Comparative Study for the Presence of Enterococcal Virulence Factors Gelatinase, Hemolysin and Biofilm Among Clinical and Commensal Isolates of Enterococcus Faecalis

INTRODUCTION

Enterococci are gram positive bacteria that normally inhabit the gastrointestinal tract of many animals including humans. However, when they colonize habitats where they are not normally found, these opportunistic bacteria can become pathogens.^[1]

Incidence of Enterococcal infections, especially hospital acquired, has dramatically increased over the last 25 years. Enterococci have been reported recently as a major cause of nosocomial infections, being increasingly detected in postoperative wound infections, blood stream and urinary tract infections (UTIs). Enterococcus faecalis is responsible for approximately 80-90% of all Enterococcal infections.

They are intrinsically resistant to or tolerant to many antibiotics and are readily able to acquire resistance to antibiotics, either by mutation or by acquisition of plasmids or transposons containing genetic sequences that confer resistance in other bacteria.^[2] A number of studies have identified different virulence factors, the most important among them being ^[3-9] gelatinase, hemolysin, enterococcal surface protein (Esp), aggregation substance (AS), MSCRAMM Ace (microbial surface component recognizing adhesive matrix molecule adhesion of collagen from Enterococci), serine protease, capsule, cell wall polysaccharide and superoxide. These factors have been associated with the virulence of E. faecalis in animal models.^[10-13] It is not clear whether the presence of these factors in E. faecalis isolates from clinical and commensal isolates contribute to the virulence in humans.

Esp or biofilm is a cell wall associated protein in E. faecalis isolates. Frequency of gene coding for Esp has been higher among clinical isolates than among commensal isolates.^[14] Esp is shown to enhance the persistence of E. faecalis in urinary bladder during experimental UTIs.

Gelatinase is a protease produced by E. faecalis. It is capable of hydrolyzing collagen, casein, hemoglobin and other peptides.^[10] Gelatinase producing strains of E. faecalis have been shown to contribute to the virulence of endocarditis in an animal model. Hemolysin is a cytolytic protein capable of lysing human, horse and rabbit erythrocytes. Hemolysin producing strains are found to be associated with increased severity of infections.^[15]

The present study evaluates E. faecalis isolates from nosocomial infections and stool samples to compare the production of these three virulence factors between clinical and commensal isolates.

MATERIALS AND METHODS

Three hundred and ninety-seven Enterococcus spp. were isolated over a period of 2 years from different clinical specimens. Two hundred isolates were confirmed as E. faecalis by biochemical reactions ^[16] and taken up for the study. Hundred commensal isolates of E. faecalis were isolated from stool samples. Isolates were grown on trypticase-soy agar for subsequent testing.

Biofilm ^[17] formation was detected by inoculating the isolates into trypticase-soy broth [TSB] with 0.5% glucose and incubated at 37ºC. After overnight incubation, the culture was diluted 1:40 in fresh TSB-0.5% glucose. Two hundred microliters of the diluted solution was added to flat-bottomed polystyrene microtiter well and incubated for 48 hours at 37ºC. Wells were gently washed three times with distilled water. After drying the plates in an inverted position at room temperature for 1 hour, the adherent biofilm was stained with 0.1% safranin and allowed to stand for 20 minutes at room temperature. Absorbance of the biofilm on the bottom surface of each well of the dried plates was determined at 490 nm in an enzyme-linked immunosorbent assay (ELISA) reader. Test was carried out in triplicate and the average of the three optical density (OD) values was taken. Culture medium without organism was taken as blank. Biofilm producing E. faecalis OG1RF was taken as positive control. Mean OD value of positive control was taken as standard. Those values above 0.2 were considered as high biofilm producers. Values below 0.081 were categorized into low or non-biofilm producers. OD values above the standard but within 0.081 and 0.2 were taken as moderate biofilm producers [Figure 1a-d].

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Figure 1:

(a) E. faecalis isolates inoculated in microtiter plates containing TSB with 0.5% glucose; (b) plates incubated with E. faecalis for 48 hours washed three times with distilled water; (c) biofilm containing plates stained with 0.1% safranin for 20 minutes; (d) wells in microtiter plates showing biofilm formation after staining with safranin

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Hemolysin production was detected by inoculating Enterococci onto freshly prepared beef heart infusion agar supplemented with 5% horse blood. Plates were incubated overnight at 37ºC in a carbon dioxide chamber and ^[2] evaluated at 24 and 48 hours. A clear zone of β-hemolysis around the streak on horse blood agar was considered to be a positive indication of hemolysin production. Gelatinase production was detected by inoculating the organism onto freshly prepared peptone-yeast extract agar containing 30 g/L of gelatine.^[2] Plates were incubated overnight at 37ºC and then cooled to ambient temperature for 2 hours. The appearance of a turbid halo or zone around the colonies was considered to be a positive indication of gelatinase production.

Testing for susceptibility to Vancomycin and Linezolid was done with the use of E test strips (Hi-Media, Mumbai, India). Minimum inhibitory concentration Multi Drug Resistant (MDR) break points recommended by Clinical and Laboratory Standards Institute (CLSI) ^[18] were used.

RESULTS

DISCUSSION

E. faecalis is an important cause of hospital borne infections. We determined and compared the prevalence of three virulence factors, biofilm, hemolysin and gelatinase, among clinical and commensal isolates. Study shows that with respect to hemolysin and gelatinase production, there is no significant difference between the clinical and commensal isolates (P ≥ 0.174).

Nosocomial strains of organisms develop various mechanisms in colonizing to cause infection. Biofilm production is an important factor which helps the organism to adhere onto surfaces, which facilitates later in invasion and causing infection. This study shows a significant difference in the production of biofilm (P<0.001).

A comparative study among the different clinical isolates with respect to production of the three virulence factors shows that biofilm production is very high among isolates grown from UTI, endotracheal tube infection and umbilical catheter tip infection as shown in Table 3.

We conclude that biofilm production in nosocomial strains of organisms is an important pathogenic factor in causing infection in the hospital environment. Other virulence factors also have a role in the occurrence of infection and in the clinical outcome. Further study on the other virulence factors will throw some light on the mechanism of pathogenesis in E. faecalis.

There was no significant relationship between virulence factors, ability to cause infection and antibiotic susceptibility to Vancomycin and Linezolid, as only one clinical isolate was resistant to Vancomycin and none for Linezolid.

Given the importance of Enterococcus as a nosocomial pathogen and increasing prevalence of MDR Enterococcus as shown by other studies, ^[19] the identification of virulence factors associated with invasiveness and disease severity has become an important subject for research. Development of other mechanisms like blocking of Enterococcal biofilm production or inhibiting the action of other virulence factors may provide an alternate method of therapy in the face of antimicrobial resistance.