Enterococci are emerging as important nosocomial pathogens due to their ability to acquire and spread genes responsible for antibiotic resistance. However, the role of other virulence factors of Enterococci in causing the disease cannot be neglected.²

Enterococci are well-adapted to areas with low redox potential, such as the oral cavity, gut, and genitourinary tract.^9,12 Enterococci first adhere to specific host tissues, invade, and exert their pathogenic effects. In unfavorable environments, the expression of various enterococcal traits ultimately contributes to virulence.¹³

With the emergence of new virulence factors in Enterococci, they evolved like other pathogenic organisms. Antibiotic resistance has been the best studied in E. faecium. In contrast, virulence traits have been studied in E. faecalis. Increased virulence is assumed to be directly associated with increased antibiotic resistance. However, cost–benefit analyses have shown that virulence and antibiotic resistance are two completely different aspects of bacterial cells; hence, increased virulence may or may not be associated with increased antibiotic resistance in bacterial cells.^13-15 Therefore, intrinsic virulence and antibiotic resistance play roles in the pathogenesis of diseases caused by Enterococci; however, in a complementary way.^7,16

In our study, of the 75 clinical Enterococci isolates, the predominant species isolated was E. faecalis (55%), followed by E. faecium (45%). The distribution ratio of E. faecalis to E. faecium is similar to that reported in other studies.^17-20

We studied biofilm formation, hemolysin, and gelatinase production in fecal and clinical isolates. We observed no significant differences in the production of hemolysin and gelatinase between the fecal and clinical isolates. However, for biofilm production, a statistically significant difference (P <0.05) was observed between the clinical and fecal isolates, similar to studies by Upadhyaya et al. and Jett et al.^3,11 Nosocomial strains are known to develop different mechanisms of colonization and cause infection. One of these mechanisms is the production of biofilms that facilitate both surface adherence and invasion of host cells by the organism causing the infection. Our results concord with those of Ira et al., who reported that gelatinase production was statistically significant among clinical isolates.²

Gelatinase production was considerably higher than biofilm and hemolysin production in E. faecalis isolates (46%) than in E. faecium (21%) (p = 0.0281). Thus, biofilm production is responsible for more infections by E. faecalis than by E. faecium. Gelatinase and hemolysin production help Enterococci spread infection, thus increasing the severity of the infection. The production of gelatinase and hemolysin helps acquire and meet Enterococci’s nutritional needs. Our results are similar to those reported by Fernandes et al. and Sood et al.^1,19

In this study, biofilm detection using the microtiter plate method was more sensitive (49%) than the Congo Red agar (23%) and tube methods (39%). In contrast, a study by Ruchi et al.²⁰ showed that the Congo Red agar method was more sensitive (40%) for biofilm detection than the tube method (37%) and microtiter plate method (27%).

The microtiter plate method is preferred because it is easy to perform, cost-effective, and assesses the biofilm-forming capacity of an isolate both qualitatively and quantitatively. The subjective error in interpreting biofilm production findings was overcome using an enzyme-linked immunosorbent assay reader. Other methods, such as epifluorescence microscopy and microscopic biofilm formation assays, have been used to study the biofilm-forming abilities of bacteria. The Congo Red agar method is a reproducible and rapid technique that is sensitive to the added advantage of test colonies on the medium remaining viable. Moreover, subjective errors during reporting were observed more frequently with the tube method than with the Congo Red agar method.^2,21

In this study, biofilm production was higher in clinical isolates than in fecal isolates. This likely plays a key role in the pathogenesis of diseases caused by Enterococci. These results are similar to those reported by Fernandes et al., Hemalatha et al., and Marothi et al.^1,16,21

Enterococci have been associated with biofilm production on various types of indwelling devices, such as prosthetic heart valves, urinary catheters, and artificial hip prostheses, and this capability to produce biofilms is considered an important virulence factor of this organism.²