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Enterococcus faecium has emerged as an important nosocomial pathogen worldwide, and this trend has been associated with the dissemination of a genetic lineage designated clonal cluster 17 (CC17). Enterococcal isolates were collected prospectively (2006 to 2008) from 32 hospitals in Colombia, Ecuador, Perú, and Venezuela and subjected to antimicrobial susceptibility testing. Genotyping was performed with all vancomycin-resistant E. faecium (VREfm) isolates by pulsed-field gel electrophoresis (PFGE) and multilocus sequence typing. All VREfm isolates were evaluated for the presence of 16 putative virulence genes (14 fms genes, the esp gene of E. faecium [espEfm], and the hyl gene of E. faecium [hylEfm]) and plasmids carrying the fms20-fms21 (pilA), hylEfm, and vanA genes. Of 723 enterococcal isolates recovered, E. faecalis was the most common (78%). Vancomycin resistance was detected in 6% of the isolates (74% of which were E. faecium). Eleven distinct PFGE types were found among the VREfm isolates, with most belonging to sequence types 412 and 18. The ebpAEfm-ebpBEfm-ebpCEfm (pilB) and fms11-fms19-fms16 clusters were detected in all VREfm isolates from the region, whereas espEfm and hylEfm were detected in 69% and 23% of the isolates, respectively. The fms20-fms21 (pilA) cluster, which encodes a putative pilus-like protein, was found on plasmids from almost all VREfm isolates and was sometimes found to coexist with hylEfm and the vanA gene cluster. The population genetics of VREfm in South America appear to resemble those of such strains in the United States in the early years of the CC17 epidemic. The overwhelming presence of plasmids encoding putative virulence factors and vanA genes suggests that E. faecium from the CC17 genogroup may disseminate in the region in the coming years.
Enterococci are now recognized as important nosocomial pathogens worldwide and in the United States are ranked as the second most common cause of nosocomial infections, after staphylococci (14). The two most common enterococcal species isolated from clinical samples are Enterococcus faecalis and E. faecium; however, the proportions of isolates of these two species have dramatically changed in the last decade. Whereas up until the early to mid-1990s E. faecalis was the overwhelmingly predominant species isolated in U.S. hospitals (37), by 2008, the proportion of nosocomial E. faecalis/E. faecium strains was ca. 1.5:1, and there was an important increase in the incidence of E. faecium nosocomial infections (14). Moreover, more than 80% of E. faecium isolates currently recovered from U.S. hospitals are resistant to vancomycin, and virtually all of them (>90%) exhibit ampicillin resistance (14). On the contrary, the prevalence of vancomycin resistance in E. faecalis remains low (<7% of isolates), and ampicillin resistance continues to be extremely rare. This change in the epidemiology of enterococcal infections has been attributed to the increased ability of a genogroup of E. faecium (designated clonal cluster 17 [CC17]) to colonize the gastrointestinal tract of humans, cause disease (37), and exhibit high levels of resistance to most antienterococcal antibiotics. Several virulence and colonizing factors have been postulated to explain this increased virulence (4, 25, 28) and include the following: (i) the presence of an intact acm gene, which encodes a collagen adhesin and which has been associated with the pathogenesis of endocarditis in members of CC17 (25); (ii) the esp gene of E. faecium (espEfm), which codes for an enterococcal surface protein, which has been shown to play a role in biofilm formation (12), and which transiently aggravates experimental urinary tract infection (18); (iii) the fms (E. faecium surface protein-encoding) genes, which encode cell wall-anchored proteins, including subunits of the enterococcal pili (13, 31); and (iv) the hyl gene of E. faecium (hylEfm; which encodes a putative glycosyl hydrolase), which is carried by transferable plasmids that have been shown to increase the ability of a laboratory strain of E. faecium to colonize the gastrointestinal tracts of mice and also enhance the virulence of a commensal strain of E. faecium in experimental peritonitis (4, 28).
In South America (Brazil and Argentina), vancomycin-resistant (VR) enterococcal infections have been described since 1998 (9, 20). In a prospective multicenter surveillance study conducted in 2003, the prevalence of VR among enterococci in Colombia was found to be lower (9.7%) than that in the United States (1), and the isolation of similar percentages of VR enterococci has been described more recently (21). Although the emergence of E. faecium CC17 has been documented in Brazil, Chile, and Paraguay (16, 19, 39), prospective studies have not been performed and limited data regarding the presence of the potential virulence determinants of CC17 E. faecium (including hylEfm-containing plasmids) in South America are available. Thus, we performed a multinational, multicenter prospective surveillance study with the aim of characterizing the population genetics of enterococci circulating in the northern region of South America. Clinical isolates (excluding colonizing isolates) were collected prospectively from 32 hospitals in four countries (Colombia, Ecuador, Perú, and Venezuela) and were further characterized at the molecular level.
(This study was presented in part at the 48th Annual Interscience Conference on Antimicrobial Agents and Chemotherapy/Infectious Diseases Society of America 46th Annual Meeting, Washington, DC, 25 to 28 October 2008, abstr. C2-1998.)
A multicenter study was performed from February 2006 through February 2008 to evaluate the molecular epidemiology of enterococci in four South American countries. Each participating tertiary-care hospital (a total of 32 hospitals, from the Latin American Network of Antimicrobial Resistance) in Colombia (22 hospitals in six cities), Ecuador (5 hospitals in one city), Perú (3 hospitals in one city), and Venezuela (2 hospitals in one city) collected consecutive enterococcal isolates (duplicate organisms from the same patient were excluded). The samples were collected by the clinical laboratory from each participating hospital and corresponded to samples from hospitalized patients in Colombia, Perú, and Venezuela. In Ecuador, the clinical laboratory also collected samples from ambulatory services and outpatient clinics. The isolates originated from the following clinical specimens: blood, urine, secretions from surgical wounds, peritoneal fluid, abdominal abscesses, joint aspirates, osteomyelitis aspirates, pleural fluid, pericardial effusion, cerebral abscesses, and cerebrospinal fluid (CSF). In an attempt to avoid isolates that likely represented colonization, enterococci recovered from sputum, rectal swabs, catheters, or skin (unless they originated in an infected surgical wound) were excluded. Each hospital identified the microorganisms by using either automated methods (performed with the Vitek or MicroScan system) or manual methods, and once the corresponding isolate was included in the study, it was sent to the reference laboratory (located in Bogotá, Colombia) via courier in transport medium (Amies; BBL). Upon arrival, the reference laboratory confirmed the purity of the isolate and confirmed the identification by molecular methods using multiplex PCR for enterococci (see below).
Susceptibility tests for enterococci were performed by an agar dilution method with ampicillin, ciprofloxacin, chloramphenicol, linezolid, vancomycin, and teicoplanin. The isolates were also evaluated for high-level resistance (HLR) to streptomycin (2,000 μg/ml) and gentamicin (500 μg/ml). All isolates from blood and CSF identified as E. faecalis were screened for the presence of the β-lactamase enzyme by use of a nitrocefin test, and all susceptibility tests were conducted by the methodology suggested by the CLSI (8). MIC determinations were carried out with the inclusion of a control reference strain (E. faecalis ATCC 29212). Species-specific identification of the enterococci and determination of the van genotype of the vancomycin-resistant isolates were performed by PCR, as described previously (2, 10) (primers for the detection of the recently described vanL genotype  were not included). E. faecium BM4147 (vanA), E. faecalis V583 (vanB), and E. gallinarum BM4174 (vanC1) were used as control strains for MIC determinations and PCR assays.
Pulsed-field gel electrophoresis (PFGE) of the vancomycin-resistant E. faecium (VREfm) isolates was performed by use of some modifications of a previously described method (22), and the banding patterns were initially interpreted by visual inspection, according to the criteria specified by Tenover et al. (35). Subsequently, cluster analysis was performed by the method of Dice and the unweighted-pair group method using average linkages (UPGMA). The band tolerance was set at 1.5%, and the threshold cutoff value was set at 85%. Representative isolates of each PFGE subtype of VREfm were further genotyped by multilocus sequence typing (MLST) by a standard protocol described previously (15, 29). Fragments of seven housekeeping genes (atpA, ddl, gdh, purK, gyd, pstS, and adk) were sequenced, the allelic profiles were obtained, and the sequence type (ST) for each unique allelic profile was designated on the basis of the information at the MLST website (http://efaecium.mlst.net).
The preparation of colony lysates on nylon membranes and hybridization under high-stringency conditions were performed as described previously (33). DNA probes for the hylEfm, espEfm, and 14 fms genes were obtained by using previously published primers (11, 27, 32); the probes were radiolabeled by use of the RadPrime DNA labeling system (Invitrogen, Carlsbad, CA). Plasmid detection in representative isolates of VREfm belonging to CC17 was performed by the use of S1 nuclease and PFGE, according to a previously described protocol (4, 5). This methodology allows the detection and estimation of the size of large bacterial plasmids in the presence of genomic DNA by PFGE (5). Plasmid bands were subsequently hybridized with probes targeting the hylEfm, fms20, fms21 (pilA), and vanA genes to determine if these genes were colocated in the same plasmid, as was reported previously (17). E. faecium TX0016 (strain DO) (23) and ERV-99 (4) were used as controls for colony hybridizations and the PFGE-S1 nuclease experiments.
A total of 760 consecutive Enterococcus sp. isolates was collected in the four countries. Thirty-seven isolates were not included in the study due to protocol violations (contamination, isolates were from the same patient, the source was not included in the protocol, or misidentification). Among the isolates included (a total of 723), Colombian hospitals contributed 309 (43%) of the isolates, Peruvian hospitals contributed 164 (23%), Ecuadorian hospitals contributed 148 (20%), and Venezuelan hospitals contributed 102 (14%). The majority (78%) were E. faecalis isolates mostly recovered from urine (54%) and blood (14%) (no β-lactamase-producing isolate was detected). E. faecium comprised 15% (n = 111) of the enterococcal isolates (Table (Table1).1). The most common clinical sources of E. faecium included urine (38%), blood (21%), and surgical wound infections (13%). Other enterococcal species also identified by PCR included E. avium, E. hirae, E. casseliflavus, E. gallinarum, and E. durans. We were unable to determine the enterococcal species for 5% (n = 35) of the isolates.
The resistance rates of the enterococci from the Andean region are shown in Table Table1.1. All E. faecalis isolates were susceptible to ampicillin and linezolid, whereas the rates of HLR to gentamicin and streptomycin were 28% and 30%, respectively; ciprofloxacin resistance was found in 29% of the E. faecalis isolates (Table (Table1).1). In contrast, the rates of resistance to ampicillin and vancomycin and HLR to gentamicin and streptomycin in E. faecium were much higher (76%, 31%, 36%, and 44%, respectively). The highest rates of resistance to ampicillin and vancomycin and HLR to gentamicin and streptomycin in E. faecium isolates were found in Perú (90%, 48%, 55%, and 65%, respectively), and the lowest were found in Venezuela (50%, 25%, 8%, and 21%, respectively). Among the ampicillin-resistant E. faecium isolates from the Andean region, a total of 51 isolates (46%) had MICs of >64 μg/ml. Linezolid was active against all the E. faecium isolates tested, and only 4% of the E. faecium isolates tested were resistant to chloramphenicol.
Thirty-five VREfm isolates were found, and all of them exhibited the VanA phenotype (high levels of resistance to both vancomycin and teicoplanin) and harbored the vanA gene cluster. Typing by PFGE revealed 11 different banding patterns (patterns A to K) (Fig. (Fig.1).1). A single PFGE banding pattern was predominant in each country (referred to as type A in Venezuela, type B in Perú, type C in Colombia, and type D in Ecuador) (Fig. (Fig.1),1), although isolates from different countries (e.g., types A and E) could be found to have related PFGE types. MLST analysis of representative isolates of each PFGE type indicated that the most frequent ST was ST412; other STs included ST17, ST18, ST125, ST203, ST280, and ST282 (all of which were associated with the CC17 genetic lineage). A representative of PFGE pattern J from a Peruvian hospital was found to belong to a novel ST (ST494) which had not been reported previously.
Only three vancomycin-resistant E. faecalis were isolated in the 2 years of the study; all three isolates were recovered in Colombia, harbored the vanB gene cluster, and were previously characterized as belonging to the same unique clonal cluster that has been circulating in that country since 2001 (ST2 by trilocus sequence typing and MLST) (3, 7).
It had previously been shown that the members of CC17 of E. faecium harbor a number of genes encoding sortase-anchoring motif (LPXTG)-containing cell wall proteins (13, 31); at least 15 of these genes encode predicted microbial surface components recognizing adhesive matrix molecule (MSCRAMMs) proteins in a U.S. strain (strain TX0016 [strain DO]) that was sequenced (32). It has been proposed that these genes function as important virulence determinants mediating adhesion to mammalian tissues, and several are enriched in the CC17 genogroup (11, 13, 31). Thus, we set out to investigate if these genes were also frequently found in CC17 isolates of E. faecium from Latin America. Figure Figure22 shows the results of colony hybridizations of isolates with several putative virulence genes (including 14 of the fms genes; acm, which may be present as an intact gene or pseudogene, was not tested since it has been detected in almost all isolates studied previously ). Indeed, the four clusters of genes encoding MSCRAMM proteins (31) were highly represented among the South American E. faecium isolates tested. The ebpAEfm-ebpBEfm-ebpCEfm (pilB) and fms11-fms19-fms16 clusters, which harbor genes encoding putative proteins with conserved pilin motifs and E-boxes of pilus proteins of Gram-positive bacteria, were present in all our South American VR isolates (Fig. (Fig.2).2). Interestingly, the scm gene, which has been shown to encode a collagen adhesin (the second collagen adhesin from E. faecium), was present in more than 90% of the isolates. The other fms genes were also frequently present at rates ranging from 67% to 97% in the VREfm isolates. The espEfm and hylEfm genes, which have previously been associated with CC17 (28, 38), were detected in South American VR E. faecium isolates (69% and 23% of isolates, respectively).
It was previously shown that hospital-associated E. faecium isolates from diverse geographical locations carry large, transferable plasmids containing the hylEfm gene (which encodes a putative family 84 glycosyl hydrolase enzyme) that, additionally, are associated with vancomycin and aminoglycoside resistance genes (4). These hylEfm-containing plasmids have emerged as important virulence and colonization determinants of CC17 of E. faecium (4, 28). More recently, one of the fms clusters (fms20-fms21 [pilA]) encoding pilus-like proteins was also found to be located in a large hylEfm-carrying plasmid in U.S. strain TX0016 (strain DO) (17). Table Table22 and Fig. Fig.33 show the results of S1 nuclease digestion; PFGE; and the hybridization of representatives of South American VR E. faecium isolates with probes targeting the fms20-fms21 (pilA) cluster, hylEfm, and vanA. When it was present, the fms20-fms21 (pilA) cluster was always encoded on plasmids ranging from ca. 60 kb to 242 kb. Of note, in some strains, fms21-containing plasmids were found not to harbor the other member of the cluster (fms20), and in three strains (all from Perú) an additional copy of fms21 was detected in a different plasmid (strains P575, P1139, and P1986; Table Table22 and Fig. Fig.3).3). Furthermore, the fms20-fms21 cluster was found to be in the same plasmid as hylEfm in three strains (estimated plasmid sizes, 190 and 230 kb) and on the same vanA-containing plasmid in three strains (Table (Table2).2). No plasmid was found to carry the fms20-fms21 cluster, hylEfm, and vanA together.
In the work described here, we performed the first prospective, multinational study evaluating the molecular epidemiology of VR enterococci in the northern region of South America. Our results indicate that the prevalence of vancomycin resistance is relatively low in the participating countries (only 6% of isolates included in this study) compared to that in the United States. In our previous multicenter study performed with isolates from 15 hospitals in Colombia in 2001 and 2002, the rate of vancomycin resistance among enterococcal isolates was 9.7%. Also, unlike in the United States, E. faecalis continues to be, by far, the most frequent enterococcal species isolated in this region (ratio of E. faecalis to E. faecium, 5:1). Vancomycin resistance in E. faecalis was found sporadically in only three isolates from one city of the participating countries, and ampicillin was active against all E. faecalis isolates, underscoring the fact that β-lactams and glycopeptides continue to be useful for the treatment of enterococcal infections in the northern region of South America. On the other hand, the rates of HLR to aminoglycosides in E. faecalis were ca. 30%, and there were important regional variations; for example, in the participating hospitals from Perú, more than 50% of the E. faecalis isolates exhibited HLR to gentamicin and 44% exhibited HLR to streptomycin, indicating that the treatment of endovascular infections with the combination of a cell wall agent and an aminoglycoside in some areas of South America may be challenging and alternative bactericidal therapies should be sought. Also, we did not find important differences in phenotypic or genotypic characteristics between VREfm isolates from urine and blood (except that the blood isolates had higher rates of resistance to chloramphenicol [20%] than those VREfm isolates that originated in urine [0%]).
Although our findings indicate that the proportion of E. faecalis/E. faecium is considerably higher in South American hospitals than in U.S. hospitals (14), the South American VREfm isolates have genotypic and phenotypic characteristics similar to those of their U.S. counterparts: (i) South American VREfm isolates mostly belong to ST412 and ST18 (the CC17 lineage), indicating that a hospital-associated genetic lineage is present in the region and supporting previous findings of studies performed in other countries of South America (16, 19, 39); (ii) the South American CC17 VREfm isolates also harbor an important number of MSCRAMM genes, including the four gene clusters encoding predicted components of the E. faecium pili (particularly the ebpEfm and the fms11-fms19-fms16 clusters) (13, 31, 32), a finding that is consistent with the fact that the presence of these genes appears to be more reliably associated with members of the CC17 genetic lineage (11) than the presence of espEfm and hylEfm; and (iii) high-level of resistance to ampicillin is commonly found in E. faecium isolates from the region, with the majority of isolates exhibiting MICs of >64 μg/ml.
Recently, a genotypic analysis of early clinical isolates of E. faecium in the United States (11) showed that CC17 E. faecium isolates have been circulating in the United States since at least 1982, and it was postulated that an ancestral genogroup of E. faecium enriched with the fms genes was able to subsequently acquire additional virulence and antibiotic resistance determinants (such as espEfm, hylEfm, HLR to ampicillin, and the van gene clusters) and establish itself as an epidemic hospital-associated pathogen in the United States. Our results suggest that the population genetics of VREfm in the northern region of South America resembles that of isolates responsible for early outbreaks in the United States (in about 1982) (11), and it is tempting to speculate that an increase in the frequency of E. faecium infections in South America may be expected in the following decade as members of the hospital-associated lineage disseminate and establish themselves as nosocomial pathogens in the region. Nonetheless, it is important to point out that the number of hospitals participating in this study differed in each country; while 22 hospitals from six different cities were represented in Colombia, a lower number of hospitals in the other three countries participated in the study. Thus, it is difficult to make countrywide generalizations of the molecular epidemiology of enterococcal isolates in Perú, Venezuela, and Ecuador. However, our data provide important information on the type of organisms currently circulating in the region.
An important goal of our study was to determine if South American VREfm isolates also contain genes encoding putative pilus proteins that are present on plasmids coexisting with hylEfm or vancomycin resistance gene clusters, as previously shown for other strains (4, 17). Enterococcal pili are important cell surface structures that have been implicated in the pathogenesis of experimental endocarditis and urinary tract infections (24, 34). These trimeric units are composed of a major backbone subunit and two minor subunits (36) with an N-terminal signal sequence and a C-terminal cell wall-sorting signal (30) encoded by different genes. Indeed, we found that one of these clusters (fms20-fms21 [pilA]) is carried by large plasmids in the majority of isolates studied. Moreover, in some strains, the fms20-fms21 (pilA) cluster coexists on the same plasmid with the hylEfm gene or with vanA, providing further evidence that the plasmid dissemination of virulence, resistance, and/or colonization determinants may be a strategy adopted by hospital-associated lineages of E. faecium worldwide. This finding also supports the hypothesis that the Andean region of South America may be in the early stages of an E. faecium epidemic and that as the fms20-fms21/hylEfm-carrying plasmids disseminate, the ability of the organisms to colonize the gastrointestinal tract of patients and cause disease may increase.
In summary, this is the first multicenter, multinational prospective study of the molecular epidemiology of VR enterococci in South America. Our results indicate that the prevalence of E. faecium is low among clinical isolates, but genotypic analysis supports the fact that the population genetics of E. faecium is similar to that seen in the United States more than 25 years ago, suggesting that the dissemination of these highly adapted E. faecium organisms in the hospital environment may occur in South America in the coming years through the clonal expansion of hospital-adapted isolates and with the horizontal transfer of virulence and colonization determinants.
This report is dedicated to the memory of Carlos Carrillo.
We thank the Universidad El Bosque for financial support and are indebted to Karen Jiménez and German Contreras for technical assistance. We also thank Maria Virginia Villegas for facilitating the coordination of the participating centers in Colombia. We are grateful to Kavindra S. Singh and Jaime Moreno for providing technical expertise and Jouko Sillanpaa for critical review of the manuscript.
The following personnel and hospitals participated in the collection of isolates: in Bogotá, Colombia, Claudia Londoño and Martha Herrera (Fundación Salud Bosque); Constanza Correa (Hospital Simón Bolívar); Norma Montoya (Clínica de Occidente); Wilson Daza and Martha Uzeta (Clínica del Niño); Narda Olarte and Martha Garzón (Hospital El Tunal); Gloria Gallo (Hospital Santa Clara); Fernando Peñaloza and Nubia Escobar (Hospital Occidente de Kennedy); Martha Ruiz (Clínica San Pedro Claver); Carlos Álvarez, Nidia Torres, and Ziomara González (Hospital San Ignacio); Clara Luz Rico (Fundación Santa Fe de Bogotá); Giovanni Rodríguez and Deise Rojas (Clínica Infantil Colsubsidio); Juan Benavides, Maritza Pérez, and Esperanza Guevara (Clínica Saludcoop Jorge Piñeros Corpas); and Patricia Arroyo (Instituto Nacional de Cancerología); in Cali, Colombia, María Virginia Villegas and Beatriz Vanegas (Centro Médico Imbanaco); María del Socorro Rojas (Clínica Saludcoop Occidente Cali); and Ernesto Martínez and Nancy Villamarín (Hospital Universitario del Valle); in Medellín, Colombia, Sergio Jaramillo and Jaime López (Hospital Pablo Tobón Uribe) and Magda Cárdenas (Clínica Saludcoop Juan Luis Londoño); in Bucaramanga, Colombia, Adriana Pinto (Clínica La Foscal and Fundación Cardiovascular); in Neiva, Colombia, Marino Cabrera and Luz Eneyda Quintero (Hospital Universitario Hernando Moncaleano Perdomo) and Carmen Elisa Llanos (Hospital Universitario San Jorge); in Ecuador, Hospital Vozandes, Hospital Eugenio Espejo, Hospital Baca Ortiz, Hospital Carlos Andrés Andrade Marín, and Hospital General de las Fuerzas Armadas; in Perú, Gene Martínez Medina and Susana Kuwae de Okuhama (Laboratorio Clínico Carlos Carrillo); Federico Yañez Rojas and Liliana Alvarado (Hospital Nacional Sergio Bernales); Greenlandia Ferreyros Brandon and María Silva (Instituto Nacional de Enfermedades Neoplásicas); and Rosa Avurio Usca and Gladys Patiño Soto (Hospital Nacional Hipólito Unanue); and in Venezuela, Adele Rizzi (Centro Médico de Caracas and Hospital Vargas de Caracas).
This work was funded in part by an independent research grant from Pfizer, SA. C.A.A. and B.E.M. are supported by a K99/R00 Pathway to Independence Award (award 4R00 AI72961) and R01 grant AI067861 from the National Institute of Allergy and Infectious Diseases, respectively. D.P. was partially funded by a graduate scholarship from the Instituto Colombiano para el Desarrollo de la Ciencia y Tecnología, Francisco José de Caldas, COLCIENCIAS; and S.R. was supported by an ASM-PAHO Infectious Disease Epidemiology and Surveillance Fellowship.
C.A.A. has received lecture fees from Pfizer, Novartis, and Merck and grant support from Pfizer. B.E.M. has had grant support from Johnson & Johnson, Astellas, Palumed, and Intercell and has served as consultant for Astellas Pharma US Inc., Theravance Inc., Cubist, Targanta Therapeutics Corporation, Johnson & Johnson, Pfizer, AstraZeneca, and Wyeth-Ayerst. J.Z. reports the receipt of research grants from Pfizer and Wyeth. M.G. has served as consultant for Pfizer, Merck and Co., Wyeth, and Becton and Dickinson.
Published ahead of print on 10 March 2010.