J Med Microbiol 53 (2004), 1069-1073; DOI: 10.1099/jmm.0.45654-0
© 2004 Society for General Microbiology
ISSN 0022-2615
Occurrence of virulence-associated genes in clinical Enterococcus faecalis strains isolated in Londrina, Brazil
Elisa Bittencourt de Marques and
Sérgio Suzart
Laboratory of Microbial Pathogenicity, Department of Microbiology, State University of Londrina, Londrina, Paraná, Brazil
Correspondence Sérgio Suzart ssuzart{at}uel.br
Received February 27, 2004
Accepted August 5, 2004
Epidemiological studies have reinforced the importance of Enterococcus faecalis in causing serious infections, and to date, our understanding of how certain virulence factors are involved in the pathogenesis of enterococcal infections is still limited. The aim of the present study was to examine the occurrence of known virulence determinants in a group of E. faecalis strains isolated from different clinical sources in Brazil. A total of 95 E. faecalis strains were investigated for the presence of nine virulence genes including aggA, cylA, cylB, cylM, eep, efaA, enlA, esp and gelE by using PCR. The data showed a relatively wide distribution of the virulence genes among the investigated strains. The clinical strains carried at least one and concomitantly up to as many as eight virulence markers, with two or three being the most common pattern. Most of the strains carried efaA (58.9 %), eep (58.9 %) and esp (57.9 %) genes, whereas the remaining virulence markers were detected in variable percentages ranging from 9.5 to 45 %. Simultaneous presence of virulence markers was observed among clinical strains regardless of their sources. In this study, the efaA+ esp+ gelE+ profile was the virulence genotype most frequently detected among E. faecalis strains. Finally, there was no significant association between virulence markers and clinical sources.
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INTRODUCTION
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Enterococci are natural inhabitants of the gastrointestinal tract of humans and animals, but are also found in other anatomical sites including the vagina and oral cavity, and in plants and insects (Devriese et al., 1992; Muller et al., 2001; Pabich et al., 2003). Several reports have documented that the two most important species, Enterococcus faecalis and Enterococcus faecium, are among the leading causes of several human infections, including bacteraemia, septicaemia, endocarditis, urinary tract infections, wound infections, neonatal sepsis and meningitis (Lautenbach et al., 1999; Giacometti et al., 2000; Higaki et al., 2002).
Numerous factors are associated with a greater risk of acquiring enterococcal infections. These factors, including antimicrobial resistance and expression of virulence factors associated with infection-derived E. faecalis strains, may account for the establishment and maintenance of this opportunistic pathogen as major community-acquired and nosocomial pathogens. E. faecalis strains possesses several putative virulence determinants, including haemolysin/bacteriocin (also called cytolysin) (Ike et al., 1990), enterolysin A (Nilsen et al., 2003), aggregation substance (AS) (Galli et al., 1990), gelatinase (Su et al., 1991), enterococcal surface protein (Esp) (Shankar et al., 1999), adhesion-associated protein EfaA (E. faecalis endocarditis antigen A) (Lowe et al., 1995), enhanced expression of pheromone (Eep) (An et al., 1999) and other factors. Currently, our knowledge about the contribution of these virulence factors to the pathogenesis of enterococcal infections is still limited.
The purpose of the present study was to assess the occurrence of putative virulence factors in 95 E. faecalis strains isolated from different clinical specimens of ambulatory and hospitalized patients seen at the Regional University Hospital of Northern Paraná in Londrina, Paraná State, Brazil.
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METHODS
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Bacterial strains, media and culture conditions.
The enterococcal strains used in the present study were isolated from 95 patients who attended the Regional University Hospital of Northern Paraná in Londrina, Paraná State, Brazil between July 1999 and July 2000. To prevent over-representation of clinical strains, only one isolate per patient was included in this study. A total of 95 E. faecalis strains from ambulatory and hospitalized patients were from the following specimen sources: 50 from urine (patients with urinary tract infection), 26 from purulent exudates (purulent umbilical cord, abdominal secretion, secretion of renal fistula, tracheal secretion, bone fragment, splenic aspirate and peritoneal fluid), and 19 from rectal swabs (patients colonized with E. faecalis, but absence of enterococcal infections). All clinical strains were routinely grown on BHI agar plates or in BHI broth (Acumedia) and then incubated under aerobic conditions, without agitation, at a constant temperature of 37 °C for 18 h.
Conventional biochemical identification.
Species identification of the clinical strains was performed using an automated MicroScan WalkAway 96 Instrument (Dade MicroScan) according to the manufacturer's instructions. Concomitantly, all strains were also confirmed by the conventional biochemical identification scheme of Facklam & Collins (1989), including colony morphology, Gram staining, tolerance to bile-aesculin, growth in 6.5 % NaCl, tolerance to 0.04 % tellurite, catalase test, pyrolidonyl arylamidase test, deamination of arginine, motility, pigmentation and several carbohydrate fermentation tests (arabinose, glycerol, lactose, mannitol, melibiose, raffinose, ribose, sorbose, sorbitol, sucrose and trehalose).
Bacterial DNA extraction.
Template DNAs for PCR were extracted by the boiling method described by Yost & Nattress (2000) with minor modifications. Briefly, a single bacterial colony grown on an agar plate was picked, inoculated in 3 ml BHI broth and incubated overnight at 37 °C. Afterwards, the late exponential phase culture was centrifuged at 11 430 g for 10 min at 4 °C and the resultant bacterial pellet suspended in 500 µl sterile MilliQ water. Cell suspensions were heated at 100 °C in a boiling water bath for 30 min to lyse cells and cellular debris was removed by centrifugation. Template DNA contained in 150 µl supernatant was stored at –20 °C until subsequent PCR amplification.
PCR primer design and amplification of putative virulence genes.
Most of the oligonucleotide primers used in this study were previously reported elsewhere (Eaton & Gasson, 2001), except for the eep, gelE and enlA virulence genes, for which primers were designed based on published DNA sequences from the NCBI database. The primer sequences, annealing temperatures and expected sizes of amplicons for each PCR assay are shown in Table 1.
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Table 1. Oligonucleotide primers and conditions used to amplify different virulence marker genes in E. faecalis strains by PCR
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PCRs were performed in a final volume of 25 µl containing 10 mM Tris/HCl (pH 8.4), 50 mM KCl, 1.5 mM MgCl2, 50 pmol of each forward and reverse primer, 200 µM of each dNTP, 2.0 U Taq DNA polymerase (Biosystems Ltda), and 10 µl template DNA (prepared as described above). The reaction mixtures were amplified in a Hybaid PCR Sprint Thermal Cycler (ThermoHybaid) with an initial denaturation at 94 °C for 5 min, followed by 30 cycles of 94 °C for 45 s, annealing at a temperature specific for each primer pair (52 to 62 °C; see Table 1) for 1 min, 72 °C for 1 min, and a final extension step at 72 °C for 3 min. The negative control was performed for each set of PCRs containing all reagents but no DNA template. All amplifications reactions were carried out in duplicate. E. faecalis OG1RF (which carried the gelE gene), and DS16 (which carried the cylM, cylB, cylA, aggA genes) was used as positive controls. These reference strains were kindly provided by Dr Sabbir Simjee, Division of Animal and Food Microbiology, Center for Veterinary Medicine, US Food and Drug Administration (FDA). E. faecalis OG1X (which carried the eep gene) and strain 594 (which carried the esp gene) were kindly provided by Dr Phillip S. Coburn, Department of Microbiology and Immunology at the University of Oklahoma, and included in the PCR assays as positive controls. The amplicons were analysed by electrophoresis in 1 % agarose gels using TBE buffer (pH 8.0). The gels were stained with ethidium bromide and visualized on a UV transilluminator (Hoefer UV-25; Pharmacia Biotech). The size of amplified products was estimated by comparison with a 1 kb DNA ladder marker (Amersham Pharmacia Biotech).
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RESULTS AND DISCUSSION
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Various studies have identified and characterized a variety of determinants of virulence in E. faecalis strains (Galli et al., 1990; Ike et al., 1990; Su et al., 1991; Lowe et al., 1995; An et al., 1999; Shankar et al., 1999; Nilsen et al., 2003); however, a limited number of these factors have been shown to contribute effectively to enterococcal pathogenicity in animal models. In Brazil, no report has been published so far concerning the occurrence of known virulence determinants in a collection of E. faecalis strains isolated from different clinical sources.
In the present study, the occurrence of several putative virulence factors of E. faecalis varied with the clinical source of the strains. Apart from cylB and cylM genes, most of the virulence genes examined were commonly found in the strains isolated from different specimens. Results in Table 2 show that more than half of the clinical strains included in our report harboured efaA and eep gene markers. On the other hand, Eaton & Gasson (2001) and Creti et al. (2004) found the efaA gene in all the clinical E. faecalis strains investigated. Furthermore, the presence of the esp gene was also investigated in this study. Our data indicate that the presence of this virulence marker was slightly higher than those reported by other authors (Creti et al., 2004; Eaton & Gasson, 2001; Franz et al., 2001).
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Table 2. Frequency of genetic virulence markers among E. faecalis strains isolated from different clinical sources
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In addition, the gelE and aggA genes were found in 45.3 and 36.8 %, respectively, whereas the remaining virulence determinants were detected in less than 17 % of the strains tested. Our study contrasts with a previous study that reported a higher percentage of E. faecalis strains carrying gelE and aggA genes (Semedo et al., 2003). We also focused our investigation on the incidence of the gene encoding enterolysin A among E. faecalis strains. This virulence determinant was found at a low rate (9.5 %) among clinical strains, with a tendency for it to be present more often among urinary strains than in purulent exudates and rectal swab strains. To our knowledge, this is the first study that has investigated the occurrence of the enlA gene in different clinical sources.
In examining the distribution of the genes that make up the cytolysin operon (cylA, cylB, cylM) in all the strains studied, we found that the genes cylA, cylB and cylM, whose products are involved in the activation, secretion and post-translational modification of cytolysin, respectively, are distributed equally in all bacterial strains, except in strains isolated from purulent exudates. Data from recent studies (Eaton & Gasson, 2001; Franz et al., 2001; Archimbaud et al., 2002; Creti et al., 2004), are contradictory with regard to the presence of these virulence markers in E. faecalis strains probably due to the presence of strains isolated only from endocarditis, bacteraemia and/or faeces of volunteers, or in some cases from a bacterial specimen of animal, food or human origin.
The distribution of the virulence determinants among E. faecalis strains isolated from different clinical sources is summarized in Table 2. When we carefully examine the results relative to the distribution of genetic markers of virulence in the different groups of clinical strains, we see that the majority of the virulence markers were more frequently found among urinary strains than in other clinical sources. Therefore, we speculated that the high incidence of multiple virulence factors in E. faecalis strains from urine samples could contribute, to a certain extent, to bacterial colonization and growth in the development of urinary infection.
Recently, Creti et al. (2004) showed that E. faecalis strains derived from different sources possessed distinct patterns of virulence factors. In the present study, the results revealed that the clinical strains carried at least one and concomitantly up to as many as eight virulence markers. The majority of the strains harboured between two and three virulence determinants (Table 3). None of the strains examined showed all of the virulence genes investigated in this study. In general, the urinary as well as the purulent exudate strains carried predominantly three virulence genes concomitantly. On the contrary, two virulence genes were found to coexist preferentially in strains derived from rectal swabs. Generally, the genotype efaA+ esp+ gelE+ was predominantly found in 20 (21 %) of the 95 clinical strains studied, where it was more prevalent in strains derived from rectal swabs and urinary strains than those isolated from purulent exudate (Table 3). Therefore, in some manner, it was observed that the efaA gene is frequently associated with esp or eep and to a lesser extent with virulence markers aggA or gelE.
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Table 3. Genetic linkage of virulence factor-related gene clusters among E. faecalis strains isolated from clinical sources
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The pathogenic role of several virulence factors identified in E. faecalis is still questionable and the meaning of these determinants in strains recovered from clinical strains is uncertain. In the near future, various studies will be conducted in our research laboratory with the aim of ascertaining the role of these virulence determinants in the mechanism of microbial pathogenesis in animal models. We aim thereby to study a combination of clinical strains containing specific virulence factors capable of contributing to enterococcal infection in an animal model different from those reported in the literature to date.
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ACKNOWLEDGEMENTS
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This research was supported by grants from CNPq (Conselho Nacional de Desenvolvimento Científico e Tecnológico) and PROPPG/UEL (Pró-Reitoria de Pesquisa e Pós-graduação da Universidade Estadual de Londrina). We are grateful to Professor Floristher Elaine Carrara of the Departamento de Patologia Aplicada, Legislação e Deontologia (PALD/UEL) for furnishing the clinical strains of Enterococcus faecalis. We thank Dr Albert Leyva for helpful discussion and for critical reading of this manuscript.
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INTRODUCTION
METHODS
