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Brazilian Journal of Microbiology

Print version ISSN 1517-8382On-line version ISSN 1678-4405

Braz. J. Microbiol. vol.37 no.3 São Paulo July/Sept. 2006

http://dx.doi.org/10.1590/S1517-83822006000300007 

MEDICAL MICROBIOLOGY

 

Virulence-associated characteristics of Enterococcus faecalis strains isolated from clinical sources

 

Características associadas à virulência de Enterococcus faecalis isolados de casos clínicos

 

 

Márcia T. FurumuraI; Patricia M.S. FigueiredoI; Gleize V. CarbonellII; Ana Lucia da Costa DariniIII; Tomomasa YanoI,*

IDepartamento de Microbiologia e Imunologia, Instituto de Biologia, Universidade Estadual de Campinas, Campinas, SP, Brasil
IISeção de Biologia Médica, Instituto Adolfo Lutz, Campinas, SP, Brasil
IIIDepartamento de Análises Clínicas, Toxicológicas e Bromatológicas, Faculdade de Ciências Farmacêuticas de Ribeirão Preto, Universidade de São Paulo, Ribeirão Preto, SP, Brasil

 

 


ABSTRACT

Thirty-two clinical isolates of Enterococcus faecalis were screened for virulence factors. Twenty-four (75%) isolates produced hemolysin on Mueller-Hinton blood agar plates with sheep erythrocytes. However, the cell free heat-stable hemolysin was detected in all isolates (100%) of E. faecalis when grown in BHI-GA (BHI medium supplemented with 1% glucose and 0.03% L-arginine), but not in BHI broth alone. Twenty-four isolates (75%) produced caseinase and 23 (71.9%) lipase, but none of the isolates produced gelatinase. Fifteen (46.9%) culture filtrates caused rounding and membrane alterations with blebbing formation followed by death in HeLa and HEp-2 cells, but not in Vero cells. Thirteen isolates (40.6%) agglutinated rabbit erythrocytes, but did not produce hemagglutination in other bloods, containing or not 1% D-manose. Sixteen (50%) E. faecalis isolates adhered to HeLa cells and thirteen (40.6%) to HEp-2 cells, but all isolates adhered to polypropylene microtiter plates, indicating that clinical E. faecalis possess the ability to form biofilm in vitro. All the isolates were resistant to the bactericidal action of normal serum and did not produce aerobactin. These findings suggest that adherence and consequently biofilm formation on ephitelial host cells are the first steps in the E. faecalis virulence and that hemolysin, lipase, caseinase and other virulence factors act as causative of human epithelial cell damages.

Key words: Enterococcus faecalis, virulence factors, hemolysin,proteases, lipase, cytotoxin, biofilm


RESUMO

Foram estudados os fatores de virulência de trinta e duas amostras de Enterococcus faecalis, isolados de casos clínicos. Vinte e quatro amostras (75%) produziram hemolisina em ágar sangue preparado com hemácias de carneiro. No sobrenadante da cultura em BHI nenhuma amostra produziu hemolisina, no entanto quando cultivadas em meio BHI suplementado com 1% de glucose e 0,03% de L-arginina (BHI-GA), 100% das amostras lisaram hemácias de carneiro. Vinte e quatro (75%) amostras produziram caseinase e 23 (71,9%) lipase, mas nenhuma amostra produziu gelatinase. Dezesseis (46,9%) causaram arredondamento e alteração na membrana das células, com formação de vesículas e, em seguida, a morte das células HeLa e HEp-2. Treze amostras (40,6) aglutinaram eritrócitos de coelhos, mas não aglutinaram outros eritrócitos na presença ou na ausência de 1% de D-manose. Dezesseis (50%) aderiram em células HeLa e 13 (40,6%) em células HEp-2, mas todas as amostras de E. faecalis aderiram na microplaca de polipropileno, indicando que E. faecalis isolados de casos clínicos possuem capacidade de formar biofilme "in vitro". Todos os isolados mostraram-se resistentes à ação bactericida do soro normal e não produziram aerobactina. Esses resultados sugerem que, inicialmente, a colonização ou infecção por E. faecalis ocorre pela aderência e formação de biofilme nas células epiteliais e a produção de hemolisina, lípase e caseinase pode atuar como fatores de virulência na infecção por E. faecalis.

Palavras-chave: Enterococcus faecalis, hemolisina, proteases, lípase, citotoxina, fatores de virulência


 

 

INTRODUCTION

Infective endocarditis, sepsis and urinary tract infections are serious infections caused by Enterococcus faecalis, a microorganism which is now a pervasive clinical problem due to its resistance to most antibiotics (21).The involvement of E. faecalis in other types of infections is less clear, partly because of its frequent occurrence in mixed infections. However, the literature (7) has indicated that enterococci are emerging as prominent nosocomial pathogens in a variety of infections, including wound and genital infections, and bacteremia. The increasing clinical significance of enterococcal infections and the emergence of strains resistant to all available therapeutic alternatives have focused interest on factors possibly associated with the colonization and pathogenesis of enterococci (9).

Enterococcal virulence factors such as hemolysin (Hly), aggregation substance (Agg or AS)and an enterococcal protease, commonly referred to as gelatinase (Gel), have been suggested as possible contributors to the virulence of E. faecalis(8,19). Some of these factors, including Cyl, Agg and Esp seem to be encoded on a large pathogenicity island (29).

Hemolysin is commonly produced by Gram-positive and Gram-negative bacteria and, in most cases, is considered to be a virulence factor, although its relative contribution to disease is variable among microbes and different host species (28). Due to the importance of Enterococcus species as nosocomial pathogens and the increasing prevalence of antimicrobial resistance among enterococci, the identification and characterization of virulence factors, associated with enterococcal invasiveness and severity of disease, are important areas of investigation.

In the present study we investigated the virulence factors in E. faecalis strains isolated from different clinical sources.

 

MATERIALS AND METHODS

Bacterial strains

Thirty-two E. faecalis isolates were included in this study. They were collected in urine, blood, wounds or catheters from admitted patients (at least for five days) to the Hospital das Clínicas, Faculty of Medicine of Ribeirão Preto, University of São Paulo (USP), a Brazilian teaching hospital with 551 beds. The identification of all isolates was performed by API 20 Strep (bioMérieux) and PCR was performed to confirm species identification according to Dukta-Malen et al. (11). Control strains were E. faecalis NCTC 775, E. faecium NCTC 7171, E. gallinarum NCTC 12359, and E. casseliflavus NCTC 12361.

Hemolytic assay

The hemolytic activity of E. faecalis was assessed on blood agar plates prepared with Mueller-Hinton agar (MHA, Biolife, Italy) containing defibrinated sheep blood (final blood concentration, 5% v/v), by observation of the hemolysis zone around colonies after incubation for 24 h at 37ºC (4).Extracellular hemolysin was assayed on microplates using 100 mL of culture supernatant from E. faecalis grown on bothBHI (Oxoid) and BHI supplemented with 1% glucose and 0.03% L-arginine (3) (BHI-GA). The supernatants were serially diluted with 5 mM phosphate buffered saline (PBS), pH 7.4, mixed with an equal volume of a 1% sheep erythrocytes suspension and incubated at 37ºC for 1 h, followed by a further overnight incubation at 4ºC.

Physico-chemical properties of the culture filtrates

Heating the samples at 45ºC, 60ºC and 100ºC for 30 min assessed the heat-stability of culture supernatants. The effect of proteolysis on activity was examined by treating the samples with trypsin, protease K and pepsin (100 mg/mL each) at 37ºC for 60 min (18). Residual hemolytic activity was measured on microplates as described above.

Protease production

Casein hydrolysis was tested on MHA containing 3% (w/v) skimmed milk, by streaking the plates with 10 mL of each suspension followed by incubation at 37ºC for 24 h. The presence of a transparent zone around the colonies indicated caseinase activity (1). Gelatinase production was detected by inoculating the enterococci onto trypticase soya broth agar (TSA) containing 3% gelatine (Oxoid). The appearance of turbid halos or zones around the colonies after incubation at 37ºC for 24 h was considered to be a positive indication of gelatinase production (33).

Lipase activity

Lipase activity was determined as described by Gunn et al. (13): 10 mL of bacterial suspension was placed into wells cut into 1% (w/v) agarose in PBS containing 1% egg yolk and incubated at 37ºC for 48h. The appearance of an opaque zone around colonies indicated lipolytic activity.

Cytotoxicity assay

Bacteria were cultured in 5 mL of BHI-GA at 37ºC for 18 h. The cultures were centrifuged (6,000 x g, 15 min, 4ºC) and the supernatants were filtered through 0.22 mm membranes (Millipore, USA) prior to testing on Vero (African green monkey kidney), HeLa (human colon adenocarcinoma), or HEp-2 (human larynx epidermoid carcinoma) cells. The cells were grown in 96-well tissue microplates with Eagle's minimal essential medium (EMEM, Nutricell) containing 10% fetal calf serum (Nutricell) (27)and incubated in a 5% CO2 atmosphere at 37ºC. The morphological changes were assessed using an inverted microscope (Nikon Instruments, Japan), after 24h incubation.

Hemagglutination tests

After centrifugation, the E. faecalis bacterial pellet was suspended in 5 mM PBS, pH 7.4, to a final concentration of 109 CFU/mL. An aliquot (50 mL) of this suspension was added to an equal volume of a 2% (v/v) suspension of horse, sheep or rabbit erythrocytes containing or not 1% D-mannose and the plates incubated at 37ºC for 1 h.

Bacterial adhesion to cultured cells

The ability of E. faecalis isolates to adhere to HEp-2 and HeLa cells was determined as previously described by Scaletsky et al. (30) with slight modifications. The cells were grown in MEM (Eagle's minimal essential medium) supplemented with 10% fetal calf serum in 24-well tissue culture microplates, in which sterile round coverslips were placed.

After 30 minutes incubation with the E. faecalis suspension at 37ºC, the monolayers were washed with sterile PBS and incubated again for 3 hours. Thereafter, the monolayers were washed with PBS, fixed with methanol for 10 min and stained by May-Grunwald and Giemsa for observation under bright field microscopy. Previously described criteria were used to determine the adhesion pattern (30).

Biofilm formation assay

E. faecalis (109 CFU/30 mL) were cultured in BHI in 96-well microtiter plates at 37ºC for 48 h. After incubation, the broth was removed and added 0.5%crystal violet for five minutes. The plates were then washed with water and 200 mL of 95% of ethanol was added. The biofilm formation was considered positive when an optical density at 570 nm (Labsystem Multiskan Bichromatic) was equal or major than 0.2 (34).

Sensitivity to the bactericidal effect of normal serum

The sensitivity of E. faecalis to the bactericidal effect of human normal serum was tested as described by Pelkonen and Finne (24). Bacteria grown in BHI for 18 h were diluted in PBS (109 bacteria/mL) and 175 mL of the bacterial suspension and 175 mL of PBS were pipetted into the wells of microtiter plates. One hundred microliters of serum (final concentration 36%) were added to the wells and the plates were incubated at 37ºC. The absorbance at 630 nm was measured at 0, 30, 60, 90, 120 and 180 min. The plates were shaken before each measurement, to avoid the influence of bacterial sedimentation on the final absorbance. The strains were classified as resistant, intermediate or sensitive as suggested by Taylor (32).

Aerobactin production

The production of aerobactin was detected by cross feeding using Escherichia coli LG 1522 as an indicator. These bacteria express receptors for the ferric-aerobactin complex. Lawns of E. coli (109 CFU/mL) were spread on supplemented M9 minimal agar plates containing a,a-dipyridil, and E. faecalis was spotted onto the lawn. Aerobactin production was detected as a satellite halo which has grown after overnight incubation at 37ºC, as described by Carbonetti et al. (5).

 

RESULTS

Hemolysin production

Twenty-four (75%) E. faecalis isolates showed hemolytic activity on Muller-Hintonagar containingsheep red blood cells (RBC) (Table 1). None of the culture supernatants from E. faecalis grown in BHI showed hemolytic activity, whereas all (100%) supernatants from bacteria grown in BHI-GA hemolysed sheep RBC.

 

 

Physico-chemical properties

The hemolytic activity of E. faecalis culture supernatants was neither affected by heating nor by treatment with papain and proteinase K (data not shown).

Lipase and Protease production

Twenty-three E. faecalis isolates (71.9%) showed lipase activity. Protease activity on skimmed milk was produced by 24 isolates (75%) but no one showed gelatinase activity (Table 1).

Cytotoxicity assay

Table 1 shows that 15 (46.9%) culture supernatant filtrates from E. faecalis grown in BHI-GA caused rounding and cell membrane alterations by blebbing formation followed by death of HeLa (Fig. 1A) and HEp-2 cells (Fig. 1B) after a 24 hours period.

 

 

Hemagglutination tests

Table 1 shows that 13 E. faecalis isolates (40%) agglutinated only rabbit erythrocytes in either absence or presence of 1% D-mannose. The hemagglutinating activity of E. faecalis isolates was completely inhibitedby proteinase K (100 mg/mL) but was unaffected by trypsin and pepsin (data not shown).

Bacterial adhesion to cultured cells and biofilm formation

Sixteen (50%) E. faecalis isolates adhered to HeLa cells (data not shown) and thirteen (40.6%) adhered to HEp-2 cells (Fig. 2). The 32 E. faecalis isolates formed biofilms on polypropylene plates (data not shown).

 

 

Sensitivity to the normal serum bactericidal effect and aerobactin production

All E. faecalis isolates were resistant to the bactericidal action of normal serum and none produced aerobactin (data not shown).

 

DISCUSSION

Enterococci are Gram-positive cocci inhabitants of the gastrointestinal tract of many mammals, including humans, and are a frequent cause of a wide variety of infections involving the urinary tract, bloodstream, endocardium, abdomen, biliary tract and burn wounds (7,21). Hemolysin-producing strains of E. faecalis are virulent in animal models and human infectionsand are associated with increasing infection severity (29).

In this study, all isolates have been previously analyzed by PFGE, the results revealing extensive heterogeneity and differentiation of all isolates into 32 types with Dice similarity coefficients between 20 and 92% (data not shown).

Of the clinical E. faecalis isolates, 75% produced the hemolytic halo around colonies on Mueller-Hinton sheep blood agar plates (Table 1). These findings differ from those of Miyazaki et al. (23) who detected hemolytic activity on several animal erythrocytes (except cow and sheep) included in BHI agar.

Vergis et al. (33) detected cell-free hemolysin on horse blood in 11% of E. faecalis. In this study, all E. faecalis isolates (100%) exhibited extracellular hemolytic activity towards sheep erythrocytes only when grown in BHI-GA.Heating at 100ºC did not abolish the hemolytic activity, indicating that the hemolysin is heat-stable. This activity was also resistant to proteolytic enzymes (data not shown). However, BHI culture supernatants did not show hemolytic activity on sheep blood suggesting that glucose and arginine are essential elements for heat-stable hemolysin production.

Gelatinase, an extracellular metalloprotease secreted by E. faecalis, hydrolyzes gelatin, collagen, and casein, and has been implicated as a virulence factor in animal models.The ability of this enzyme to hydrolyze collagens and certain bioactive peptides suggests its participation in the initiation and propagation of inflammatory processes involving E. faecalis (35). Vergis et al. (33) showed that 64% of E. faecalis isolated from patients with bacteremia, produced gelatinase, although no activity was found in the E. faecalis isolates described in this report. However, the data does suggest that the gelatin hydrolyzing activity is different from caseinase, detected in 75% of E. faecalis isolates (Table 1).

A variety of bacterial lipases, enzymes able to hydrolyze or synthesize triacylglycerols, have been described (15) but data relating these lipases to bacterial virulence factors are scarce. Among bacterial lipases, the enzymes from Staphylococcus epidermidis and Pseudomonas are well known but are yet to be defined as bacterial virulence factors (31).

The most prominent role of microorganism extracellular lipases may be the digestion of host cellular lipids for nutrient acquisition, which results in sticking to host tissue and neighboring cells (31).For Staphylococcus aureus it has been postulated that its lipase enhances adhesion by degrading host surface molecules, thereby liberating new receptors. Additionally, released free fatty acids (FFAs) might increase unspecific hydrophobic interactions, as it is assumed for Propionicbacterium acnes (22),the causative agent of acne vulgaris, a condition quite common during puberty. Undoubtedly, the biological role of lipases in infections by many microorganisms might be considered the most important step in bacterial infections (6,15,31).

In this study, 23 E. faecalis clinical isolates (71.8%) presented lipolytic activity. This frequency may be significant and suggests an important virulence property of E. faecalis in causing infections. However, no studies relating E. faecalis to lipases production were found in the literature.

Culture supernatants of some E. faecalis isolates(Table 1) caused rounding, a loss of intracellular junctions and the appearance of morphological features associated with apoptosis (Fig. 1). As a result, the cells became spherical and showed extensive surface blebbing. Similar morphological changes were reported by Falcón et al. (12), who observed that the cytotoxic enterotoxin produced by Aeromonas hydrophila caused cell detachment, cytoplasmatic disorganization and blebbing. Epithelial cell death by apoptosis may contribute to the action of this hemolysin. Therefore, further studies of this and other virulence factors in E. faecalis are necessary to establish their role in infections caused by the pathogen.

As shown in Table 1, only 13 (40.6%) of the 32 E. faecalis isolates examined caused agglutination of rabbit erythrocytes in absence or presence of 1% D-mannose; this hemagglutination was inhibited by proteinase K (data not shown). Kurl et. al. (18) reported that hemagglutination caused by strains of Streptococcus suis was completely abolished by incubation with proteases and temperature-dependent. Activity was greater at 0ºC than at 22ºC or 37ºC, and rapidly lost at high temperatures. In contrast, as shown here, hemagglutination by isolates was unaffected by heating at 45ºC, 60ºC and 100ºC (data not shown).

Potential enterococcal adherence factors involved in virulence include cell surface carbohydrates, homologues of cell surface adhesins found on a number of enterococcal species (20),that share characteristics with cell surface proteins of Gram-positive bacteria and can act as a collagen adhesin (26) or as an aggregation substance (Agg). Bacterial adherence to host cells is the initial event in many infections (2). However, little information is available on the factors that promote adhesion of E. faecalis to host tissues (1). Kreft et al. (17) showed that 27.6% of E. faecalis strains can adhere to human urinary tract epithelial cells. Guzmàn et al. (14) also demonstrated that clinical isolates of E. faecalis have a greater capacity to adhere to urinary tract epithelial cells and that ability of bacteria to adhere to host epithelial cells is an essential step in the pathogenicity of infections. In our study, sixteen E. faecalis isolates(50%) adhered to HeLa cells (data not shown) and thirteen isolates (40.6%) adhered to HEp-2 cells (Fig. 2) but, did not show the pattern of adherence described for diarrheagenic Escherichia coli (30).

The ability to adhere to biomaterials is an important bacterial property, commonly implicated in the colonization and infection of catheters, but microbial adherence to non-biological surfaces depends on bacterial surface characteristics and on the nature of the inert material (15). Among the factors involved in this type of adherence are physico-chemical forces, such as polarity, van der Waal's forces and hydrophobic interactions (10) but factors involved in enterococcal adherence to biomaterials have not been well defined (19).

In the present study, we observed that all isolates of clinical E. faecalis showed ability to form biofilms on polystyrene plates, according to the methodologies described by Wakimoto et al. (34).

Bacteria associated with septicemia are frequently resistant to serum. Since the main role of the serum bactericidal system is to prevent microorganisms from invading and persisting in the blood, differences in the degree of bacterial susceptibility to serum determine whether a microorganism can infect and persist in a given organism (25). All the E. faecalis strains examined in this investigation were resistant to serum. The growth of bacteria in host tissue is limited, not only by host defense mechanisms, but also by the availability of iron, an essential factor for bacterial growth that functions mainly as a redox catalyst for proteins participating in oxygen and electron transport (25). E. faecalis did not produce aerobactin, indicating that these strains were not iron-bound to host proteins or cell-dependent.

Our results suggest that the main virulent property of clinical E. faecalis is adherenceto epithelial host cells, leading to biofilm formation and consequently to the production of hemolysin, caseinase, lipase and other virulence factors, causative of human epithelial cell damages.

Further studies about virulence properties of E. faecalis are necessary to fully understand the pathogenicity of the bacteria. However, the findings in this study provide some new information in this direction.

 

ACKNOWLEDGMENT

The authors thank Ana Stella Menegon Degrossoli for technical assistance. We thank the Microbiology Laboratory of the Hospital das Clínicas de Ribeirão Preto, Universidade de São Paulo, Ribeirão Preto, Brazil, for kindly providing the E. faecalis strains. Financial support was provided by Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) and Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq), Brazil.

 

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Submitted: September 26, 2005; Returned to authors for corrections: January 11, 2006; Approved: April 19, 2006

 

 

* Corresponding author. Mailing address: Departamento de Microbiologia e Imunologia, IB, Universidade Estadual de Campinas. Caixa Postal 6109. 13083-862, Campinas, SP, Brasil. Tel.: (+5519) 3788-6254, Fax: (+5519) 3788-6276 E-mail: tyano@unicamp.br

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