tedium AP, Novais C, Ruiz-Garbajosa P, Werner G, Laverde-Gomez JÁ, et al. Global spread of the hylEfm colonization-virulence gene in megaplasmids of the Enterococcus faecium CC17 polyclonal subcluster. Antimicrob Agents Chemother 2010;45:2660–5.
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Diagnostic Microbiology and Infectious Disease j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / d i a g m i c r o b i o
Genetic features and molecular epidemiology of Enterococcus faecium isolated in two university hospitals in Brazil☆ Leila Priscilla Pinheiro da Silva a, André Pitondo-Silva a, Roberto Martinez b, Ana Lúcia da Costa Darini a,⁎ a b
Departamento de Análises Clínicas, Toxicológicas e Bromatológicas, Faculdade de Ciências Farmacêuticas de Ribeirão Preto, Universidade de São Paulo, São Paulo, Brazil Departamento de Clínica Médica, Faculdade de Medicina de Ribeirão Preto, Universidade de São Paulo, São Paulo, Brazil
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Article history: Received 28 May 2012 Accepted 30 July 2012 Available online 6 September 2012 Keywords: CC17 E. faecium MLST PFGE Tn1546 Virulence factors VREs
a b s t r a c t The global emergence of vancomycin-resistant Enterococcus faecium (VREfm) has been characterized by a clonal spread of strains belonging to clonal complex 17 (CC17). Genetic features and clonal relationships of 53 VREfm isolated from patients in 2 hospitals in Ribeirao Preto, São Paulo, Brazil, during 2005–2010 were determined as a contribution to the Brazilian evolutionary history of these nosocomial pathogens. All isolates were daptomycin susceptible, vancomycin-resistant, and had the vanA gene. The predominant virulence genes were acm and esp. Only 5 VREfm isolated in 2005–2006 had intact Tn1546, while 81% showed Tn1546 with deleted left extremity and insertion of IS1251 between the vanS and vanH genes. Multilocus sequence typing analysis permitted the identification of 9 different sequence types (STs), with 5 being new ones (656, 657, 658, 659, and 660). Predominant STs were ST412 and ST478, all belonging to CC17, except ST658. This is the first report of the ST78 in Brazil. © 2012 Elsevier Inc. All rights reserved.
1. Introduction Enterococci, formerly considered as part of the microbiota in the gastrointestinal tract of humans and animals, have emerged as important nosocomial pathogens in the last 2 decades. Enterococcus faecalis and E. faecium are the species most frequently isolated from human clinical specimens. A recent significant increase in the incidence of nosocomial infections caused by E. faecium has been reported (Top et al., 2008). The change in the epidemiology of enterococcal infections has been attributed to the presence of a genetic lineage of E. faecium, inserted in clonal complex 17 (CC17) as determined by multilocus sequence typing (MLST), that has spread globally (Willems et al., 2005). This lineage is characterized by resistance to ampicillin and quinolone and by the presence of putative virulence genes such as esp and hyl (Freitas et al., 2010; Willems et al., 2001). Although it is recognized that enterococci cause serious infections such as endocarditis and sepsis, they are generally considered lowvirulence microorganisms. Virulence factors in E. faecium are not well known, but some have been pointed out as possible contributors: aggregation substance (agg gene), cytolysin (cyl gene), collagen
☆ This study was supported by the Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP, grants 2010/01463-6 and 2011/18574-8). The funding source had no role in the study design; in the collection, analysis, and interpretation of data; in the writing of the manuscript; or in the decision to submit the manuscript for publication. ⁎ Corresponding author. Tel.: +55-1636024290; fax: +55-1636024725. E-mail address: [email protected] (A.L. da Costa Darini). 0732-8893/$ – see front matter © 2012 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.diagmicrobio.2012.07.012
binding protein (acm gene), enterococcal surface protein (espfm gene), gelatinase (gel gene), and hyaluronidase (hyl gene) (Billsstrom et al., 2008; Top et al., 2008). Currently, 7 phenotypes of acquired vancomycin resistance (types VanA/B/D/E/G/L/M) are known in enterococci, but VanA and VanB are reported most frequently (Werner et al., 2008). The VanA phenotype is characterized by the presence of intact Tn1546 or VanA element, which may result from mutations, deletions, and/or insertion sequences. Therefore, molecular characterization of the Tn1546 structure is important because it provides epidemiologic information about the spread of resistance determinants by horizontal transfer and also about transmission of vancomycin resistance between different ecologic niches (López et al., 2010). In Brazil, vancomycin-resistant Enterococcus faecium (VREfm) with vanD phenotype was first described in 1996 in the city of Curitiba, State of Parana, whereas VanA E. faecium was isolated in 1997 from a meningitis case in São Paulo, SP (Dalla Costa et al. 2000; Zanella et al., 1999). Afterwards, vancomycin-resistant enterococci (VRE) were detected at hospitals in various cities in the country, including São Paulo, Marilia, Campinas, Rio de Janeiro, Uberlândia, Porto Alegre, and others (Camargo et al., 2006; D'azevedo et al., 2000; Palazzo et al., 2011). The recent enterococci epidemiology in Brazil has shown great changes, but very limited data are available regarding clonal profiles characterized by MLST. This study was designed to determine the enterococcal genetic features of isolates from 2 hospitals in the city of Ribeirao Preto, State of São Paulo, as a contribution to the evolutionary history of these nosocomial pathogens in Brazil.
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2. Materials and methods Of the 53 VREfm isolates included in this study, 43 were from different patients hospitalized at the University Hospital of the Faculty of Medicine of Ribeirao Preto, University of São Paulo (HCFMRP-USP) between September 2008 and September 2010. Of these, 29 were isolated from surveillance cultures (rectal swabs), 13 from different infected patients (urine, 6; blood, 3; ear, 1; surgical wounds, 2; and peritoneal cavity, 1), and only 1 VREfm from an unknown origin. Five VREfm (HC-61, HC-62, HC-63, HC-64, and HC-65), which were isolated at the same hospital between 2005 and 2006, were also included. Of the remaining 5 VREfm isolated in another university hospital, from July 2009 to mid-2010, 4 were from infections (urine, 2; bile, 1; and abdominal secretion, 1) and 1 from an unknown origin. The identification and antimicrobial susceptibility of VREs isolated at HCFMRP-USP were determined by the Vitek2® system (bioMérieux Vitek Systems, Hazelwood, MO, USA). Conventional biochemical tests were used to identify the VREs from the other hospital. Minimum inhibitory concentrations (MICs) of vancomycin and daptomycin were determined by E-test® (AB Biodisk, Solna, Sweden), and MIC values of susceptibility to both antibiotics were based on the guidelines issued by the Clinical and Laboratory Standards Institute (CLSI, 2009). The total genomic DNA of the isolates was extracted as described by Bolano et al. (2001) with some modifications. Briefly, cells recovered by centrifugation from 5 mL of Brain Heart Infusion broth of an 18- to 20-h bacterial culture, washed once with saline, and centrifuged during 5 min at 2.655 × g were mixed with glass beads and lysis buffer and kept under stirring for 10 min. Addition of phenol chloroform was followed by centrifugation for 20 min at 15.294 × g. After the addition of absolute isopropanol, the tubes were maintained at −20 °C for 18–20 h. DNA was purified and recovered by ethanol precipitation. The genomic DNA obtained was used as template in polymerase chain reactions (PCR), which were performed to confirm species and to investigate the presence of vanA, vanB, and vanC genes in the VREs investigated (Dutka-Malen et al., 1995; Woodford et al., 1993). Amplification of VanA elements was by the long-PCR 1 reaction using primer (P1) complementary to the inverted repeats flanking the transposon. Long-PCR 2/3 reaction was used for amplification of vanRSHAX genes, using specific primers (P2 and P3). Long PCRs were performed using a commercial kit (Long PCR Enzyme Mix, Fermentas, Foster City, CA, USA) according to the manufacturer's recommendations. DdeI restriction endonuclease was used to digest the enterococci vanRSHAX gene clusters that produced long-PCR 2/3 products different from the prototype Tn1546. The long PCR and restriction enzyme cleavage products were analyzed in 1% agarose gel electrophoresis and visualized with ethidium bromide under ultraviolet light (Palepou et al., 1998). PCR overlapping was performed for all enterococci showing Tn1546 different from the prototype (Woodford and Stigter, 1998). The IS1251 insertion element was investigated by PCR using primers described in the literature, and the MegaBace™ 1000 DNA Sequencing System (GE Healthcare, Chalfont St Giles, Buckinghamshire, UK) was used to sequence the PCR products of some isolates to determine the exact location of the insertion sequence in the Tn1546 (Camargo et al., 2004). The following virulence genes were analyzed by PCR: genes codifying collagen-adhesin protein (acm), aggregation substance (agg), cytolysin (cylLL, cylLS, cylA), enterococcal surface protein (esp), gelatinase (gel), and hyaluronidase (hyl). Positive and negative controls from our collection were used in all PCR assays (Camargo et al., 2006). Pulsed-field gel electrophoresis (PFGE) was performed after SmaI digestion of genomic DNA of the 53 VREfm in a Gene Navigator apparatus (Amersham, Uppsala, Sweden), following the protocol described by Kaulfmann (1998). Among all 53 E. faecium in the study, 31 VREfm selected according to PFGE results were characterized by MLST. Those with different
pulsotypes were selected, but others with the same pulsotype were also chosen based on some characteristics as date of isolation, type of clinical specimens, isolation hospital, and hospital sector. E. faecium MLST was performed using the scheme proposed by Homan et al. (2002), and internal fragments of 7 housekeeping genes (adk, atpA, ddl, gyd, gdh, purK, and pstS) were amplified with specific primers, according to conditions described in the online MLST database (http://efaecium.mlst.net/). All amplified fragments were purified using GFX PCR (GE Healthcare) and were sequenced using both forward and reverse primers with the MegaBace™ 1000 DNA Sequencing System. An allelic number corresponding to a sequence already in the database was assigned to each housekeeping gene sequenced, or a new one was assigned by the curator of the database. Based on the 7 allelic numbers assigned, each isolate was allocated to a specific ST. Phylogenetic analyses were performed using the online eBURST v3 algorithm (http://eburst.mlst.net/) following the criteria described by Pitondo-Silva et al. (2009). 3. Results All 53 isolates included in the study were identified as E. faecium, harbored the vanA gene, and showed vancomycin MICs N256 μg/mL, whereas all VREfm were daptomycin susceptible with MICs varying from ≤0.5 to 2 μg/mL. Only the first 5 VREfm isolated showed an intact Tn1546, whereas in 81% of the remaining enterococci (n = 43), Tn1546 had the deleted left extremity and an insertion of IS1251 between the vanS and vanH genes. The sequencing of IS1251 showed that it was inserted downstream of the 5.820 nucleotide of the VanA element in an inverted position, beginning 17 nucleotides beyond the stop codon of the vanS gene. Only 1 isolate (HC-42) showed a Tn1546-like element without either extremity, although it had the IS1251 insertion (Fig. 1). Digestion of the vanRSHAX gene cluster produced 3 extra bands (964, 851, and 558 bp) indicating the enterococci, which had a base-pair variant (T nucleotide instead of G) in vanX at position 8234 and was interfering in the DdeI cleavage site (data not shown). Virulence genes codifying subunits of cytolysin and hyaluronidase were not detected in VREfm isolates. In contrast, among all E. faecium analyzed, 100% (n = 53) showed amplified fragments of the esp gene, 98.1% (n = 52) of the acm gene, 20.8% (n = 11) of the agg gene, and 18.9% (n = 10) of the gel gene. Most VREfm isolated in 2010 had acm and esp genes as well as agg and gel genes, in contrast to the 2008 and 2009 isolates with only esp and acm genes. According to the PFGE dendrogram, the strains belonged to 4 predominant pulsotypes (A, D, F, and H) among a total of 14, each containing 6 or more isolates, which were considered of the same clonal type by a criterion of above 90% genomic similarity (Fig. 2). The first 5 VREfm isolated in HCFMRP showed pulsotypes (M, M1, and M2), which, although predominant in a period when only a few isolates were obtained, were not detected in later isolates, suggesting the occurrence of clonal disseminations of some PFGE profiles at specific times. PFGE indicated high genomic similarity among strains from both hospitals (Fig. 2). MLST of 31 VREfm showed 9 different STs (Fig. 3), all belonging to CC17, except for ST658, which was a singleton. Among the 9 STs identified, 5 are first reported in this study (656, 657, 658, 659, and 660). ST659 and ST660 were detected owing to the presence of new alleles for the ddl gene, identified as 52 and 53, respectively. According to eBURST analysis, among the first 5 E. faecium isolated at the HCFMRP, 3 showed ST78, a single-locus variant (SLV) of ST17, and the other 2 showed ST659 and ST660, SLVs of ST78. ST78 is a worldwide-spreading clone first identified in Brazil by this study (strains HC-61, HC-64, and HC-65). Although ST78 was not detected in later years, most isolates from 2008 to 2010 showed genetic variations like double-locus (DLV) or triple-locus variants (TLV) of ST78, generating ST412, ST478, and ST656. The major STs identified,
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Fig. 1. Schematic representation showing intact Tn1546 (gray arrows) and different VanA elements with their deletions and the site of insertion of IS1251 of the VREfm studied. The deletions are represented and delimited by dotted and dashed lines, respectively. The position and orientation of IS1251 are indicated by black arrows.
Fig. 2. Pulsed-field gel electrophoresis (PFGE) of SmaI-digested DNA of VREfm in the study. PFGE profiles are represented by uppercase letters, followed by an identification number of the VREfm, hospital sector, date of isolation, and ST (MLST). HC-HCFMRP = University Hospital of the Faculty of Medicine of Ribeirao Preto; SCMRP = initials of the other university hospital in the study; HEM = hematology; GAM = gastroenterology; IMU = immunology; TMO = bone marrow transplantation; ORT = orthopaedics; CTI = intensive care unit; CTIP = pediatric intensive care unit; arrows indicate isolates from patients with infection (15, 16, 17, 22, 26, 27, 33, 34, 37, 38, 39, 43, 47, 54, 55, 56, and 57).
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Fig. 3. Application of eBURST algorithm to MLST data, which includes E. faecium collected in this study and existing isolates in the database. Each ST is represented by a dot in a size proportional to the number of E. faecium involved, considering all isolates in the database. ST related VREfm strains from Brazil are indicated by arrows. The broken line indicates STs found in this study.
ST412 and ST478, were found in 38.7% (n = 12) and 32.3% (n = 10) of the strains, respectively. ST17 is a worldwide-spreading epidemic clone, but the only VREfm showing ST17 (HC-07) did not spread in the hospitals involved in the study. Results obtained by MLST showed that VREfm strains isolated in both hospitals had a clonal relationship, showing the same STs (412 and 478). 4. Discussion The genotypic characterization of glycopeptide resistance for all VREfm isolates showed that only the first 5 had an intact Tn1546, whereas most of the other isolates showed the same alteration in the VanA element characterized by deletion of the Tn1546 left extremity and insertion of IS1251 between the vanS and vanH genes. This insertion sequence (IS1251) was first described in the United States and, more recently, in Taiwan (Handwerger et al., 1995; Hsieh et al., 2010; Jensen et al., 1998; Willems et al., 1999). In Brazil, IS1251 was first described in a VanA element of VRE in 2004 and was also detected during an outbreak, which occurred during 2007 in a university hospital in Campinas, São Paulo (Camargo et al., 2004; Palazzo et al., 2011). The enterococci in this study showed a base-pair variant (T nucleotide instead of G) in vanX which was first reported in Brazil in 2004 by Camargo et al. (2004). For many years, the prevalent characteristic among determinants of VREfm glycopeptide resistance in Brazil was the presence of the Tn1546 prototype (Palazzo et al., 2006), but the scenario is being modified owing to the alterations found in VanA elements in VREs isolated in the last few years (Camargo et al., 2004; Darini et al., 2000; Palazzo et al., 2011). It could be inferred that the enterococci in the present study initially showed intact transposon but, over time, acquired insertion sequences and deletions, thus showing polymorphisms among variants of Tn1546. The presence of polymorphisms in different Tn1546 subtypes of enterococci has been widely debated by several authors worldwide. Furthermore, some researchers suggested a presumptive association among specific VanA elements, geographic areas, and environmental sources (Novais et al., 2008; Woodford et al., 1998).
Enterococci are considered microorganisms of low virulence, but some genes that codify virulence factors can contribute to enhancing their capacity to cause infections. Predominant virulence genes among enterococci studied were the esp gene, which is involved in the initial adhesion and biofilm formation in E. faecium, and the acm gene, which has also been involved in the adhesion of E. faecium (Van Wamel et al., 2007; Werner et al., 2008). In contrast with previous findings, none of the isolates showed amplified fragments for the hyl gene (Billsstrom et al., 2008; Freitas et al., 2010; Palazzo et al., 2011). Since VREfm isolated in 2010 showed more virulence factors than those from 2008 and 2009, further investigations are needed to elucidate whether these differences influenced the pathogenesis of E. faecium infections. Both PFGE profiles and MLST data indicated that there was a clonal relationship between VREfm isolates at both hospitals, an evidence of horizontal transmission between them, possibly through patients hospitalized in both hospitals, or transferred from one to other, or even through employees who work in both institutions. PFGE could not determine a common ancestry, but MLST results indicated that VREfm in this study could be direct descendants of or could have evolved from a common ancestor, since most isolates from 2008 to 2010 showed ST412, ST478, and ST656, closely linked to ST78, which was identified in 3 isolates among the first 5 VREfm isolated in HCFMRP. ST78 in Brazil was identified for the first time in this study. According to the literature and to information deposited in the E. faecium MLST database, several countries such as France, Taiwan, Italy, Korea, Japan, Portugal, Paraguay, Germany, China, Hungary, Austria, Latvia, and the Netherlands have identified enterococci with ST78 in hospitalized patients (Fallico et al., 2011; Freitas et al., 2009; Hsieh et al., 2010; Khan et al., 2010; Ko et al., 2005; Libisch et al., 2008; Qu et al., 2007; Watanabe et al., 2009; Werner et al., 2008, 2011). These data should be taken as a warning, which indicates an increase in the worldwide distribution of enterococci pertaining to ST78 clone. A comparative study involving Brazilian VREfm included all isolates characterized by MLST. It was observed that most VREfm identified in outbreaks reported until recently showed ST114, which is not inserted in CC17 (Camargo et al., 2006). VREfm strains also
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belonging to CC17 were identified in some cases in Brazil (ST50, ST115, ST97, ST281), and ST17 in 2 strains isolated in 1998 (São Paulo, SP) and in 2 strains from Rio de Janeiro (1 in 2000 and another in 2001) (Camargo et al., 2006). According to MLST results concerning VREfm strains in this study, 9 different STs were found, with 5 being new ones (656, 657, 658, 659, and 660). All STs identified belonged to clonal complex 17 (CC17), except for ST658, a singleton, showing changes in the VREfm profiles causing outbreaks in Brazil and suggesting that VREfm strains belonging to CC17 are beginning to predominate in the country, a fact that was suggested in a previous study (Palazzo et al., 2011). ST412 and ST478, both belonging to CC17, predominated during the study period and were identified in the various wards of the 2 hospitals. Panesso et al. (2010) showed that ST412 was also the most frequent among VREs isolated in 4 countries in South America, indicating the presence of strains associated with the hospital environment. New STs, 659 and 660, were detected in this study owing to the presence of new alleles for the ddl gene. Mutations in the ddl gene can produce vancomycin dependence which has been recently reported in Brazil (Kerbauy et al., 2011). Although the 2 new alleles of the ddl gene of the VRE studied have shown 2 mutations previously identified (GGC → AGC in position 615 and TGT → CGT in position 739), those mutations were not enough to determine the vancomycin-dependent phenotype in the isolates studied. This study confirms that VREfm isolated in recent years in Brazil belong to CC17 and have genetic determinants, such as virulence genes and antimicrobial resistance, contributing as a selective advantage for these organisms to adapt to the hospital environment. Acknowledgments The authors are grateful to W.A. Silva, Jr., A.A., Marques, and C. Ayres for DNA sequencing; to J.C. Ferreira for technical assistance; and to I.L.B.C. Camargo for suggestions in some experiments. References Billsstrom H, Lund B, Sullivan A, Nord CE. Virulence and antimicrobial resistance in clinical Enterococcus faecium. Int J Antimicrob Agents 2008;32:38–49. Bolano A, Stinchi S, Preziosi R, Bistoni F, Allegrucci M, Baldelli F, et al. Rapid methods to extract DNA and RNA from Cryptococcus neoformans. FEMS Yeast Res 2001;1: 221–4. Camargo ILBC, Del Peloso PF, Da Costa CF, Goldman GH, Darini ALC. Identification of an unusual VanA element in glycopeptide-resistant Enterococcus faecium in Brazil following international transfer of a bone marrow transplant patient. Can J Microbiol 2004;50:767–70. Camargo ILBC, Gilmore MS, Darini ALC. Multilocus sequence typing and analysis of putative virulence factors in vancomycin-resistant and vancomycin-sensitive Enterococcus faecium isolates from Brazil. Clin Microbiol Infect 2006;12:1123–30. Clinical and Laboratory Standards Institute (CLSI). Zone diameter and MIC interpretive standards for Enterococcus spp. v. 29, n. 3; approved standard, M2 M7. Wayne, PA: CLSI; 2009. D'azevedo PA, Kacman SB, Schmalfuss T, Silva A, Rodrigues LF. Primeiro caso de Enterococcus resistente a vancomicina isolado em Porto Alegre, RS. J Bras Pat 2000;36:258. Dalla Costa LM, Reynolds PE, Souza HAPHM, Souza DC, Palepou MFI, Woodford N. Characterization of a divergent van D-type resistance element from the first glycopeptide-resistant strain of Enterococcus faecium isolated in Brazil. Antimicrob Agents Chemother 2000;44:3444–6. Darini ALC, Palepou MF, Woodford N. Effects of the movement of insertion sequences on the structure of VanA glycopeptide resistance elements in Enterococcus faecium. Antimicrob Agents Chemother 2000;44:1362–4. Dutka-Malen S, Evers S, Courvalin P. Detection of glycopeptides resistance genotype and identification to the species level of clinically relevant enterococci by PCR. J Clin Microbiol 1995;33:24–7. Fallico L, Boldrin C, Grossato A, Franchin E, De Canale E, Tommasini T, et al. Molecular epidemiology of Enterococcus faecium isolates from an Italian hospital. Infect 2011;39:127–33. Freitas AR, Novais C, Ruiz-Garbajosa P, Coque TM, Peixe L. Dispersion of multidrugresistant Enterococcus faecium isolates belonging to major clonal complexes in different Portuguese settings. Appl Environ Microbiol 2009;75:4904–8. Freitas AR, Tedium AP, Novais C, Ruiz-Garbajosa P, Werner G, Laverde-Gomez JÁ, et al. Global spread of the hylEfm colonization-virulence gene in megaplasmids of the Enterococcus faecium CC17 polyclonal subcluster. Antimicrob Agents Chemother 2010;45:2660–5.
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