Journal of Laboratory Physicians
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Year : 2010  |  Volume : 2  |  Issue : 2  |  Page : 100-104 Table of Contents   

Comparative study for the presence of enterococcal virulence factors gelatinase, hemolysin and biofilm among clinical and commensal isolates of Enterococcus faecalis

1 Department of Microbiology, Kempegowda Institute of Medical Sciences, Bangalore, Karnataka, India
2 Department of Microbiology,PGI-ESIMSR, Model Hospital, Bangalore, Karnataka, India

Date of Web Publication29-Oct-2010

Correspondence Address:
P M Giridhara Upadhyaya
Department of Microbiology, Kempegowda Institute of Medical Sciences, Bangalore, Karnataka
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Source of Support: None, Conflict of Interest: None

DOI: 10.4103/0974-2727.72159

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Background : Biofilm production, gelatinase and hemolysin are the potential virulence factors of Enterococci. Gelatinase and hemolysin producing strains of Enterococcus faecalis have been shown to cause severe infections in animal models. Biofilm production has been shown to enhance the persistence of E. faecalis in urinary bladder and other medical indwelling devices infections.
Aims : To compare the presence of gelatinase, hemolysin and biofilm formation among clinical and commensal isolates and to study the co-relation between virulence factors with respect to different clinical specimens.
Settings and Design : During the study period of 2 years from July 2004 to July 2006, 200 clinical isolates from nosocomial infections and 100 commensal isolates of E. faecalis were taken for the study.
Materials and Methods : The clinical and commensal isolates were tested for the presence of gelatinase, hemolysin and biofilm and compared. The presence of these virulence factors among different clinical isolates was also studied.
Statistical Analysis : Chi-square and likelihood ratio analysis were carried out using SSPS version 5.1 software.
Results : The clinical isolates produced 39, 16.5 and 32.5% of gelatinase, hemolysin and biofilm, respectively, as compared to 31, 19 and 16% produced by the commensal isolates, respectively. Endotracheal tube infection, urinary tract infection, umbilical catheter tip infected isolates produced 60.8, 86.6 and 100% biofilm, respectively.
Conclusion : Significant difference in the production of biofilm (P<0.001) was noted between clinical and commensal isolates. Organism isolated from medically indwelling devices produced high amount of biofilm, confirming its role in colonization and causing nosocomial infections.

Keywords: Biofilm, Enterococcus, gelatinase, hemolysin, virulence factors

How to cite this article:
Giridhara Upadhyaya P M, Umapathy B L, Ravikumar K L. Comparative study for the presence of enterococcal virulence factors gelatinase, hemolysin and biofilm among clinical and commensal isolates of Enterococcus faecalis. J Lab Physicians 2010;2:100-4

How to cite this URL:
Giridhara Upadhyaya P M, Umapathy B L, Ravikumar K L. Comparative study for the presence of enterococcal virulence factors gelatinase, hemolysin and biofilm among clinical and commensal isolates of Enterococcus faecalis. J Lab Physicians [serial online] 2010 [cited 2020 Jan 25];2:100-4. Available from:

   Introduction Top

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],[4],[5],[6],[7],[8],[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],[11],[12],[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 Top

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 1]a-d.
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 Top

Production of biofilm, hemolysin and gelatinase

Seventy-eight (39%) clinical isolates and 31 (31%) commensal isolates were gelatinase producing and 33 (16.5%) clinical and 19 (19%) commensal isolates produced hemolysin.

Sixty-five (32.5%) clinical isolates were positive for biofilm production, out of which 23 (11.5%) were high biofilm producers and the remaining 42 (21%) were moderate biofilm producers.

Among the commensal isolates, 16 (16%) were biofilm producers with 4 producing high and 12 producing moderate amounts of biofilm [Table 1].
Table 1 :Gelatinase, hemolysin and biofilm production between clinical and commensal isolates

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[Table 2] shows the age, sex and clinical cases (no. and %) from which E. faecalis was isolated.
Table 2 :Demographic and clinical characteristics of 200 patients with nosocomial infection due to E. faecalis

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Production of combination of hemolysin, gelatinase and biofilm

Eighteen (9%) clinical isolates produced all the three virulence factors. Fifteen (7.5%) isolates produced biofilm and gelatinase. Seven (3.5%) isolates produced biofilm and hemolysin. Six (3%) isolates produced gelatinase and hemolysin. Twenty-five (12.5%) isolates produced only biofilm. Thirty-nine (19.5%) and two (1%) isolates were positive for only gelatinase and hemolysin production, respectively.

Among the commensal isolates, 6 (6%) were positive for all the three factors. Two (2%) were positive for biofilm and gelatinase, and one (1%) was positive for biofilm and hemolysin production. Seven (7%) isolates produced only biofilm. Twenty-three (23%) and 12 (12%) were positive for only gelatinase and hemolysin production, respectively.

Relationship between clinical characteristics and virulence factors

The relationship between clinical characteristics and virulence factors is shown in [Table 3] and [Table 4].
Table 3 :Production of the three virulence factors in isolates from different clinical conditions

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Table 4 :Production of combination of different virulence factors among clinical isolates

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Relationship between virulence factors and antibiotic susceptibility

Among the E. faecalis clinical isolates tested, one (0.5%) was resistant for Vancomycin and none of the isolates were resistant to Linezolid. All the commensal isolates were sensitive to Vancomycin and Linezolid. Clinical isolate which was resistant to Vancomycin was positive for biofilm production.

   Discussion Top

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.

   References Top

1.Tendulkar PM, Baghdayan AS, Shankar N. Pathogenic Enterococci: New developments in the 21 st Century. Cell Mol Life Sci 2003;60:2622-36.  Back to cited text no. 1
2.Vergis EN, Shankar N, Chow JW, Hayden MK, Snydman DR, Zervos MJ, et al. Association between the presence of Enterococcal virulence factors gelatinase, haemolysin and enterococcal surface protein and mortality among patients with bacteremia due to Enterococcus faecalis. Clin Infect Dis 2002;35:570-5.  Back to cited text no. 2
3.Hancock LE, Gilmore MS. Pathogenicity of Enterococci. In: Fischetti V, Novick R, Ferretti J, Portnoy D, Rood J, editors,. Gram positive pathogens. Washington DC:Am Soc Microbiol 2000;251-8.   Back to cited text no. 3
4.Garsin AD, Sifri CD, Mylonakis E, Qin X, Singh KV, Murray BE, et al. A simple model host for identifying gram positive virulence factors. Proc Natl Acad Sci U S A 2001;98:10892-7.   Back to cited text no. 4
5.Jett BD, Huycke MM, Gilmore MS. Virulence of Enterococci. Clin Microbiol Rev 1994;7:462-78.   Back to cited text no. 5
6.Nallapareddy SR, Qin X, Weinstock GM, Hook M, Murray BE. Enterococcus faecalis adhesion, Ace mediates attachment to extracellular matrix proteins collagen type IV and laminin as well as collagen Type I. Infect Immun 2000;68:5218-24.   Back to cited text no. 6
7.Sartingen S, Rozdzinski E, Muscholl-Silberhorn A. Aggregation substances increases adherence and internalization but not translocation of Enterococcus faecalis through different intestinal epithelial cells invitro. Infect Immun 2000;68:6044-7.   Back to cited text no. 7
8.Hass W, Shepard BD, Gilmore MS. Two-component regulator of Enterococcus faecalis cytolysin responds to quorum-sensing autoinduction. Nature 2002;415:84-7.   Back to cited text no. 8
9.Huycke MM, Abrams V, Moore DR. Enterococcus faecalis produces extracellular superoxide and hydrogen peroxide that damages colonic epithelial cell DNA. Carcinogenesis 2002;23:529-6.  Back to cited text no. 9
10.Upadhyaya PG, Ravikumar KL, Umapathy BL. Review of virulence factors of Enterococcus: An emerging nosocomial pathogen. Indian J Med Microbiol 2009;27:301-5.  Back to cited text no. 10
11.Coque TM, Patterson JE, Steckleberg JM, Murray BE. Incidence of hemolysin, gelatinase, and aggregation substance among Enterococci isolated from patients with endocarditis and other infections and from feces of hospitalized and community-based persons. J Infect Dis 1995;171:1233-9.  Back to cited text no. 11
12.Jett BD, Jensen HG, Nordquist RE, Gilmore MS. Contribution of the pAD1-encoded cytolysin to the severity of experimental Enterococcus faecalis endophthalmitis. Infect Immun 1992;60:2445-52.   Back to cited text no. 12
13.Ike Y, Hashimoto H, Clewell DB. Hemolysin of Streptococcus faecalis subspecied zymogenes contributes to virulence in mice. Infect Immun 1984;45:528-30.  Back to cited text no. 13
14.Shankar V, Baghdayan AS, Huycke MM, Lindahl G, Gilmore MS. Infection-derived Enterococcus faecalis strains are enriched in esp, a gene encoding a novel surface protein. Infect Immun 1999;67:193-200.  Back to cited text no. 14
15.Chow JW, Thal LA, Perri MB, Vazquez JA, Donabedian SM, Clewell DB. Plasmid-associated hemolysin and aggregation substance production contribute to virulence in experimental enterococcal endocarditis. Antimicrob Agents Chemother 1993;37:2474-7.  Back to cited text no. 15
16.Ross PW. Streptococcus and Enterococcus. In: Collee JG, Fraser AG, Marmion BP, Simmons A, editors. Practical medical microbiology. New Delhi: Curchill Livingstone; 2006. p. 263-73.  Back to cited text no. 16
17.Jayanthi S, Ananthasubramanian N, Appalaraju B. Assessment of pheromone response in biofilm forming clinical isolates of high level gentamycin resistant Enterococcus faecalis. Indian J Med Microbiol 2008;23:248-51.  Back to cited text no. 17
18.Clinical and Laboratory Standards Institute (CLSI). Performance standards for antimicrobial susceptibility testing, M100-S16. Wayne, PA: CLSI; 2006.  Back to cited text no. 18
19.Gilmore MS, Huycke MM, Daniel FS. Multidrug-resistant Enterococci. The nature of the problem and an agenda for the future. Emerg Infect Dis 1998;4:239-49.  Back to cited text no. 19


  [Figure 1]

  [Table 1], [Table 2], [Table 3], [Table 4]

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